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Background: Homozygous mutant mice with a defect of klotho gene expression (kl/kl) show multiple age-related disorders and premature death from unknown causes., Methods and Results: The kl/kl mice subjected to 20-hour restraint stress showed a high rate (20/30) of sudden death, which was associated with sinoatrial node dysfunction (conduction block or arrest). Heart rate and plasma norepinephrine of kl/kl mice, unlike those of wild-type (WT) mice, failed to increase during the stress. Intrinsic heart rate after pharmacological blockade of autonomic nerves in kl/kl mice was significantly lower than that in WT mice (380+/-33 versus 470+/-44 bpm; n=7). The sinus node recovery time after an overdrive pacing (600 bpm, 30 seconds) in kl/kl mice was significantly longer than in WT mice (392+/-37 versus 233+/-24 ms; n=6). In isolated sinoatrial node preparations, the positive chronotropic effect of isoproterenol was significantly less, whereas the negative chronotropic effect of acetylcholine was significantly greater in kl/kl than in WT mice. There was no degenerative structural change in the sinoatrial node of kl/kl mice. The precise localization of klotho was analyzed in newly prepared klotho-null mice with a reporter gene system (kl(-geo)). Homozygous kl(-geo) mice showed characteristic age-associated phenotypes that were almost identical to those of kl/kl mice. In the kl(-geo) mice, klotho expression was recognized exclusively in the sinoatrial node region in the heart in addition to parathyroid, kidney, and choroid plexus., Conclusions: In the heart, klotho is expressed solely at the sinoatrial node. klotho gene expression is essential for the sinoatrial node to function as a dependable pacemaker under conditions of stress.

Background: Recent clinical electrophysiology studies and successful results of radiofrequency catheter ablation therapy suggest that high-frequency focal activity in the pulmonary veins (PVs) plays important roles in the initiation and perpetuation of atrial fibrillation, but the mechanisms underlying the focal arrhythmogenic activity are not understood., Methods and Results: Extracellular potential mapping of rabbit right atrial preparations showed that ryanodine (2 micromol/L) caused a shift of the leading pacemaker from the sinoatrial node to an ectopic focus near the right PV-atrium junction. The transmembrane potential recorded from the isolated myocardial sleeve of the right PV showed typical atrial-type action potentials with a stable resting potential under control conditions. Treatment with ryanodine (0.5 to 2 micromol/L) resulted in a depolarization of the resting potential and a development of pacemaker depolarization. These changes were enhanced transiently after an increase in the pacing rate: a self-terminating burst of spontaneous action potentials (duration, 33.6+/-5.0 s; n=32) was induced by a train of rapid stimuli (3.3 Hz) applied after a brief rest period. The pacing-induced activity was attenuated by either depletion of the sarcoplasmic reticulum of Ca2+ or blockade of the sarcolemmal Na+-Ca2+ exchanger or Cl- channels and potentiated by beta-adrenergic stimulation., Conclusions: PV myocardial sleeves have the potential to generate spontaneous activity, and such arrhythmogenic activity is uncovered by modulation of intracellular Ca2+ dynamics.

Background: The ionic basis of acquired QT prolongation and torsade de pointes (TdP) unrelated to drugs is not fully understood., Methods and Results: We created a rabbit model with chronic complete atrioventricular block (AVB) (n=34), which showed prominent QT prolongation (by 120%), high incidence of spontaneous TdP (71%), and cardiac hypertrophy. Patch-clamp experiments were performed in left ventricular myocytes from 9 rabbits (8 with TdP, 1 without TdP) at approximately 21 days of AVB and from 8 sham-operated controls with sinus rhythm. Action potential duration was prolonged in AVB myocytes compared with control (+61% at 0.5 Hz, +21% at 3 Hz). Both rapidly and slowly activating components of the delayed rectifier K(+) current (I(Kr) and I(Ks)) in AVB myocytes were significantly smaller than in control by 50% and 55%, respectively. There was no significant difference in Ca(2+)-independent transient outward current (I(to1)). L-type Ca(2+) current (I(Ca,L)) in control and AVB myocytes was similar in peak amplitude, but the half voltage for activation was shifted to the negative direction (5.9 mV) in AVB myocytes. Voltage dependence of I(Ca,L) inactivation was not different in control and AVB myocytes. The inward rectifier K(+) current (I(K1)) significantly increased in AVB myocytes compared with control., Conclusions: In the rabbit, chronic AVB leads to prominent QT prolongation and high incidence of spontaneous TdP. Downregulation of both I(Kr) and I(Ks) in association with altered I(Ca,L) activation kinetics may underlie the arrhythmogenic ventricular remodeling.

Background: Anisotropic conduction properties may provide a substrate for reentrant arrhythmias. We investigated the age-dependent changes of structural and functional anisotropy in isolated right atria from infant (1 to 2 months), young (6 to 12 months), and old (6 to 10 years) dogs., Methods and Results: The histology of the mapped atrial tissues (a small subepicardial area, 2.8x4.2 mm) was characterized by an age-dependent increase of myofiber width and fat cell infiltration between myofibers. Cx43 was distributed homogeneously over the entire cell surface in infant dogs, whereas it progressively polarized to the cell termini with increasing age. The activation sequences were analyzed by high-resolution optical mapping using a voltage-sensitive dye. Activation fronts from the pacing site proceeded more rapidly along fiber orientation (longitudinal) than across it (transverse). Infant dogs showed "elliptical" isochrones with a smooth transition between longitudinal and transverse propagation, whereas old dogs had a "square" pattern with a sharp transition. Conduction block occurred predominantly during longitudinal propagation in infant dogs but during transverse propagation in old dogs. The shape of the wave front and the degree of lateral uncoupling seemed to decide the preferential direction of block. A zigzag activation causing an extremely slow transverse conduction was observed only in old dogs., Conclusions: Along with the age-dependent structural anisotropy, the preferential direction of block changed from longitudinal to transverse in association with a change in the wave front configuration. A zigzag propagation based on lateral uncoupling would predispose the elderly to multiple reentry and a higher incidence of atrial fibrillation.

