Masayasu Hiraoka

The positive dynamic current and its inactivation properties in cardiac Purkinje fibres

1. The positive dynamic current in sheep cardiac Purkinje fibres was studied using ‘voltage clamp’ technique. The major portion of this current was carried by Cl− ions.

A novel SCN5A mutation associated with idiopathic ventricular fibrillation without typical ECG findings of Brugada syndrome.

Mutations in the human cardiac Na+ channel α subunit gene (SCN5A) are responsible for Brugada syndrome, an idiopathic ventricular fibrillation (IVF) subgroup characterized by right bundle branch block and ST elevation on an electrocardiogram (ECG). However, the molecular basis of IVF in subgroups lacking these ECG findings has not been elucidated. We performed genetic screenings of Japanese IVF patients and found a novel SCN5A missense mutation (S1710L) in one symptomatic IVF patient that did not exhibit the typical Brugada ECG. Heterologously expressed S1710L channels showed marked acceleration in the current decay together with a large hyperpolarizing shift of steady‐state inactivation and depolarizing shift of activation. These findings suggest that SCN5A is one of the responsible genes for IVF patients who do not show typical ECG manifestations of the Brugada syndrome.

Novel Mechanism of HERG Current Suppression in LQT2: Shift in Voltage Dependence of HERG Inactivation

In a Xenopus oocyte heterologous expression system, we characterized the electrophysiology of 3 novel missense mutations of HERG identified in Japanese LQT2 families: T474I (within the S2-S3 linker), A614V, and V630L (in the outer mouth of pore-forming region). For each of the 3 mutations, injection of mutant cRNA alone did not express detectable currents. Coinjection of wild-type (WT) along with each mutant cRNA (T474I/WT, A614V/WT, and V630L/WT) suppressed HERG current in a dominant-negative manner, and the order of magnitude of current suppression was V630L/WT>A614V/WT>T474I/WT. In addition to decreases in slope conductance for all 3 mutants, the voltage dependence of steady-state inactivation was shifted to negative potentials for V630L/WT and A614V/WT. Consequently, channel availability at positive potentials was diminished, and inward rectification was enhanced for these 2 mutants. Thus, missense mutations of HERG caused dominant-negative suppression through multiple mechanisms. The shift in voltage dependence of HERG inactivation and the resulting enhanced inward rectification in A614V/WT and V630L/WT provide a novel mechanism for suppression of the HERG current carrying outward current during the repolarization phase of the action potential.

Increased QT dispersion in patients with vasospastic angina.

BACKGROUND The risk factors for ventricular arrhythmias in patients with coronary vasospasm have not been identified. We evaluated QT dispersion in patients with vasospastic angina and its relation to susceptibility to ventricular arrhythmias during myocardial ischemia and reperfusion. METHODS AND RESULTS We assessed the corrected QT (QTc) dispersion before induction of coronary artery spasm by intracoronary injection of acetylcholine (baseline) and 30 minutes after administration of isosorbide dinitrate in 50 patients with vasospastic angina and 50 patients with atypical chest pain. The baseline QTc dispersion was significantly greater in patients with vasospastic angina than in patients with atypical chest pain (mean+/-SD: 69+/-24 versus 44+/-19 ms, 95% confidence interval of mean difference [CI]: 16 to 33 ms; P<0.001). QTc dispersion decreased significantly, to 48+/-15 ms (CI: 15 to 26 ms; P<0.001 versus baseline), after administration of isosorbide dinitrate in patients with vasospastic angina but did not change significantly in patients with atypical chest pain (mean+/-SD: 41+/-17 ms, CI: -3 to 9 ms). During the provocation test, 24 of 50 patients with vasospastic angina experienced ventricular arrhythmias. The baseline QTc dispersion was significantly greater in patients with than without ventricular arrhythmias (mean+/-SD: 77+/-23 versus 61+/-19 ms, CI: 4 to 26 ms; P<0.05). CONCLUSIONS Patients with vasospastic angina exhibited an increased baseline QTc dispersion compared with patients with atypical chest pain, which suggests that inhomogeneity of repolarization and susceptibility to ventricular arrhythmias are increased in patients with vasospastic angina, even when asymptomatic. The association between increased QTc dispersion and ventricular arrhythmias during the provocation test suggests that measurement of QT dispersion may help predict which patients with vasospastic angina are at high risk for ventricular arrhythmias during ischemia.

