Left ventricular hypertrophy decreases slowly but not rapidly activating delayed rectifier potassium currents of epicardial and endocardial myocytes in rabbits.

BACKGROUND: Delayed rectifier K(+) currents are critical to action potential (AP) repolarization. The present study examines the effects of left ventricular hypertrophy (LVH) on delayed rectifier K(+) currents and their contribution to AP repolarization in both epicardial (Epi) and endocardial (Endo) myocytes. METHODS AND RESULTS: VH was induced in rabbits by a 1-kidney removal, 1-kidney vascular clamping method. Slowly (I(Ks)) and rapidly (I(Kr)) activating delayed rectifier K(+) currents were recorded by the whole-cell patch-clamp technique, and APs were recorded by the microelectrode technique. In normal rabbit left ventricular myocytes, I(Ks) densities were larger in Epi than in Endo (1.1+/-0.1 versus 0.43+/-0.07 pA/pF), whereas I(Kr) density was similar between Epi and Endo (0.31+/-0.05 versus 0.36+/-0.07 pA/pF) at 20 mV. LVH reduced I(Ks) density to a similar extent (approximately 40%) in both Epi and Endo but had no significant effect on I(Kr) in either Epi or Endo. Consequently, I(Kr) was expected to contribute more to AP repolarization in LVH than in control. This was confirmed by specific I(Kr) block with dofetilide, which prolonged AP significantly more in LVH than in control (31+/-3% versus 18+/-2% in Epi; 53+/-6% versus 32+/-4% in Endo at 2 Hz). In contrast, L-768,673 (a specific I(Ks) blocker) prolonged AP less in LVH than in control. The very small I(Ks) density in Endo with LVH is consistent with the greater incidence of early afterdepolarizations induced in this region by dofetilide. CONCLUSIONS: LVH induces a decrease in I(Ks) density and increases the propensity to develop early afterdepolarizations, especially in Endo.

Synthesis and SAR of 6-substituted purine derivatives as novel selective positive inotropes.

A series of purine derivatives was prepared and examined for selective inotropic activity in vitro and in vivo. Thioether-linked derivatives were superior to their oxygen and nitrogen isosteres. Substitution of electron-withdrawing groups on the benzhydryl moiety of these agents increased potency. The best compound of the study, 17 (carsatrin), was examined further and demonstrated selective oral activity as a positive inotrope. These compounds are presumed to act by affecting the kinetics of the cardiac sodium channel by analogy to the prototypic agent DPI 201106 (1). Their high selectivity for increasing contractile force and dP/dt without affecting blood pressure or heart rate is consistent with this mechanism. Carsatrin (17) was selected as a potential development candidate.

Blockade of HERG channels by the class III antiarrhythmic azimilide: mode of action.

1. The class III antiarrhythmic azimilide has previously been shown to inhibit I(Ks) and I(Kr) in guinea-pig cardiac myocytes and I(Ks) (minK) channels expressed in Xenopus oocytes. Because HERG channels underly the conductance I(Kr), in human heart, the effects of azimilide on HERG channels expressed in Xenopus oocytes were the focus of the present study. 2. In contrast to other well characterized HERG channel blockers, azimilide blockade was reverse use-dependent, i.e., the relative block and apparent affinity of azimilide decreased with an increase in channel activation frequency. Azimilide blocked HERG channels at 0.1 and 1 Hz with IC50s of 1.4 microM and 5.2 microM respectively. 3. In an envelope of tail test, HERG channel blockade increased with increasing channel activation, indicating binding of azimilide to open channels. 4. Azimilide blockade of HERG channels expressed in Xenopus oocytes and I(Kr) in mouse AT-1 cells was decreased under conditions of high [K+]e, whereas block of slowly activating I(Ks) channels was not affected by changes in [K+]e. 5. In summary, azimilide is a blocker of cardiac delayed rectifier channels, I(Ks) and HERG. Because of the distinct effects of stimulation frequency and [K+]e on azimilide block of I(Kr) and I(Ks) channels, we conclude that the relative contribution of block of each of these cardiac delayed rectifier channels depends on heart frequency. [K+]e and regulatory status of the respective channels.