Background: Class III antiarrhythmic agents commonly exhibit reverse frequency-dependent prolongation of the action potential duration (APD). This is undesirable because of the danger of bradycardia-related arrhythmias and the limited protection against ventricular tachyarrhythmias. The effects of blockade of separate components of delayed rectifier K(+) current (I(K)) may help to develop agents effective at high heart rate., Methods and Results: We assessed the density and kinetics of the 2 components of the delayed rectifier K(+) current, I(Kr) and I(Ks), in rabbit and guinea pig ventricular myocytes. The effects of their specific blockers (chromanol 293B for I(Ks) and E-4031 for I(Kr)) on the action potential was studied at different heart rates by use of whole-cell patch-clamp techniques. In guinea pig ventricular myocytes only, blockade of I(Ks) causes APD prolongation in a frequency-independent manner, whereas blockade of I(Ks) in rabbit ventricular myocytes shows reverse frequency dependence, as does blockade of I(Kr) in both species. This result can be explained primarily by the higher density of I(Ks) in guinea pig ventricle and by its slow deactivation kinetics, which allows I(Ks) to accumulate at high heart rate because little time is available for complete deactivation of it during diastole., Conclusions: Density and kinetics of components of I(K) explain why blockade of I(Ks) is more effective at high heart rate in the guinea pig ventricle than in the rabbit ventricle, without adverse effects at low heart rate.

Background: Amiodarone is the most promising drug for the treatment of life-threatening tachyarrhythmias in patients with structural heart disease. The pharmacological effects of amiodarone on cardiac ion channels are complex and may differ for short-term and long-term administration., Methods and Results: The delayed rectifier K(+) current (I(K)) of ventricular myocytes isolated from rabbit hearts was recorded with the whole-cell voltage-clamp technique. I(K) was separated into 2 components by use of specific blockers for either I(Ks) (chromanol 293B, 30 micromol/L) or I(Kr) (E-4031, 10 micromol/L). Short-term application of amiodarone caused a concentration-dependent decrease in I(Kr) with an IC(50) of 2.8 micromol/L (n=8) but only a minimal reduction in I(Ks). The short-term effects of amiodarone were also determined in Xenopus oocytes expressing the cloned human channels that conduct I(Kr) and I(Ks) (HERG and KvLQT1/minK). HERG current in oocytes was reduced by amiodarone (IC(50)=38 micromol/L), whereas KvLQT1/minK current was unaffected by 300 micromol/L amiodarone. To study the effects of long-term drug administration, rabbits were treated for 4 weeks with oral amiodarone (100 mg. kg(-1). d(-1)) before cell isolation. Long-term administration of amiodarone decreased I(K) to 55% (n=10) in control rabbits and altered the relative density of I(Kr) and I(Ks). The majority (92%) of current was I(Kr). mRNA levels of rabbit ERG,KVLQT1, and minK in left ventricular myocardium did not differ between control and long-term amiodarone., Conclusions: Amiodarone has differential effects on the 2 components of I(K), depending on the application period; short-term treatment inhibits primarily I(Kr), whereas long-term treatment reduces I(Ks).

Background: Methanesulfonanilide derivatives, selective inhibitors of the rapidly activating component (I(Kr)) of the delayed rectifier potassium current (I(K)), prolong action potential duration (APD) of cardiac muscles with reverse frequency dependence, which limits their clinical use because of proarrhythmia. Vesnarinone, a quinolinone derivative developed as a cardiotonic agent, has complex pharmacological properties, but its clinical efficacy is explained in part by I(K) reduction. Therefore, we investigated the mode of I(K) block by vesnarinone., Methods and Results: I(K) of the rabbit ventricular myocyte was activated by voltage-clamp steps applied from a holding potential to various depolarizing levels. The development of I(K) block at depolarization (+10 mV) and its recovery process at hyperpolarization (-75 mV) were compared between vesnarinone and E-4031. The I(K) block by vesnarinone (3 micromol/L) developed and recovered monoexponentially, with time constants of 361 ms (n=5) and 1.87 seconds (n=4), respectively. I(K) block by E-4031 (0.3 micromol/L) developed instantaneously, with no recovery from the block at hyperpolarization. The I(K) block by vesnarinone, estimated by I(K) tail after a train of depolarizing pulses (for 30 seconds at 0.2 to 2 Hz), was increased with increasing frequency (twofold at 2 from 0.2 Hz), but that by E-4031 was unchanged. In rabbit papillary muscles, vesnarinone (10 micromol/L) prolonged APD at stimulation frequencies >0.2 Hz, whereas E-4031 (0.3 micromol/L) prolonged that in a reverse frequency-dependent manner., Conclusions: Vesnarinone may prolong the repolarization of human cardiac muscle without reverse frequency dependence, because I(Kr) is expressed in humans as well as in the rabbit. Thus, this drug may be a model for an ideal class III drug without the risk of proarrhythmia.