Junji Toyama

Cellular electropharmacology of amiodarone

The complex profile of amiodarone actions on the electrophysiological properties of cardiac cells reviewed in this article may be summarized as follows. As acute effects, amiodarone inhibits both inward and outward currents. The inhibition of inward Na+ and Ca2+ currents is enhanced in a use- and voltage-dependent manner, resulting in suppression of excitability and conductivity in both iNa- and iCa-dependent cardiac tissues. The inhibition is greater in the tissues stimulated at higher frequencies, and in those with less negative resting (or diastolic) membrane potentials. As outward currents, iK (iKr and iKs), iK,ACh and iK,Na are inhibited by acute amiodarone, iKl could also be inhibited at high concentrations of amiodarone. Acute effects of amiodarone on i(to) remain unclear. Previous reports on the acute effects of amiodarone on APD are conflicting, presumably because different ionic currents are responsible for the repolarization of action potential in different animal species, cardiac tissues and experimental conditions. APD would be shortened if the inhibitory action of amiodarone on the inward current is greater than on the outward current, and vice versa in the opposite case. The major and consistent chronic effect of amiodarone is a moderate APD prolongation with minimal frequency-dependence. This prolongation is most likely due to a decrease in the current density of iK and i(to). Chronic effects of amiodarone are modulated by tissue accumulation of amiodarone and DEA. Variable suppression of excitability and conductivity of the heart by chronic amiodarone might reflect direct acute effects of the parent drug and/or its active metabolite (DEA) retained at the sites of action. Chronic amiodarone was shown to cause a down-regulation of Kv1.5 mRNA in rat hearts, suggesting a drug-induced modulation of potassium channel gene expression. Electrophysiological changes in the heart induced by chronic amiodarone resemble those induced by hypothyroidism. Three mechanisms have been proposed to explain this hypothyroid-like action of amiodarone. Amiodarone and/or DEA may inhibit peripheral conversion from T4 to T3, cellular uptake of T4 and T3, and T3 binding to nuclear receptors (TR). The second and third mechanisms are considered to be more important than the first. Amiodarone or DEA could antagonize T3 action on the heart at a cellular or subcellular level. Two distinct characteristics in the cellular electropharmacology or amiodarone are different from those of other antiarrhythmic drugs. First, it acts on many different types of molecular targets including Na+, Ca2+, and K+ channels as well as adrenoceptors. Second, it may cause antiarrhythmic remodeling of cardiac cells, probably through a modulation of gene expression of ion channels and other functional proteins. We hypothesize that this remodeling is mediated most likely by cellular or subcellular T3 antagonism. Nevertheless, much remains to be studied as ot the acute and especially chronic effects of amiodarone on ionic currents, transporters, receptors and other molecules in cardia cells. The role of the cardiac hypothyroid state in the genesis of antiarrhythmic activity is still a matter of considerable controversy among investigators. Recently, two amiodarone analogues (SR 33589 and ATI-2001) showing a potent acute antiarrhythmic activity in animal models, have been developed [37,87,88,131]. These new compounds are not known to exhibit chronic antiarrhythmic potential or cardiac hypothyroidism activity. Unraveling these tissues will be required to understand the exact molecular and cellular mode of action of amiodarone and to find a new direction for the development of the ideal antiarrhythmic drugs of the future.

Amiodarone: Ionic and Cellular Mechanisms of Action of the Most Promising Class III Agent

Amiodarone is the most promising drug in the treatment of life-threatening ventricular tachyarrhythmias in patients with significant structural heart disease. The pharmacologic profile of amiodarone is complex and much remains to be clarified about its short- and long-term actions on multiple molecular targets. This article reviews electrophysiologic effects of amiodarone based on previous reports and our own experiments in single cells and multicellular tissue preparations of mammalian hearts. As acute effects, amiodarone inhibits both inward and outward currents. The inhibition of inward sodium and calcium currents (I(Na), I(Ca)) is enhanced in a use- and voltage-dependent manner, resulting in suppression of excitability and conductivity of cardiac tissues especially when stimulated at higher frequencies and in those with less-negative membrane potential. Both voltage- and ligand-gated potassium channel currents (I(K), I(K,Na), I(K,ACh)) are also inhibited at therapeutic levels of drug concentrations. Acutely-administered amiodarone has no consistent effect on the action potential duration (APD). The major and consistent long-term effect of the drug is a moderate APD prolongation with minimal frequency dependence. This prolongation is most likely due to a decrease in the current density of I(K) and I(to). Chronic amiodarone was shown to cause a down-regulation of Kv1.5 messenger ribonucleic acid (mRNA) in rat hearts, suggesting a drug-induced modulation of potassium-channel gene expression. Tissue accumulation of amiodarone and its active metabolite (desethylamiodarone) may modulate the chronic effects, causing variable suppression of excitability and conductivity of the heart through the direct effects of the compounds retained at the sites of action. Amiodarone and desethylamiodarone could antagonize triiodothyronine (T3) action on the heart at cellular or subcellular levels, leading to phenotypic resemblance of long-term amiodarone treatment and hypothyroidism.