In vivo acetylcholine turnover in rat heart.

The in vivo uptake of choline (Ch) and synthesis of acetylcholine (ACh) in rat heart were studied using a pyrolysis mass fragmentography (PMF) method. Deuterium labeled Ch was pulse injected (i.v.) into anesthetized rats. Labeled and unlabeled Ch and ACh were measured by PMF in hearts at various times following injection. From these data we calculated the specific activities of Ch and ACh, rate constants for ACh and turnover rates of ACh. After an initial equilibration period of approximately 2 min, the specific activities of Ch and ACh decayed in a parallel manner with half-times of 28.2 and 28.8 min respectively. Between 2 and 60 min the calculated ACh turnover rate was 0.144 nmol/g/min. Unlike brain Ch, heart Ch levels are very stable with time following sacrifice. No advantage was found in using microwave irradiation to stabilize heart ACh and Ch content.

The distribution of acetylcholine and choline in guinea pig heart.

The regional distributions of acetylcholine (ACh) and choline (Ch) in the guinea pig heart were investigated with a pyrolysis-mass fragmentography technique. Using ACh as a marker for cholinergic neurons, we have described a pattern of parasympathetic innervation in the guinea pig heart. This distribution is very similar to that suggested by studies using several different cholinergic indicators in various species. Atrial areas receive richer parasympathetic innervation than ventricular areas, with the right portions receiving more than the left. The nodal areas were the most abundantly innervated regions examined. Ch content is not a good indicator for cholinergic innervation as the regional distribution of ACh and Ch throughout the guinea pig heart are not strongly associated.

"Fade" of hyperpolarizing responses to vagal stimulation at the sinoatrial and atrioventricular nodes of the rabbit heart.

Previous studies have suggested that maintained vagal stimulation or acetylcholine infusion results in a fade of responses in the sinoatrial node but not in the atrioventricular node, implying different muscarinic receptor subtypes in the two regions. We investigated this hypothesis in 23 isolated rabbit atrial preparations made quiescent by continuous superfusion with verapamil (1 microgram/ml). Transmembrane potentials were recorded simultaneously from cells in the sinoatrial pacemaker region and from the "N" region of the atrioventricular node. Postganglionic vagal stimulation was achieved by the application of trains of pulses (50-150 microseconds; 10-20 V; 200 Hz). Simultaneous application of long-lasting (1-10 sec) vagal trains produced hyperpolarizations which were nearly identical for both nodal regions. Maximal hyperpolarizations (approximately or equal to 24 mV for sinoatrial node; 26 mV for atrioventricular node) were reached about 500 msec after initiation of the vagal train. Thereafter, hyperpolarizations faded, following a biphasic time course, and thus displaying two different time constants, one fast (tau fast = 580 msec for sinoatrial node; 550 msec for atrioventricular node), and one slow (tau slow = 9.2 sec for both sinoatrial and atrioventricular nodes). Hyperpolarizations during brief (200-msec) but repetitive vagal trains also faded biphasically, but approached a steady state much more rapidly than responses to long-lasting trains. Recovery from hyperpolarization decay occurred rather slowly and was linear. Our results demonstrate that the membrane potential responses to vagal stimulation in the atrioventricular node are indistinguishable from those in the sinoatrial node, and suggest that similar muscarinic receptors are operative in both regions. These phenomena may play an important role in the response of the cardiac conducting system to direct or reflexly mediated vagal input.

Effects of dantrolene sodium on the electrophysiological properties of canine cardiac Purkinje fibers.

