Frequency- and voltage-dependent effects of disopyramide in canine Purkinje fibers

The voltage- and frequency-dependent blocking actions of disopyramide were assessed in canine Purkinje fibers within the framework of concentrations, membrane potentials, and heart rates which have relevance to the therapeutic actions of this drug. Vmax was used to assess the magnitude of sodium channel block. Disopyramide produced a concentration- and rate-dependent increase in the magnitude and kinetics of Vmax depression. Effects on activation time (used as an estimate of drug effect on conduction) were exactly analogous to effects on Vmax. A concentration-dependent increase in tonic block was also observed. Despite significant increases in tonic block at more depolarized potentials, rate-dependent block increased only marginally with membrane potential over the range of potentials in which propagated action potentials occur. Increases in extracellular potassium concentration accentuated drug effect on Vmax but attenuated drug effect on action potential duration. Recovery from rate-dependent block followed two exponential processes with time constants of 689 +/- 535 ms and 15.7 +/- 2.7 s. The latter component represents dissociation of drug from its binding site and the former probably represents recovery from slow inactivation. A concentration-dependent increase in the amplitude of the first component suggested that disopyramide may promote slow inactivation. There was less than 5% recovery from block during intervals equivalent to clinical diastole. Thus, depression of beats of all degrees of prematurity was similar to that of basic drive beats. Prolongation of action potential duration by therapeutic concentrations of drug following a long quiescent interval was minimal. However, profound lengthening of action potential duration occurred following washout of drug effect at a time when Vmax depression had reverted to normal, suggesting that binding of disopyramide to potassium channels may not be readily reversed. Variable effects on action potential duration may thus be attributed to a block of the window current flowing during the action potential being partially or over balanced by block of potassium channels. Purkinje fiber refractoriness was prolonged in a frequency-dependent manner. Disopyramide did not significantly alter the effective refractory period of basic beats but did increase the effective refractory period of sequential tightly coupled extra stimuli. The results can account for the antiarrhythmic actions of disopyramide during a rapid tachycardia and prevention of its initiation by programmed electrical stimulation.     

Electrophysiological effects of encainide and its metabolites in normal canine Purkinje fibers and Purkinje fibers surviving infarction

In this study, we assessed the effects of O-demethyl encainide (0.5 microM), the most active metabolite of encainide, and the combination with 3-methoxy-O-demethyl encainide (0.5 microM) and encainide (0.1 microM) on cardiac action potential characteristics in normal canine Purkinje fibers and Purkinje fibers surviving 24 h of myocardial ischemia. O-demethyl encainide decreased Vmax and conduction in normal Purkinje fibers and Purkinje fibers surviving infarction. Further decreases were observed with the combination. Action potential duration at both 50 and 95% repolarization was decreased by O-demethyl encainide. The combination of O-demethyl encainide, 3-methoxy-O-demethyl encainide, and encainide had no further effect. The combination of O-demethyl encainide, 3-methoxy-O-demethyl encainide, and encainide produced a smaller change in effective refractory period than O-demethyl encainide in normal Purkinje fibers and in Purkinje fibers surviving infarction. O-demethyl encainide and the combination shifted the membrane responsiveness curve to more negative potentials in both normal Purkinje fibers and Purkinje fibers surviving infarction. It is apparent from this study that there are differences in the effects of O-demethyl encainide and the combination of O-demethyl encainide, 3-methoxy-O-demethyl encainide, and encainide in normal Purkinje fibers compared with Purkinje fibers surviving infarction. Also, the combination used in this study had different electrophysiological effects than those of O-demethyl encainide alone.     

Reassessment of the electrophysiological effects of the antiarrhythmic agent quinidine in canine Purkinje fibers

Microelectrode techniques were used to re-examine the direct effects of quinidine on isolated canine Purkinje fibers. Our results confirmed the previous findings that quinidine in a dose-related manner suppressed spontaneous activity, prolonged effective refractory period and depressed the maximal rate of upstroke (dv/dt). Quinidine shifted the membrane response curves to the right of the voltage axis regardless of external K⁺ concentrations. The drug diminished the phase 2 plateau of the action potential indicating a possible inhibitory effect on calcium influx. Quinidine generally lengthened the action potential duration (ADP) but the extent of the increase varied with the site of recording along the Purkinje conduction system. The shorter APDs were lengthened far more than the longer APDs, thus dissimilar APDs became more uniform. Following the alteration of external K⁺ between 2 mM and 6 mM, the effects of quinidine on transmembrane characteristics, including the depression of dv/dt, remain constant with the exception of APD.     

