Electrophysiologic effects of amiloride in canine Purkinje fibers: evidence for a delayed effect on repolarization

Amiloride, a potassium-sparing diuretic, inhibits Na+ transport, Na+-H+ exchange and possibly Na+-Ca++ exchange in a variety of cellular and epithelial tissues. Similar membrane ion transport mechanisms exist in cardiac tissue, yet there are little data on possible interference by amiloride with ion transport in the heart. Given recent evidence for a delay in amiloride uptake into erythroid cells, we studied the electrophysiologic effects of amiloride after prolonged drug exposure in canine Purkinje fibers using standard microelectrode techniques. Amiloride (1-10 microM) led to a progressive lengthening of action potential duration with a tau of 1.8 +/- 0.5 hr (n = 15). At long cycle lengths (greater than or equal to 2000 msec) early afterdepolarizations and oscillations around the plateau were seen. To determine the etiology of the afterdepolarizations, Purkinje fibers treated for 2 hr with 10 microM amiloride were then exposed to tetrodotoxin, manganese and nisoldipine. Tetrodotoxin (7.8 X 10(-7) M) reversed completely all amiloride effects rapidly and reversibly. MnCl2 (4 mM) increased the afterdepolarizations, and arrest occurred at the plateau potential routinely. Nisoldipine (10(-6) M), a more selective blocker of slow inward current, shortened action potential duration somewhat but did not reverse fully the effects of amiloride. We conclude that amiloride has a pronounced effect on repolarization in the canine Purkinje fiber and this effect is manifest only after prolonged exposure to the drug.     

Electrophysiological effects of ACC-9358, a novel class I antiarrhythmic agent, on isolated canine Purkinje fibers and ventricular muscle

Class I antiarrhythmic agents are heterogeneous with respect to their cardiac electrophysiological effects and have been subdivided into three categories: la, lb and lc. The purpose of the present study was to determine the classification and investigate the mechanism of action of ACC-9358 [4-hydroxy-N-phenyl-3,5-bis (1-pyrrolidinyl-methyl)benzamide], a novel class I antiarrhythmic agent currently under clinical investigation. The effects of ACC-9358 on action potentials from isolated canine Purkinje fibers and ventricular muscle were examined using standard microelectrode techniques. In Purkinje fibers, ACC-9358 (1-50 microM) exerted a dose-dependent reduction in maximum upstroke velocity (Vmax) and action potential duration at 50 and 90% repolarization (APD50 and APD90). The reduction of Vmax was voltage-dependent (greater at an extracellular potassium concentration of 6 mM than at 2.7 mM), frequency-dependent (greater at a basic cycle length of 500 than at 2000 msec) and very slow in onset (rate constant of 0.017 action potentials-1) and offset (recovery half-time of 66.9 sec). In Purkinje fibers, ACC-9358 attenuated the action potential shortening effects of lidocaine but not that of nicardipine or nicorandil and shortened APD50 to a greater extent at a basic cycle length of 2000 than at 500 msec. In ventricular muscle, ACC-9358 (1-50 microM) exerted a dose-dependent reduction in Vmax and prolongation of APD50 and APD90.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiologic and voltage clamp analysis of the effects of sotalol on isolated cardiac muscle and Purkinje fibers

The effect of dl-, d- and I-sotalol on electrophysiologic characteristics of guinea-pig papillary muscles, sheep and rabbit Purkinje fibers was studied. Standard electrophysiologic and voltage clamp techniques were used. At concentrations between 10(-6) and 10(-4) M, the main effect of sotalol consisted of prolongation of the action potential duration. In voltage clamp experiments this effect correlated with a substantial reduction of the time-dependent K current activated during the plateau of the action potential and a small reduction of the background K current. At concentrations above 10(-4) M, a secondary shortening of the action potential concomitant with a fall in maximal rate of depolarization was seen. In voltage clamp experiments this effect correlated with a decrease of a slowly inactivating Na current. In the absence of catecholamines d- and I-sotalol exerted identical effects on action potentials and voltage clamp currents.     