Effects of estrogen on action potential and membrane currents in guinea pig ventricular myocytes.

To explore a possible ionic basis for the prolonged Q-T interval in women compared with that in men, we investigated the electrophysiological effects of estrogen in isolated guinea pig ventricular myocytes. Action potentials and membrane currents were recorded using the whole cell configuration of the patch-clamp technique. Application of 17beta-estradiol (10-30 microM) significantly prolonged the action potential duration (APD) at 20% (APD(20)) and 90% repolarization (APD(90)) at stimulation rates of 0. 1-2.0 Hz. In the presence of 30 microM 17beta-estradiol, APD(20) and APD(90) at 0.1 Hz were prolonged by 46.2 +/- 17.1 and 63.4 +/- 11.7% of the control (n = 5), respectively. In the presence of 30 microM 17beta-estradiol the peak inward Ca(2+) current (I(CaL)) was decreased to 80.1 +/- 2.5% of the control (n = 4) without a shift in its voltage dependence. Application of 30 microM 17beta-estradiol decreased the rapidly activating component of the delayed outward K(+) current (I(Kr)) to 63.4 +/- 8% and the slowly activating component (I(Ks)) to 65.8 +/- 8.7% with respect to the control; the inward rectifier K(+) current was barely affected. The results suggest that 17beta-estradiol prolonged APD mainly by inhibiting the I(K) components I(Kr) and I(Ks).

Voltage-shift of the current activation in HERG S4 mutation (R534C) in LQT2

OBJECTIVE Recently, a novel missense mutation (R534C) in the S4 region of human ether-a-go-go-related gene (HERG) was identified in one Japanese LQT2 family. The S4 region presumably functions as a voltage sensor. However, it has not yet been addressed whether the S4 region of HERG indeed functions as a voltage sensor, and whether these residues play any role in abnormal channel function in cardiac repolarization. METHODS We characterized the electrophysiological properties of the R534C mutation using the heterologous expression system in Xenopus oocytes. Whole cell currents were recorded in oocytes injected with wild-type cRNA, R534C cRNA, or a combination of both. RESULTS Clinical features--QTc intervals of all affected patients with R534C mutation in HERG are prolonged ranging from 460 to 680 ms (averaged QTc interval > 540 ms). One member of this family had experienced sudden cardiac arrest, and other suffered from recurrent palpitation. Electrophysiology--Oocytes injected with R534C cRNA did express functional channels with altered channel gating. Kinetic analyses revealed that the R534C mutation shifted the voltage-dependence of HERG channel activation to a negative direction, accelerated activation and deactivation time course, and reduced steady-state inactivation. Quantitative analyses revealed that this mutation did not cause apparent dominant-negative suppression. Computer simulation--Incorporating the kinetic alterations of R534C, however, did not reproduce prolonged action potential duration (APD). CONCLUSIONS The data revealed that arginine at position 534 in the S4 region of HERG is indeed involved in voltage-dependence of channel activation as a voltage sensor. Our examination indicated that HERG current suppression in R534C mutation was the least severe among other mutations that have been electrophysiologically examined, while affected patients did show significant QT prolongation. This suggest that another unidentified factor(s) that prolong APD might be present.