Dantrolene sodium at concentrations between 8.8 and 35.1 microM produced rather selective changes in the electrophysiological properties of isolated dog Purkinje fibers. The action potential duration at 90% repolarization and effective refractory period of normally polarized fibers were increased in a dose- and frequency-dependent fashion. The plateau phase of the action potential was significantly depressed and this effect coincided with a marked decrease in the strength of contraction. Dantrolene sodium had no significant effect on resting membrane potential, upstroke velocity of phase O, conduction velocity or pacemaker activity of Purkinje fibers. Dantrolene diminished or abolished "slow response" action potentials produced by superfusion with 18 mM K+ Tyrode's solution containing 10(-7) M isoproterenol. Drug effects took 10 to 15 min to become apparent, reached a steady-state after 45 to 60 min and were not reversible even after 2 hr of washout with drug-free Tyrode's solution. In contrast, increasing the [Ca++]0 produced nearly a complete reversal of the dantrolene-induced changes. These results suggest that dantrolene produces its effects by interfering with the slow inward current. Thus, dantrolene may be similar in action to other slow channel blocking agents, such as verapamil, and may be useful as an antiarrhythmic agent.

Basis for tetrodotoxin and lidocaine effects on action potentials in dog ventricular myocytes.

We studied the effects of tetrodotoxin (TTX) and lidocaine on transmembrane action potentials and ionic currents in dog isolated ventricular myocytes. TTX (0.1-1 x 10(-5) M) and lidocaine (0.5-2 x 10(-5) M) decreased action potential duration, but only TTX decreased the maximum rate of depolarization (Vmax). Both TTX (1-2 x 10(-5) M) and lidocaine (2-5 x 10(-5) M) blocked a slowly inactivating toward current in the plateau voltage range. The voltage- and time-dependent characteristics of this current are virtually identical to those described in Purkinje fibers for the slowly inactivating inward Na+ current. In addition, TTX abolished the outward shift in net current at plateau potentials caused by lidocaine alone. Lidocaine had no detectable effect on the slow inward Ca2+ current and the inward K+ current rectifier, Ia. Our results indicate that 1) there is a slowly inactivating inward Na+ current in ventricular cells similar in time, voltage, and TTX sensitivity to that described in Purkinje fibers; 2) both TTX and lidocaine shorten ventricular action potentials by reducing this slowly inactivating Na+ current; 3) lidocaine has no additional actions on other ionic currents that contribute to its ability to abbreviate ventricular action potentials; and 4) although both agents shorten the action potential by the same mechanism, only TTX reduces Vmax. This last point suggests that TTX produces tonic block of Na+ current, whereas lidocaine may produce state-dependent Na+ channel block, namely, blockade of Na+ current only after Na+ channels have already been opened (inactivated-state block).

Effects of quinidine on action potentials and ionic currents in isolated canine ventricular myocytes.

We examined the effects of quinidine (5-20 microM) on transmembrane action potentials and ionic currents of isolated canine ventricular myocytes. Collagenase treatment of canine ventricular tissue produced a yield of 40-60% healthy cells. Myocytes had normal resting and action potentials as measured using conventional microelectrodes. Quinidine decreased Vmax, amplitude, overshoot, and the duration of action potentials stimulated by passage of brief current pulses through the recording pipette. Recovery was complete after washout except that action potential duration was prolonged compared with control. A discontinuous single microelectrode voltage ("switch") clamp was used to measure ionic currents. Quinidine irreversibly reduced steady-state outward current as measured with three different voltage clamp protocols. Quinidine reversibly decreased peak calcium current as well as the slowly inactivating and/or steady-state inward currents in the plateau voltage range, presumably both "late" sodium (tetrodotoxin-sensitive) and calcium (tetrodotoxin-insensitive) currents. The effect on calcium current showed both tonic and use-dependent block. Thus, quinidine has a multitude of actions on both inward and outward currents, which combine to produce the net effect of quinidine on action potential configuration.

Effects of 4-aminopyridine on rate-related depression of cardiac action potentials.