Adenosine attenuation of the electrophysiological effects of isoproterenol on canine cardiac Purkinje fibers

Adenosine is known to have effects on electrophysiologic parameters of the sinus node, AV node and atrium and to antagonize isoproterenol-induced increased inotropy in the ventricle. However, the effects on cardiac Purkinje fibers are not well established. Therefore, the purpose of the present experiments was to examine the effects of adenosine alone and adenosine on isoproterenol-treated canine cardiac Purkinje fibers. Microelectrode techniques were used to record transmembrane action potentials. Adenosine alone (10(-7) to 10(-4) M) produced no effects on action potential characteristics of paced fibers. Adenosine in concentrations of 10(-7), 10(-6), 10(-5) and 10(-4) M produced a 15, 24, 44* and 72*% attenuation of isoproterenol (10(-6) M)-induced action potential duration shortening, respectively (*P less than .001). In 7 of 7 fibers depolarized with 22 mM K+, adenosine (10(-4) M) ablated calcium-dependent action potentials restored with isoproterenol (10(-6) M). These effects were antagonized by theophylline (5 X 10(-5) M) and adenosine deaminase (1 U/ml). Action potential shortening due to superfusion of high calcium Tyrode's solution and calcium-dependent action potentials generated in Na+ free-high Ca++ Tyrode's solution were not antagonized by adenosine. Adenosine (10(-5) M) produced a negative chronotropic effect, increasing escape intervals from 2669 +/- 647 to 3702 +/- 717** msec in control fibers and from 1864 +/- 329 to 2658 +/- 399** msec in tyramine (10(-4) M)-treated fibers (**P less than .05), but failed to produce a negative chronotropic response in fibers pretreated with propranolol (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological effects of cesium and tetraethylammonium in canine cardiac Purkinje fibers

Cesium (Cs) and tetraethylammonium (TEA) have been shown to increase action potential duration. However, action potential duration is known to be influenced by the rate of stimulation. In this study, the effect of stimulation rate on action potential characteristics was studied in Cs-treated and TEA-loaded canine Purkinje fiber preparations. Action potentials of Purkinje fibers from Cs-treated and TEA-loaded preparations had longer durations than action potentials of Purkinje fibers from normal preparations. Greater prolongation of action potential duration was observed when the rate of stimulation was reduced in Purkinje fibers from Cs-treated and TEA-loaded preparations than those from normal preparations. Whereas the increase in action potential duration of Purkinje fibers from Cs-treated preparations was accompanied by a significant membrane depolarization, no change in membrane potential was observed in Purkinje fibers from TEA-loaded preparations. In some Cs-treated and TEA-loaded preparations, the prolonged duration observed at slow stimulation rates was associated with the appearance of early afterdepolarizations. Lidocaine and cromakalim, agents known to reduce action potential duration in normal Purkinje fibers, also shortened action potential duration in Purkinje fibers from both Cs-treated and TEA-loaded preparations. However, lidocaine and cromakalim caused a significant membrane depolarization in Cs-treated Purkinje fibers but not in TEA-loaded Purkinje fibers. Our results suggested that although Cs and TEA are capable of producing rate-dependent prolongation of action potential duration and the occurrence of bradycardia-dependent early afterdepolarization, differences exist in Cs-treated Purkinje fibers in terms of the appearance of membrane depolarization at reduced stimulation rate and in the presence of lidocaine and cromakalim.     

Relationship between use-dependent effects of antiarrhythmic drugs on conduction and Vmax in canine cardiac Purkinje fibers

The purpose of these experiments was to evaluate the relationship between interval dependent effects of antiarrhythmic drugs on conduction time and Vmax in canine cardiac Purkinje fibers. Standard microelectrode techniques were used to monitor action potential characteristics at two sites along a canine cardiac false tendon and to measure interelectrode conduction time. The maximum rate of voltage rise during phase 0 (Vmax) and conduction time were independent of diastolic interval under control conditions. In the presence of local anesthetic drugs, recovery from drug-induced depression of Vmax and conduction were first order processes with recovery time constants (mean +/- S.D. in seconds) of 0.14 +/- 0.02 (for Vmax) and 0.15 +/- 0.04 (for conduction time) for lidocaine; 0.17 +/- 0.04 and 0.18 +/- 0.05, respectively, for mexiletine; 0.26 +/- 0.05 and 0.27 +/- 0.07 for amitriptyline; and 1.01 +/- 0.31 and 1.00 +/- 0.32 for procainamide. The kinetics of onset of block were studied using a 30-sec pause, followed by a pacing cycle length of 300 msec (for procainamide) or 1 sec (for quinidine). The onset time constants averaged 2.66 +/- 0.53 pulses (for Vmax) and 2.49 +/- 0.42 pulses (for conduction time) in the presence of procainamide; and 4.02 +/- 1.33 pulses (for Vmax) and 3.86 +/- 1.22 pulses (for conduction time) in the presence of quinidine. These experiments show that local anesthetic drugs produce use dependent changes in conduction time in vitro with time constants comparable to simultaneously measured time constants for effects on Vmax. They imply that the use dependence of drug effects on cardiac conduction can be studied quantitatively in vivo by studying the response to changes in activation frequency.     

