Noritsugu Tohse

Effects of MS-551, a new class III antiarrhythmic drug, on action potential and membrane currents in rabbit ventricular myocytes.

1 Electrophysiological effects of MS‐551, a new class III antiarrhythmic drug, were examined and compared with those of (+)‐sotalol in rabbit ventricular cells. 2 In rabbit ventricular muscles stimulated at 1.0 Hz, MS‐551 (0.1–10 μm) and (+)‐sotalol (3–100 μm) prolonged action potential duration (APD) and effective refractory period without affecting the maximum upstroke velocity of phase 0 depolarization . The class III effect of MS‐551 was approximately 30 times more potent than that of (+)‐sotalol. 3 Class III effects of MS‐551 and (+)‐sotalol showed reverse use‐dependence, i.e., a greater prolongation of APD at a longer cycle length. 4 In rabbit isolated ventricular cells, 3 μm MS‐551 and 100 μm sotalol inhibited the delayed rectifier potassium current (IK) which was activated at more positive potentials than −50 mV and saturated around + 20 mV. 5 MS‐551 at a higher concentration of 10 μm decreased the transient outward current (Ito) and the inward rectifier potassium current (IK1) although 100 μm sotalol failed to inhibit these currents. 6 MS‐551 is a non‐specific class III drug which can inhibit three voltage‐gated K+ channels in rabbit ventricular cells.

Cyclic GMP-mediated inhibition of L-type Ca2+ channel activity by human natriuretic peptide in rabbit heart cells.

1 Effects of atrial natriuretic peptide (ANP) on the L‐type Ca2+ channels were examined in rabbit isolated ventricular cells by use of whole‐cell and cell‐attached configurations of the patch clamp methods. ANP produced a concentration‐dependent decrease (10–100 nm) in amplitude of a basal Ca2+ channel current. 2 The inactive ANP (methionine‐oxidized ANP, 30 nm) failed to decrease the current. 3 8‐Bromo‐cyclic GMP (300 μm), a potent activator of cyclic GMP‐dependent protein kinase (PKG), produced the same effects on the basal Ca2+ channel current as those produced by ANP. The cyclic GMP‐induced inhibition of the Ca2+ channel current was still evoked in the presence of 1‐isobutyl‐3‐methyl‐xanthine, an inhibitor of phosphodiesterase. ANP failed to produce inhibition of the Ca2+ channel current in the presence of 8‐bromo‐cyclic GMP. 4 In the single channel recording, ANP and 8‐bromo‐cyclic GMP also inhibited the activities of the L‐type Ca2+ channels. Both agents decreased the open probability (NP0) without affecting the unit amplitude. 5 The present results suggest that ANP inhibits the cardiac L‐type Ca2+ channel activity through the intracellular production of cyclic GMP and then activation of PKG.

Effects of ATP‐sensitive K+ channel blockers on the action potential shortening in hypoxic and ischaemic myocardium

1 In order to determine whether activation of adenosine triphosphate (ATP)‐sensitive K+ channels exclusively explains the hypoxia‐ and ischaemia‐induced action potential shortening, effects of tolbutamide and glibenclamide on changes in action potential duration (APD) during hypoxia, metabolic blockade or experimental ischaemia were examined in guinea‐pig and canine isolated myocardium by standard microelectrode techniques. 2 With use of patch clamp techniques, activity of ATP‐sensitive K+ channels was recorded from open cell‐attached patches of guinea‐pig isolated ventricular myocytes. The probability of opening of the K+ channels was decreased by 2 mm tolbutamide and 20 μm glibenclamide to almost the same extent, whereas it was increased by 100 μm pinacidil. 3 In guinea‐pig papillary muscles a marked shortening of the action potential produced by 100 μm pinacidil was completely antagonized by 2 mm tolbutamide or 20 μm glibenclamide. 4 In guinea‐pig papillary muscles exposed to hypoxic, glucose‐free solution or dinitrophenol (10 μm)‐containing, glucose‐free solution, APD declined gradually and twitch tension decreased. Pretreatment with glibenclamide partially but significantly inhibited the action potential shortening, whereas tolbutamide failed to improve it during hypoxia or metabolic blockade. 5 When in canine isolated myocardium, experimental ischaemia was produced by the cessation of coronary perfusion, APD was gradually shortened. The action potential shortening was partially but not completely inhibited by pretreatment with 20 μm glibenclamide. 6 These results suggest that changes in membrane current(s) other than the outward current through ATP‐sensitive K+ channels also contribute to the action potential shortening in hypoxic or ischaemic myocardium.

Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes

OBJECTIVE The aim of this study was to characterize the spatio-temporal dynamics of [Ca(2+)](i) in rat heart in the fetal and neonatal periods. METHODS Using confocal scanning laser microscopy and the Ca(2+) indicator fluo-3, we investigated Ca(2+) transients and Ca(2+) sparks in single ventricular myocytes freshly isolated from rat fetuses and neonates. T-tubules were labeled with a membrane-selective dye (di-8-ANEPPS). Spatial association of dihydropyridine receptors (DHPR) and ryanodine receptors (RyR) was also examined by double-labeling immunofluorescence. RESULTS Ca(2+) transients in the fetal myocytes were characterized by slower upstroke and decay of [Ca(2+)](i) compared to those in adult myocytes. The magnitude of fetal Ca(2+) transients was decreased after application of ryanodine (1 microM) or thapsigargin (1 microM). However, Ca(2+) sparks were rarely detected in the fetal myocytes. Frequent ignition of Ca(2+) sparks was established in the 6-9-day neonatal period, and was predominantly observed in the subsarcolemmal region. The developmental change in Ca(2+) sparks coincided with development of the t-tubule network. The immunofluorescence study revealed colocalization of DHPR and RyR in the postnatal period, which was, however, not observed in the fetal period. In the adult myocytes, Ca(2+) sparks disappeared after disruption of t-tubules by glycerol incubation (840 mM). CONCLUSIONS The sarcoplasmic reticulum (SR) of rat ventricular myocytes already functions early in the fetal period. However, ignition of Ca(2+) sparks depends on postnatal t-tubule formation and resultant colocalization of DHPR and RyR.

Alpha 1-adrenoceptor stimulation enhances the delayed rectifier K+ current of guinea pig ventricular cells through the activation of protein kinase C.

The effect of alpha 1-adrenoceptor stimulation on the delayed rectifier K+ current (IK) was examined in isolated guinea pig ventricular cells by use of the patch-clamp method. IK was evoked by a 3-second depolarizing pulse from a holding potential of -30 mV in a Na(+)- and K(+)-free solution containing 3 microM nifedipine. Phenylephrine (30 microM) in the presence of propranolol (1 microM) produced an increase in IK. In five cells, phenylephrine increased the tail current of IK by 23 +/- 5%. This effect of phenylephrine was blocked by prazosin (0.3 microM), a selective alpha 1-blocker. Phenylephrine produced only a small effect on the voltage and time dependence of IK. Pretreatment with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 10 microM) abolished the phenylephrine-induced increase in IK. In addition, pretreatment with a maximally effective concentration of 12-O-tetradecanoylphorbol 13-acetate (100 nM) abolished the phenylephrine-induced increase in IK. In conclusion, alpha 1-adrenoceptor stimulation increases IK in guinea pig cardiomyocytes. This alpha 1-adrenoceptor-mediated response may be related to an activation of protein kinase C. The increase in IK may explain a shortening of action potential duration observed after alpha 1-adrenoceptor stimulation in guinea pig cells.

A de novo missense mutation of human cardiac Na+ channel exhibiting novel molecular mechanisms of long QT syndrome

Mutations in a human cardiac Na+ channel gene (SCN5A) are responsible for chromosome 3‐linked congenital long QT syndrome (LQT3). Here we characterized a de novo missense mutation (R1623Q, S4 segment of domain 4) identified in an infant Japanese girl with a severe form of LQT3. When expressed in oocytes, mutant Na+ channels exhibited only minor abnormalities in channel activation, but in contrast to three previously characterized LQT3 mutations, had significantly delayed macroscopic inactivation. Single channel analysis revealed that R1623Q channels have significantly prolonged open times with bursting behavior, suggesting a novel mechanism of pathophysiology in Na+ channel‐linked long QT syndrome.