Three-year follow-up of patients with right bundle branch block and ST segment elevation in the right precordial leads: Japanese registry of Brugada syndrome

Abstract OBJECTIVES We sought to determine the prevalence of right bundle branch block (RBBB) and ST segment elevation in the working Japanese population, as well as the event rate during a three-year prospective follow-up period. BACKGROUND A poor prognosis of RBBB and ST segment elevation has been reported in Europe and South America, even in asymptomatic patients; however, a large population of asymptomatic patients with sporadic RBBB and ST segment elevation has not been studied. METHODS Ten thousand 12-lead electrocardiograms (ECGs) were obtained during annual check-ups of working adults in the Tokyo area. This three-year prospective follow-up study consisted of 105 patients, including 20 with ventricular fibrillation, 18 with syncope and 67 who were asymptomatic. They were registered from 46 institutions in Japan. RESULTS The prevalence of ECG abnormalities in working adults was 0.16%. A coved-type ST segment elevation was related to a history of cardiac events, and 18% of registered patients had PR prolongation and 9.5% had left-axis deviation. The cumulative cardiac event-free rate was 67.6% in the symptomatic group and 93.4% in the asymptomatic group (p = 0.0004) after three years. CONCLUSIONS The recurrence rate of cardiac events in symptomatic patients was similar to that reported previously, but it was very low in sporadic asymptomatic patients. The ECG findings may help us to select patients for further examination and more accurate evaluation of their prognoses.

Correlation between electrical activity and the size of rabbit sino-atrial node cells.

1. Single cells were isolated from rabbit sino‐atrial (SA) node by enzymatic dissociation. Spontaneous action potentials and membrane currents were recorded using the whole‐cell patch clamp technique to study the relationship between electrical activity and the size of the cells. 2. The size of SA node cells was estimated by measuring the cell capacitance. The cell capacitance of SA node cells ranged from 21.8 to 61.5 pF with a mean +/‐ S.E.M. of 38.2 +/‐ 1.3 pF (n = 61). 3. The action potential amplitude, maximum diastolic potential, take‐off potential and action potential upstroke velocity were greater in larger cells. The rate of diastolic depolarization was greater and the intrinsic spontaneous activity was faster in larger cells. 4. The density of hyperpolarization‐activated current (i(f)) was greater in larger cells, whereas the density of L‐type calcium current was not correlated with the size of SA node cells. 5. TTX‐sensitive sodium current (iNa) was absent in small cells with a capacitance of less than approximately 25 pF, and the density of iNa was greater in larger cells. 6. The greater density of iNa in larger cells may explain the higher upstroke velocity of the action potential in large cells, and the greater density of i(f) and iNa could be responsible for the faster intrinsic spontaneous activity of large cells. These results suggest that the SA node consists of electrophysiologically heterogeneous pacemaker cells with different electrical membrane properties.

Heterogeneous distribution of the two components of delayed rectifier K+ current: a potential mechanism of the proarrhythmic effects of methanesulfonanilideclass III agents