Developmental changes in the muscarinic stimulation of canine Purkinje fibers

Acetylcholine (ACh) hyperpolarizes adult canine Purkinje fibers and induces a decrease in their automaticity. In Purkinje fibers from young dogs, there is a biphasic effect on automaticity, which increases at low and decreases at high ACh concentrations. We used standard microelectrode techniques to study these actions of ACh. In fibers from young dogs, 10(-10) to 10(-9) M ACh increased automaticity and 10(-5) M ACh decreased automaticity. The decrease was blocked by the M2 muscarinic blocker AFDX-116, whereas the increase was blocked by the predominant M1 blocker pirenzepine. The M2 agonist oxotremorine never increased automaticity. Rather, it decreased automaticity and hyperpolarized adult and young fibers, the former more than the latter. The hyperpolarization and biphasic effect on automaticity of ACh in fibers from young dogs failed to occur after treatment with pertussis toxin, suggesting that these effects are dependent on a pertussis toxin-sensitive G protein. These electrophysiologic studies suggest that postsynaptic M1 and M2 muscarinic processes modulate the automatic response of Purkinje fibers from young dogs and that the postsynaptic M1 pathway is no longer seen in the adult.     

Phospholipase C modulates automaticity of canine cardiac Purkinje fibers

Alpha-1 adrenergic agonists increase cardiac Purkinje fiber automaticity and elevate D-myo-inositol-trisphosphate (IP3) levels. To learn about the relationship between phosphoinositide metabolism and the modulation of cardiac rhythm, we used phospholipase C to activate phosphoinositide hydrolysis in an alpha-1 receptor-independent fashion and determined whether this intervention modulated automaticity. We used standard microelectrode techniques to study automaticity in adult Purkinje fiber bundles, fluorescence microscopy to study fura-2 fluorescence in isolated Purkinje and ventricular myocytes and standard biochemical techniques to measure inositol phosphate production in ventricular myocytes. Phospholipase C increased Purkinje fiber automaticity, a process that was enhanced by 10 mM lithium (which had no effect alone) and suppressed by verapamil or ryanodine (both 10 microM). Superfusion with 12-O-tetradecanoyl-phorbol-13-acetate phorbol ester, phospholipase D and A2, as well as L-alpha-phosphatidic acid, trypsin and D-myo-inositol-1-phosphate, D-myo-inositol-1,4-bisphosphate, IP3 and D-myo-inositol-1,4,5,6-tetrakisphosphate did not affect automatic rate or transmembrane potentials. Biochemical studies of ventricular myocytes demonstrated a phospholipase C-induced increase in intracellular and extracellular IP3, D-myo-inositol-1,4-bisphosphate and D-myo-inositol-1-phosphate at 3 min, with the extracellular increase persisting thereafter. Fluorescence microscopy with fura-2 revealed that phospholipase C increased systolic-free calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Frequency-dependent antiarrhythmic drug effects on postrepolarization refractoriness and ventricular conduction time in canine ventricular myocardium in vivo