Canine cardiac Purkinje fibers and atrial trabeculae and rat and cat papillary muscles superfused with a hyperkalemic, hypoxic, and acidotic Tyrode solution were depolarized to membrane potentials (-70 to -60 mV) at which action potential amplitude declined as the coupling intervals of pacing stimuli were prolonged from 500 to 4,500 ms. The rate-related decline of action potential amplitude appeared to be due to time-dependent recovery of the early outward current rather than to a decrease in inward calcium current, since it was prevented by 4-aminopyridine (1.0 mM), but not by isoproterenol (1.0 microM), caffeine (5.0 mM), or CsCl (5-20 mM) and it was accompanied by an exponential increase of developed tension. Experiments using Purkinje fibers mounted in a single sucrose gap chamber demonstrated that the rate-related decline of action potential amplitude was maximal at membrane potentials between -70 and -40 mV and was negligible at less negative or more negative membrane potentials. These results may pertain to the mechanism for deceleration-dependent bundle branch block.

Rate-related suppression and facilitation of conduction in isolated canine cardiac Purkinje fibers.

Previous studies have shown that antegrade conduction through damaged His Purkinje tissue may be suppressed following rapid ventricular pacing (overdrive suppression of conduction). We studied this phenomenon using isolated Purkinje fibers placed in a three-chamber bath. Superfusates for the left, middle, and right segments of the fiber were altered to produce action potentials that resembled those of normal bundle branch, damaged His bundle, and normal His bundle, respectively. To produce anisotropic conduction, the left segment of the fiber was adjusted to be three to four times longer than the right segment. Pacing the right segment at intermediate rates produced maximal action potential amplitude in the middle segment and 1:1 right-to-left conduction, whereas pacing at faster or slower rates reduced action potential amplitude and produced block. Pacing the left segment at fast or slow rates also reduced action potential amplitude in the middle segment, but conduction was maintained (anisotropy). After rapid or slow left segment pacing, action potential amplitude in the middle segment remained low during subsequent right segment pacing at intermediate rates, and transient block occurred (overdrive or underdrive suppression of conduction). With time, action potential amplitude normalized and conduction resumed. In other more severely depressed preparations, conduction block occurred even at intermediate right segment pacing rates prior to left segment pacing. Under these conditions, pacing the left segment at intermediate rates increased action potential amplitude in the middle segment and temporarily permitted 1:1 conduction at intermediate right segment pacing rates (overdrive facilitation of conduction).(ABSTRACT TRUNCATED AT 250 WORDS)

A model for early afterdepolarizations: induction with the Ca2+ channel agonist Bay K 8644.

Early afterdepolarizations (EADs) are one mechanism proposed to cause certain cardiac arrhythmias. We studied the effect of the Ca2+ channel agonist Bay K 8644 (1 x 10(-8) to 5 x 10(-5) M) on normally polarized sheep and canine cardiac Purkinje fiber short segments. EADs occurred with higher Bay K 8644 concentrations and had an average take-off potential of -34 mV. The initiation of EADs was preceded by lengthening of action potential duration and flattening of the plateau. Induction of EADs with Bay K 8644 was enhanced by low stimulation frequencies, lowering of [K]o, addition of tetraethylammonium chloride, or application of depolarizing constant current pulses during the plateau. EADs were abolished by increasing stimulation frequency, raising [K]o, the addition of tetrodotoxin, lidocaine, ethmozin, verapamil, and nitrendipine, or application of repolarizing constant current pulses. Using current pulses to modify the action potential plateau, a steep inverse relationship was found between the EAD peak voltage and its take-off potential, and EADs could be initiated over only a narrow range of take-off potentials. Thus, interventions that suppressed EADs shortened action potential duration or shifted the plateau away from the voltage range needed to initiate EADs. These observations suggest that mechanisms dependent on both time and voltage underlie EADs, and provide a unifying hypothesis for the induction of the EADs. We propose that induction of EADs requires 1) lengthening of action potential duration within a plateau voltage range where 2) recovery from inactivation and reactivation of an inward current possibly carried through Ca2+ channels can occur.

Synthesis and class III type antiarrhythmic activity of 4-aroyl (and aryl)-l-aralkylpiperazines.