OBJECTIVE To elucidate the regional difference of the K+ current blocking effects of methanesulfonanilide class III agents. METHODS Regional differences in action potential duration (APD) and E-4031-sensitive component (IKr) as well as -insensitive component (IKs) of the delayed rectifier K+ current (IK) were investigated in enzymatically isolated myocytes from apical and basal regions of the rabbit left ventricle using the whole-cell clamp technique. RESULTS At 1 Hz stimulation, APD was significantly longer in the apex than in the base (223.1 +/- 10.6 vs. 182.7 +/- 14.5 ms, p < 0.05); application of 1 microM E-4031 caused more significant APD prolongation in the apex than in the base (32.5 +/- 6.4% vs. 21.0 +/- 8.8%, p < 0.05), resulting in an augmentation of regional dispersion of APD. In response to a 3-s depolarization pulse to +40 mV from a holding potential of -50 mV, both IK tail and IKs tail densities were significantly smaller in apical than in basal myocytes (IK: 1.56 +/- 0.13 vs. 2.09 +/- 0.21 pA/pF, p < 0.05; IKs: 0.40 +/- 0.15 vs. 1.43 +/- 0.23, p < 0.01), whereas IKr tail density was significantly greater in the apex than in the base (1.15 +/- 0.13 vs. 0.66 +/- 0.11 pA/pF, p < 0.01). The ratio of IKs/IKr for the tail current in the apex was significantly smaller than that in the base (0.51 +/- 0.21 vs. 3.09 +/- 0.89; p < 0.05). No statistical difference was observed in the voltage dependence as well as activation and deactivation kinetics of IKr and IKs between the apex and base. Isoproterenol (1 microM) increased the time-dependent outward current of IKs by 111 +/- 8% during the 3-s depolarizing step at +40 mV and its tail current by 120 +/- 9% on repolarization to the holding potential of -50 mV, whereas it did not affect IKr. CONCLUSIONS The regional differences in IK, in particular differences in its two components may underlie the regional disparity in APD, and that methanesulfonanilide class III antiarrhythmic agents such as E-4031 may cause a greater spatial inhomogeneity of ventricular repolarization, leading to re-entrant arrhythmias.

Murine cardiac progenitor cells require visceral embryonic endoderm and primitive streak for terminal differentiation

Cardiac progenitor cells in avian and amphibian embryos are known to commit to cardiac lineage during gastrulation or early neurulation. These cells require cell interaction with anterior endoderm for their differentiation into cardiomyocytes. However, little is known about cell interaction in mammalian cardiogenesis. We investigated the staging of murine cardiomyocyte commitment and the role of cell interaction in differentiation of cardiac progenitor cells into cardiomyocytes, using cultures of various embryonic regions at 7.25 and 7.5 days post coitum (p.c.), respectively. To evaluate the terminal differentiation of cardiac progenitor cells, we employed three parameters; expression of spontaneous beating, myosin heavy chain (MHC) protein, and cardiac‐specific genes (α myosin heavy chain, Csx/Nkx2.5 and myosin light chain 2V genes). mRNAs of cardiac‐specific genes were detected in 7.25‐day p.c. mesoderm by RT‐PCR, suggesting that the genetic specification to cardiac lineage initiated in the mesoderm by 7.25 days p.c. The 7.25‐day p.c. isolated mesoderm in 48 hr culture, however, failed to differentiate into spontaneous beating cardiomyocytes and exhibited non‐organized MHC protein in 19% of these culture. In contrast, all of the 7.5‐day p.c. isolated mesoderm differentiated into beating cardiomyocytes even in 24 hr culture. The 7.25‐day p.c. mesoderm associated with primitive streak increased MHC protein expression in 93% of these cultures, although they formed beating foci in 3%. The 7.25‐day p.c. explants containing both visceral embryonic endoderm and primitive streak succeeded in terminal differentiation into spontaneous beating cardiomyocytes. Our study suggests that cardiac progenitor cells obtain the potency to complete terminal differentiation autonomously at 7.5 days p.c., as a consequence of the multistep induction by cell interactions with both the primitive streak and visceral embryonic endoderm, following the genetic specification to cardiac lineage in the early gastrula stage. Dev. Dyn. 1997;210:344–353.

Effects of activation sequence and anisotropic cellular geometry on the repolarization phase of action potential of dog ventricular muscles.

The influence of activation sequences on action potential configuration, especially in the repolarization phase, was examined in isolated canine ventricular muscles. Action potentials were recorded from the epicardial surface in the center of a preparation having nearly uniform fiber orientation (25 X 25 mm). Stimuli applied just adjacent to the recording site produced nearly centrifugal propagation. An activation sequence either parallel (longitudinal) or perpendicular (transverse) to the long axis of the muscle fibers was produced by peripheral stimulation. Action potential duration at -60 mV (APD-60 mV) during centrifugal propagation was significantly longer than that during longitudinal propagation. Further shortening of APD-60 mV was observed during transverse propagation. When a collision of longitudinal or transverse wavefronts (longitudinal or transverse collision) was produced at the action potential recording site, the shortest APD was recorded. During centrifugal propagation, action potential mapping around the stimulating electrodes revealed that APD-60 mV shortened gradually as the recording site was moved further from the stimulation site. The spatial gradient of APD was steeper in the transverse than in the longitudinal direction, causing a distortion in the repolarization sequence and the recovery of excitability near the center of the tissue. Premature stimuli applied to an area near the central stimulation site induced one-way block and circus movement of the wavefront, indicating reentry of excitation. We concluded that the activation sequence and anisotropic cellular geometry substantially affect APD, and that such a change contributes to the spatial inhomogeneity of refractoriness leading to reentrant arrhythmias.