Both conduction time (CT) and effective refractory period (ERP), absolute and relative to action potential duration (APD), are major determinants of re-entry arrhythmia circuits. We compared the effects of 3 commonly used class I antiarrhythmic agents, lidocaine, mexiletine and quinidine, and of the combination of the latter 2, on APD, ERP, ERP/APD ratio and interventricular CT in 26 in vivo canine hearts. To assess also the frequency dependence of these effects, each measurement was made at multiple steady-state cycle lengths ranging from 600 to 250 msec. A modified contact electrode technique was used to measure both APD and ERP simultaneously and at the same left ventricular site. Interventricular CT was measured as the interval from the stimulus of right ventricular paced beats to the upstroke of the ensuing left ventricular action potential. Lidocaine did not change APD, ERP and ERP/APD ratio significantly at any basic cycle length. In contrast, both mexiletine and quinidine increased the ERP/APD ratio, with progressively greater effects toward shorter cycle lengths. The quinidine/mexiletine combination increased the ERP/APD ratio significantly more than either drug alone (cycle length 350 msec: 8.3 +/- 2.2% quinidine; 17.6 +/- 7.0% mexiletine; 35.3 +/- 9.6% mexiletine/quinidine combination, P less than .01 vs. quinidine, P less than .05 vs. mexiletine). CT increased only with quinidine but not with mexiletine or lidocaine. Combination of mexiletine and quinidine caused no further slowing of conduction as compared to quinidine alone. Thus, although both ERP/APD ratio and CT are related to sodium channel conductance, drug effect on one parameter does not necessarily imply quantitatively similar effects on the other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of amiloride on pH regulation in canine cardiac Purkinje fibers

Myocardial cells utilize membrane transport systems for proton extrusion as well as internal buffers to preserve pH homeostasis. Our laboratory had shown previously that amiloride (0.01-1.0 mM) causes a time- and dose-dependent increase in action potential duration, early after depolarizations and enhanced automaticity. Ion-selective microelectrode technique was used to evaluate whether the observed electrophysiologic effects of amiloride are linked to inhibition of Na/H exchange and subsequent inability of the myocardial cell to maintain steady-state intracellular pH (pHi), either under normal physiological conditions or in the presence of an imposed acid load. We analyzed different components of intracellular pH transients that occur in response to NH4Cl exposure and washout, which allowed us to quantitatively describe the effects of Na/H exchange inhibition in a multicellular preparation. Amiloride (0.01-1.0 mM) did not change the steady-state pHi, but did cause a dose-dependent increase in both the time for the pHi to reach a minimum value (time-to-peak) during washout of NH4Cl as well as in the absolute minimum value of pHi (peak acid). The effects of amiloride on pHi transients are rapidly reversible and antagonized by physiologic values of extracellular sodium activity. We conclude that Na/H exchange inhibition by amiloride does not cause intracellular acidosis under normal physiologic conditions, despite the dramatic changes in action potential characteristics. However, amiloride affected the time-to-peak and the peak acid value of the pHi transient during NH4Cl washout at concentrations that had no discernible effect on the overall time course of pHi recovery.     

Interval-dependent effects of lidocaine on conduction in canine cardiac Purkinje fibers: experimental observations and theoretical analysis

Whereas the interval-dependence of antiarrhythmic drug effects on Vmax is known, the corresponding time-dependence of drug-induced changes in cardiac conduction is not established. The purpose of these experiments was to study the relationship between the time-dependence of lidocaine-induced changes in Vmax and in conduction time. Standard microelectrode techniques were used to monitor conduction and action potential characteristics of Purkinje fibers within free-running canine false tendons. Lidocaine-induced alterations in conduction and Vmax were related to drug concentration and to the preceding diastolic recovery time. At concentrations of 18 to 74 microM, changes in both Vmax and conduction time were an exponential function of diastolic interval, with recovery time constants averaging 123 to 150 msec for conduction time and 138 to 150 msec for Vmax. With higher lidocaine concentrations, changes in Vmax continued to be an exponential function of recovery interval, whereas changes in conduction time consistently deviated from the terminal exponential relationship at short diastolic intervals. These observations are consistent with the predictions of a model based on linear cable theory. Calculations using this model suggest that recovery from moderate drug-induced conduction slowing should proceed with a time course similar to changes in Vmax, whereas recovery from more severe conduction slowing should occur more rapidly than changes in Vmax. These observations suggest that the time dependence of drug effects on conduction in vivo can be analyzed quantitatively in relationship to observations on Vmax in vitro.     