The synthesis and in vitro Class III antiarrhythmic activity of several 4-aroyl (and aryl)-1-aralkylpiperazine and piperidine derivatives are described. Among several potent compounds identified in the series, RWJ-28810 (3), with its EC20 of 3 nM, ranks as one of the most potent (in vitro) compounds reported.

Vagal control of pacemaker periodicity and intranodal conduction in the rabbit sinoatrial node.

Effects of brief vagal bursting on pacemaker periodicity and intranodal conduction were studied by recording transmembrane potentials in isolated rabbit sinoatrial preparations. Postganglionic vagal terminals were activated by applying brief (50- to 150-msec) trains of pulses (duration, 100 mu sec; frequency, 200 Hz) to the endocardial surface of the node. Sympathetic effects were prevented by superfusion with Tyrode's solution containing propranolol (1 microgram/ml). Vagal trains applied singly every 10 seconds produced brief hyperpolarizations in "true" pacemaker and transitional cells, and induced phasic changes in periodicity and conduction. These changes were out of phase with each other, and were dependent on the magnitude and duration of the vagal train, as well as on its position within the pacemaker period. When similar trains were applied repetitively at cycle lengths (200-1200 msec) that were independent of the pacemaker period, complex patterns of vagus-sinoatrial node interactions resulted. Hence, depending on the vagal stimulus cycle length, the sinoatrial pacemaker was forced to beat at stable and predictable harmonic (i.e., 1:1, 1:2, 2:1, etc.), subharmonic (3:2, 4:3, etc.), or more complex entrainment ratios. At some of these ratios, arrhythmic sinus patterns coexisted with intranodal conduction disturbances, including first- and second-degree block, Wenckebach phenomena, or combinations thereof. At other entrainment ratios, arrhythmias occurred in the absence of conduction changes. At still other ratios, conduction disturbances developed in the presence of apparently undisturbed sinus rhythm. These results provide insight into the mechanism of the dynamic vagal control of sinoatrial periodicity and conduction, and may have clinical implications as well.

Dantrolene sodium: effects on isolated cardiac tissues.

The effects of dantrolene sodium on dog Purkinje fibers, cat atrial and ventricular muscles were studied. Action potential duration was significantly increased and contractility was significantly decreased by dantrolene in all three types of tissue. The plateau phase of Purkinje fiber and occasionally atrial action potential was slightly depressed. Dantrolene sodium had no significant effect on resting membrane potential, action potential amplitude or upstroke velocity of phase 6. The negative inotropic effects were most pronounced in Purkinje fibers, followed by atrial muscle while papillary muscles were least sensitive. Contractile force of Purkinje fibers was decreased by relatively the same amount at all frequencies of stimulation. At faster rates, atrial and ventricular muscle contractility was depressed relatively more than at slower rates. Slow response action potentials in cat papillary muscle were diminished slightly, but this effect was not significant. All drug effects took 10 to 15 min to develop, reached a steady state after 30 to 40 min, and were irreversible upon washout. Increasing the extracellular calcium concentration reversed the dantrolene-induced changes. These findings suggest that effects of dantrolene are mediated in part by a decrease in the intracellular free calcium concentration in cardiac tissue.

Amantadine-induced diastolic depolarization and automaticity in ventricular muscle.

We studied the cardiac effects of amantadine, an antiviral and anti-Parkinson drug. Amantadine hydrochloride (100--800 microM) produced significant changes in the electrophysiological properties of isolated ventricular muscle preparations from frog, rabbit, cat, dog, and calf. At relatively low concentrations (100--300 microM), the drug increased action potential duration, decreased action potential amplitude and maximum diastolic potential, and induced phase 4 depolarization. Amantadine also caused subthreshold diastolic depolarizations, apparent upon cessation of stimulation in all preparations studied. The amplitude of the diastolic depolarizations increased as a function of time and/or concentration of drug, eventually reached threshold, and spontaneous activity ensued. In the steady state, amantadine-induced spontaneous activity was rather stable, and the rate was dependent upon the amantadine and external potassium concentrations, as well as the membrane potential. In the absence of stimulation, amantadine-induced spontaneous activity occurred abruptly or could be triggered by a single stimulus, often occurring in a "bursting" fashion that appeared to originate from multiple discrete foci. All actions of amantadine were rapidly reversed upon washout. Propranolol had no effect on the actions of the drug. Amantadine-induced spontaneous activity was unaffected by lidocaine, diminished by TTX, and reduced or abolished by verapamil. The results indicate that amantadine can directly alter the membrane properties of ventricular muscle, possibly due to an effect on potassium conductance. Furthermore, amantadine can be used as a tool to study the ionic basis of ventricular automaticity and to model cellular mechanisms of ventricular rhythm disturbances.