THE TETRAVALENT ORGANIC CATION SPERMINE CAUSES THE GATING OF THE IRK1 CHANNEL EXPRESSED IN MURINE FIBROBLAST CELLS

1. The activation kinetics of the IRK1 channel stably expressed in L cells (a murine fibroblast cell line) were studied under the whole‐cell voltage clamp. Without polyamines or Mg2+ in the pipettes, inward currents showed an exponential activation on hyperpolarization. The steep inward rectification of the currents around the reversal potential (Erev) could be described by the open‐close transition of the channel with first‐order kinetics. 2. When the tetravalent organic cation spermine (Spm) was added in the pipettes, the activation kinetics changed; this was explicable by the increase in the closing rate constant. The activation of the currents observed without Spm or Mg2+ in the pipettes was ascribed to the unblocking of the ‘endogenous‐Spm block’. 3. In the presence of the divalent cation putrescine (Put) or of Mg2+ in the pipettes, a different non‐conductive state suppressed the outward currents on depolarization; the channels instantaneously changed to the open state on repolarization. As the depolarization was prolonged, this non‐conductive state was replaced by the non‐conductive state that shows an exponential activation on repolarization. This phenomenon was attributed to the redistribution of the channels from the Put‐ or Mg(2+)‐blocked state to the ‘endogenous Spm‐blocked state’ during depolarization. 4. In the presence of the trivalent cation spermidine (Spd) in the pipettes, two different non‐conductive states occurred, showing a faster and a slower activation on repolarization. The rectification around Erev was mainly due to the non‐conductive state showing a faster activation, which appeared to be the Spd‐blocked state. During depolarization, redistribution of the channels to the ‘endogenous Spm‐blocked state’ also occurred. 5. In the presence of Spd, Put or Mg2+ in the pipettes, the voltage dependence of the activation time constant reflecting the unblocking of the ‘endogenous Spm’ was shifted in the hyperpolarizing direction. 6. Our results suggest that the ‘intrinsic gating’ that shows the time‐dependent activation on repolarization, and that is responsible for the inward rectification around Erev, reflects the blocking kinetics of the tetravalent Spm.

Characterization of S818L mutation in HERG C-terminus in LQT2

We examined the mechanism(s) for HERG channel dysfunction in an S818L mutation in the HERG C‐terminus using the heterologous expression system in Xenopus oocytes. Injection of S818L cRNA alone did not produce expressed currents. Coinjection of an equal amount of S818L cRNA with wild‐type (WT) cRNA into oocytes did not exhibit apparent dominant‐negative suppression. However, coinjection of excess amounts of S818L cRNAs with WT cRNA into oocytes decreased HERG current amplitudes and shifted the voltage dependence of activation to negative potentials, accelerated its activation and deactivation. The data suggest that S818L alone cannot form functional channels, whereas S818L subunits can, at least in part, coassemble with WT subunits to form heterotetrameric functional channels, and imply that the HERG C‐terminus may contain a domain involving the activation–deactivation process of the channel. These findings may provide new insights into the structure–function relationships of the HERG C‐terminus.

Interaction between external Na+ and mexiletine on Na+ channel in guinea-pig ventricular myocytes

To assess the modulation of Na+ channel block with local anaesthetics by the change of external Na+ concentration ([Na+]o), we examined the block by mexiletine at different [Na+]o using the whole-cell and the cell-attached configurations of the patch-clamp technique. Lowering [Na+]o increased the degree of use dependent block of the whole-cell Na+ current. The external Na+ dependence of the Na+ current block was caused by the interaction of mexiletine with the activated Na+ channel, but not with the inactivated channel. In single-Na+ channel current recordings at a reduced [Na+]o of 70 mM, mexiletine shortened the mean open time of the channels (1.32±0.06 ms in the control vs. 0.86±0.12 ms with the drug, P<0.05) without changes in the unitary current amplitude, whereas the drug did not affect mean open time at a [Na+]o of 140 mM. Moreover, the open time distributions during drug exposure at the reduced [Na+]o were better fitted to a double exponential than to a single exponential in four out of six experiments. These data suggest that mexiletine induces two conductive states: the native open state and a state representing the first step of open channel block. The transition from the former to the latter is dependent on [Na+]o, suggesting an antagonistic interaction of external Na+ with mexiletine.

Effects of low temperature on the positive dynamic current of cardiac Purkinje fibres

1. The effects of low temperature on the positive dynamic current (mainly Cl− current) of sheep cardiac Purkinje fibres were studied using the ‘voltage clamp’ technique.