Effect of lidocaine on the electrophysiological properties of ventricular muscle and Purkinje fibers

Preparations of right ventricular papillary muscle and false tendon (Purkinje fiber) were obtained from dog hearts, placed in a bath perfused with Tyrode solution, and observed both under control conditions and during exposure to lidocaine in concentrations from 1 x 10(-7) to 5 x 10(-4) mole/liter. Transmembrane voltages were recorded from both ventricular muscle (VM) and Purkinje fibers (PF) of spontaneously beating and electrically driven preparations. Low concentrations (1 x 10(-6) and 1 x 10(-5) mole/liter) attenuated or abolished phase 4 (diastolic) depolarization and spontaneous firing in PF without decreasing their diastolic excitability. Concentrations of 1 x 10(-5) mole/liter produced maximal shortening of both action potential duration (APD) and effective refractory period (ERP) and made the ERP long relative to APD; the latter alteration was more prominent in VM. At concentrations /= 1 x 10(-4) mole/liter) did not cause further shortening of APD or ERP in either VM or PF but did produce a decrease in peak V(max) of phase 0 and membrane responsiveness. In most cases, these concentrations also caused a decrease in RP or DTMV(max) and action potential amplitude, with progression to bizarre action potential depolarization and inexcitability. These properties of lidocaine are strikingly different from those of quinidine or procaine amide. The mechanisms responsible for lidocaine's in vivo antiarrhythmic action are discussed.     

The effects of calcium entry blockade on the vulnerability of infarcted canine myocardium toward ventricular fibrillation

The effects of the calcium entry blockers diltiazem, KB-944 [diethyl 4-(benzothiazol-2-yl)benzylphosphonate] and bepridil on the vulnerability of ischemically injured myocardium toward fibrillation were determined in urethane-anesthetized dogs 4 to 7 days after anterior myocardial infarction. Diltiazem (3.0-30.0 micrograms/kg/min X 30 min), KB-944 (0.3-3.0 mg/kg) and bepridil (1.0-10.0 mg/kg) were administered i.v. to produce equivalent increases in atrioventricular nodal effective and functional refractory periods as a measure of slow calcium channel blockade. At dosages producing equivalent increases in atrioventricular nodal refractoriness, diltiazem and KB-944 failed to increase the electrical current threshold required to produce ventricular fibrillation, whereas bepridil elevated the fibrillation threshold from 4.2 +/- 0.5 mA predrug to 14.7 +/- 2.2 mA postdrug (P less than .01). Increases in atrial (128 +/- 6-185 +/- 29 msec, P less than .01) and ventricular (156 +/- 4-175 +/- 6 msec, P less than .05) refractory periods accompanied the increase in fibrillation threshold with bepridil. These findings suggest that calcium entry blockade per se does not reduce ventricular vulnerability toward fibrillation in the setting of recent myocardial infarction.     

Frequency-dependent actions of phenytoin in adult and young canine Purkinje fibers

Phenytoin has been reported to be particularly effective in the treatment of postoperative ventricular arrhythmias in children. The authors used standard microelectrode techniques to examine the developmental changes in the action of phenytoin on the transmembrane action potential of neonatal and adult canine Purkinje fibers. Their goals were to test whether developmental differences in phenytoin action on the action potential might explain the clinical observations and to evaluate the contribution of use-dependent reduction of Vmax and effects on slow responses to the antiarrhythmic action of phenytoin. In Tyrode's solution with [K+]0 = 4 mM, phenytoin at 5 and 10 micrograms/ml (concentrations comparable to therapeutic plasma levels) had no major effects on action potential characteristics or use dependence at either age. At [K+]0 = 6 mM, on decreasing the drive cycle length from 1300 to 300 msec, phenytoin reduced Vmax significantly and in a concentration-dependent manner. The magnitude of this action was similar at both ages. Conduction times were also significantly prolonged. The time constants for onset of (tau o) and recovery from (tau r) use-dependent block were similar in neonates and adults. The effects of phenytoin on slow responses were significant, although modest, at both ages, but there was no significant effect on conduction. This study indicates that in K+-depolarized Purkinje fibers, use-dependent reduction of the fast Na+ current is a major determinant of the antiarrhythmic action of phenytoin. In contrast to lidocaine and quinidine, no age-related changes in phenytoin action were found, underscoring the different developmental effects of individual antiarrhythmic drugs.