Effects of sympathetic tone on vagally induced phasic changes in heart rate and atrioventricular node conduction in the anesthetized dog.

We examined the effects of stellate ganglia stimulation on the phase-dependent chronotropic and dromotropic responses to brief vagal bursts in open-chest anesthetized dogs. Stellate stimulation affected the phasic vagal effects on heart rate by shortening the latent period, shifting the phase at which maximum decrease in heart rate occurred to earlier phases, and reducing the maximum decrease in heart rate. These effects were due primarily to an increase in the basic heart rate. No significant sympathetic-parasympathetic interaction occurred for heart rate, indicating that accentuated antagonism did not occur with brief vagal bursts. Stellate stimulation primarily decreased the amplitude of the phasic vagal effects on atrioventricular nodal conduction, regardless of the underlying heart rate, and a significant sympathetic-parasympathetic interaction was associated with this effect. The peak of the phase-dependent vagal effects on heart rate and atrioventricular nodal conduction were phase-shifted with one another. From these findings, we postulate the small changes in sympathetic tone might shift the predominant phase-dependent vagal effect from one on heart rate to one on atrioventricular nodal conduction. Furthermore, our results suggest that dynamic vagal control of heart rate and atrioventricular node conduction involves both phase-dependent and phase-independent factors. Sympathetic activity appears to affect only the phase-independent factor(s) in the control of heart rate, whereas it affects both phase-dependent and phase-independent factors in the control of atrioventricular node conduction.

Mechanism of action potential prolongation by RP 58866 and its active enantiomer, terikalant. Block of the rapidly activating delayed rectifier K+ current, IKr.

BACKGROUND: The class III antiarrhythmic agent RP 58866 and its active enantiomer, terikalant, are reported to selectively block the inward rectifier K+ current, IK1. These drugs have demonstrated efficacy in animal models of cardiac arrhythmias, suggesting that block of IK1 may be a useful antiarrhythmic mechanism. The symmetrical action potential (AP)-prolonging and bradycardic effects of these drugs, however, are inconsistent with a sole effect on IK1. METHODS AND RESULTS: We studied the effects of RP 58866 and terikalant on AP and outward K+ currents in guinea pig ventricular myocytes. RP 58866 and terikalant potently blocked the rapidly activating delayed rectifier K+ current, IKr, with IC50S of 22 and 31 nmol/L, respectively. Block of IK1 was approximately 250-fold less potent; IC50S were 8 and 6 mumol/L, respectively. No significant block of the slowly activating delayed rectifier, IK1, was observed at < or = 10 mumol/L. The phenotypical IKr currents in mouse AT-1 cells and Xenopus oocytes expressing HERG were also blocked 50% by 200 to 250 nmol/L RP 58866 or terikalant, providing further conclusive evidence for potent block of IKr. RP 58866 < or = 1 mumol/L and dofetilide increased AP duration symmetrically, consistent with selective block of IKr. Only higher concentrations (> or = 10 mumol/L) of RP 58866 slowed the rate of AP repolarization and decreased resting membrane potential, consistent with an additional but substantially less potent block of IK1. CONCLUSIONS: These data demonstrate that RP 58866 and terikalant are potent blockers of IKr and prompt a reinterpretation of previous studies that assumed specific block of IK1 by these drugs.