Frequency, predictors and outcome of stent fracture after sirolimus-eluting stent implantation

BACKGROUND Recently, stent fracture (SF) of sirolimus-eluting stents (SES) has been shown to be associated with an increased risk of in-stent restenosis. We sought to evaluate the incidence, predictors and clinical outcome of SF after SES implantation in comparable unselected lesions. METHODS A total of 430 lesions of 382 patients treated with SES were analyzed. SF was defined as single or multiple stent strut fracture as well as complete separation of stent segments. RESULTS At follow-up, SF was identified in 33 of 430 lesions (7.7%). In lesions with SF, the in-stent restenosis was observed more frequently than non-SF lesions (15.2% vs. 4.0%, P=0.004). At 450 days, however, the cumulative rate of major cardiac events was not significantly different between lesions with and without SF (9.1% vs. 7.1%, P=0.722). The risk of SF was independently associated with total stent length (OR 2.22; 95% CI, 1.25 to 3.95; P=0.007), the change in the angulation of the lesion after stenting (OR 1.55; 95% CI, 1.07 to 2.25; P=0.020), and the right coronary artery lesions (OR 3.26; 95% CI, 1.18 to 8.96; P=0.022). CONCLUSIONS The occurrence of SF after SES implantation, was found to be relatively common in the particular population, however, did not lead to an increased risk of adverse cardiac events at 450 days, despite a higher incidence of in-stent restenosis.

Body surface isopotential mapping in Wolff-Parkinson-White syndrome: Noninvasive method to determine the localization of the accessory atrioventricular pathway

The body surface isopotential maps of 22 patients with WPM syndrome were obtained from the 85 unipolar lead ECGs using the on-line minicomputer system newly devised by the authors group. The map patterns were classified into three types-I, II, and III (Type I, eight; Type II, seven; Type III, three; and unclassified, four cases). In Type I, the back surface displayed the negative potential throughout the entire ventricular activation, and at the terminal stage the lower precordial area displayed the positive potential and the upper precordial area, the negative one. Type II was characterized by two longitudinal lines, one staying at its place on the back and the other moving right to left on the precordial area following the process of ventricular activation. In Type III, the right precordial area displayed negative potential in the early stage, and in the terminal stage the upper part of the right side of chest surface displayed positive potential and the lower part, negative potential. It was surmised from these patterns that the pre-excited area was located at the posterior region of the ventricles in Type I, at the right ventricle in Type II, and the right ventricular base near the posterior margin of the ventricular septum in Type III. Type A patients in the conventional ECG classification fell under Type I; Type C patients, under Type III; Type B patients under either Type I or Type II.

ATP directly affects junctional conductance between paired ventricular myocytes isolated from guinea pig heart.

Effects of ATP on junctional conductance (gj) were investigated in paired ventricular myocytes isolated from guinea pig hearts. One cell of the pair was voltage-clamped with a single-patch pipette, and gj was measured after the perforation of the nonjunctional membrane of the partner cell. The current-voltage relation of gj was linear between -30 and +30 mV. The control gj at 5.0 mM ATP in 88 pairs of cells ranged from 100 to 1,055 nS (average, 268 nS). ATP within the range from 0.1 to 5.0 mM increased gj in a dose-dependent manner. The Hill coefficient was 2.6, and the half-maximum effective concentration of ATP was 0.68 mM. Adenylylimidodiphosphate (2 mM) caused a transient increase in gj in the presence of 0.5 mM ATP, but forskolin (30 microM), cyclic AMP (50 microM), catalytic subunit of cyclic AMP-dependent protein kinase (1 microM), and ADP (10 mM) had no significant effect on gj. The temperature coefficient of gj in the presence of 5.0 mM ATP was 1.29. These findings suggest that gj in paired ventricular myocytes is directly regulated by ATP probably through a specific ligand-receptor interaction between ATP and gap junctional channel protein.