Regression of Left Ventricular Hypertrophy Prevents Ischemia-Induced Lethal Arrhythmias Beneficial Effect of Angiotensin II Blockade

To evaluate the preventive effect of regression of left ventricular hypertrophy (LVH) on sudden cardiac death (SCD), the incidence of ventricular tachycardia or ventricular fibrillation (VT/Vf) after left coronary artery occlusion in Langendorff preparations was studied in the following five groups: (1) spontaneously hypertensive rats (SHR) without treatment (SHR-N), (2) SHR treated with captopril (SHR-C), (3) SHR treated with the angiotensin II receptor antagonist TCV-116 (SHR-A), (4) SHR treated with hydralazine (SHR-H), and (5) Wistar-Kyoto (WKY) rats. Although blood pressure was equally lowered in all treated groups, SHR-C and SHR-A but not SHR-H showed regression of LVH. The incidence of VT/Vf was 5% in WKY rats, 63% in SHR-N (P < .005 versus WKY rats), 0% in SHR-C, 10% in SHR-A, and 45% in SHR-H (P < .05 versus WKY rats). Further evaluation of the effect of TCV-116 revealed that SHR treated with a low dose of TCV-116 (1 mg/kg per day) showed a decrease in left ventricular mass with only a little decrease in blood pressure and that the incidence of VT/Vf was reduced in association with the degree of regression of LVH. Electrophysiological study using microelectrode techniques revealed that in the LVH groups (SHR-N and SHR-H), the action potential duration (APD) of the left ventricular papillary muscle was more prolonged than in WKY rats, whereas APD shortened to a greater extent during superfusion with a hypoxia/no-glucose solution. APD showed no difference in the regression groups (SHR-C and SHR-A) compared with the WKY group.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect mediated by α1-adrenoceptors on transient outward current in isolated rat ventricular cells

In order to clarify the underlying ionic mechanism(s) by whichα1-adrenoceptor stimulation prolongs the action potential duration (APD), single rat ventricular cells were voltage-clamped under a Na+-free condition using patch pipettes. Depolarizing pulses from a holding potential of -77 mV induced a 4-aminopyridine-sensitive transient outward current (Ito). Phenylephrine, in the presence of theβ-blocker propranolol (1μM), inhibited Ito in a concentration-dependent fashion and the maximum inhibition of Ito (42.5±10.0%, n=5) was produced by 30μM phenylephrine. The inhibitory effect of phenylephrine on Ito was almost abolished by 1μM. prazosin, a selectiveα1-blocker, indicating that the Ito inhibition is mediated byα1-adrenoceptors. On the other hand, phenylephrine had little influence on the Ca2+ current in the presence of 4-aminopyridine. In isolated rat papillary muscles, both theα1-adrenoceptor-mediated APD prolongation and positive inotropic response were markedly attenuated by pretreatment with 1.5 mM 4-aminopyridine. These results suggest that the inhibition of Ito is a primary cause of the prolongation of APD produced byα1-adrenoceptor stimulation and that the Ito inhibition may be causally related to the positive inotropic effect mediated byα1-adrenoceptors.

Mechanism of the membrane depolarization induced by oxidative stress in guinea-pig ventricular cells.

Mechanism of the membrane depolarization induced by oxidative stress was examined using ion-selective microelectrode and patch clamp techniques. In guinea-pig papillary muscles stimulated at 0.5 Hz, cumene hydroperoxide (CH) at a concentration of 300 microM decreased the resting membrane potential and shortened the action potential, concomitantly with muscle contracture. The membrane depolarization was not associated with a significant decrease in intracellular potassium ion activity, indicating that the depolarization is not due to a decrease in potassium equilibrium potential resulting from leak of intracellular K+. In isolated guinea-pig ventricular cells. CH (10-30 microM) consistently decreased the inward rectifier potassium current and slightly decreased the calcium current. In cell-attached patches CH inhibited the opening of the inward rectifier K+ channel without affecting the unit amplitude of the single channel current. Thus, the depolarization of the resting membrane induced by oxidative stress is, at least in part, due to the inhibition of the inward rectifier K+ channel activity, and may play an important role in the genesis of reperfusion-induced arrhythmias.

Cyclic GMP Regulation of Calcium Slow Channels in Cardiac Muscle and Vascular Smooth Muscle Cells

The effects of cAMP and cGMP on the slow Ca2+ channels in cardiac muscle, VSM, and skeletal muscle fibers are summarized in Table V. As shown, in cardiac muscle, cAMP stimulates and cGMP inhibits. In VSM, both cAMP and cGMP inhibit. In skeletal muscle, both cAMP and cGMP stimulate.