Membrane current following activity in canine cardiac Purkinje fibers

Membrane current following prolonged periods of rapid stimulation was examined in short (less than 1.5 mm) canine cardiac Purkinje fibers of radius less than 0.15 mm. The Purkinje fibers were repetitively stimulated by delivering trains of depolarizing voltage clamp pulses at rapid frequencies. The slowly decaying outward current following repetitive stimulation ("post-drive" current) is eliminated by the addition of 10(-5) M dihydro-ouabain. The post-drive current is attributed to enhanced Na/K exchange caused by Na loading during the overdrive. Depolarizing voltage clamp pulses initiated from negative (- 80 mV) or depolarized (-50 mV) holding potentials can give rise to post- drive current because of activation of tetrodotoxin-sensitive or D600- sensitive channels. The magnitude of the post-drive current depends on the frequency of voltage clamp pulses, the duration of each pulse, and the duration of the repetitive stimulation. The time constant of decay of the post-drive current depends on extracellular [K] in accordance with Michaelis-Menten kinetics. The Km is 1.2 mM bulk [K], [K]B. The mean time constant in 4 mM [K]B is 83 s. Epinephrine (10(-5) M) decreases the time constant by 20%. The time constant is increased by lowering [Ca]o between 4 and 1 mM. Lowering [Ca]o further, to 0.1 mM, eliminates post-drive current following repetitive stimulation initiated from depolarized potentials. The latter result suggests that slow inward Ca2+ current may increase [Na]i via Na/Ca exchange.     

The effects of acidosis and bicarbonate on action potential repolarization in canine cardiac Purkinje fibers

Studies were performed on canine cardiac Purkinje fibers to evaluate the effects of acidosis and bicarbonate (HCO3) on action potential repolarization. Extracellular pH (pHe) was reduced from 7.4 to 6.8 by increasing carbon dioxide (CO2) concentration from 4 to 15% in a HCO3-buffered solution or by NaOH titration in a Hepes-buffered solution. Both types of acidosis produced a slowing of the rate of terminal repolarization (i.e., period of repolarization starting at about -60 mV and ending at the maximum diastolic potential) with an attendant increase in action potential duration of 10--20 ms. This was accompanied by a reduction in the maximum diastolic potential of 2--8 mV. In contrast, if the same pH change was made by keeping CO2 concentration constant and lowering extracellular HCO3 from 23.7 to 6.0 mM, in addition to the slowing of terminal repolarization, the plateau was markedly prolonged resulting in an additional 50- to 80-ms increase in action potential duration. If pHe was held constant at 7.4 and HCO3 reduced from 23.7 mM to 0 (Hepes-buffered solution), the changes in repolarization were nearly identical to those seen in 6.0 mM HCO3 except that terminal repolarization was unchanged. This response was unaltered by doubling the concentration of Hepes. Reducing HCO3 to 12.0 mM produced changes in repolarization of about one-half the magnitude of those in 6.0 mM HCO3. These findings suggest that in Purkinje fibers, HCO3 either acts as a current that slows repolarization or modulates the ionic currents responsible for repolarization.     

Membrane currents following activity in canine cardiac Purkinje fibers.

Recent experiments in canine Pukinje fibers (Gadsby and Cranefield, 1979) have shown that following a period of sodium loading in K+-free solution a slowly decaying outward current is observed. This current has been attributed to the activity of the electrogenic Na+-K+ exchange pump. In the present paper we show that similar slowly decaying outward currents are observed following prolonged periods of overdrive with action potentials or with brief depolarizing voltage clamp pulses. The dependent of the prolonged outward current on the duration and frequency of the preceding period of overdrive and on the potential following overdrive is reported. We also present results which indicate that a large portion of this current can be induced by phasic Na+ loading through the fast-inward channel.     

Slow inactivation of a tetrodotoxin-sensitive current in canine cardiac Purkinje fibers.

We used the two-microelectrode voltage clamp technique and tetrodotoxin (TTX) to investigate the possible occurrence of slow inactivation of sodium channels in canine cardiac Purkinje fibers under physiologic conditions. The increase in net outward current during prolonged (5-20 s) step depolarizations (range -70 to +5 mV) following the application of TTX is time dependent, being maximal immediately following depolarization, and declining thereafter towards a steady value. To eliminate the possibility that this time-dependent current was due to inadequate voltage control of these multicellular preparations early during square clamp pulses, we also used slowly depolarizing voltage clamp ramps (range 5-100 mV/s) to ensure control of membrane potential. TTX-sensitive current also was observed with these voltage ramps; the time dependence of this current was demonstrated by the reduction of the peak current magnitude as the ramp speed was reduced. Reducing the holding potential within the voltage range of sodium channel inactivation also decreased the TTX-sensitive current observed with identical speed ramps. These results suggest that the TTX-sensitive time-dependent current is a direct measure of slow inactivation of canine cardiac sodium channels. This current may play an important role in modulating the action potential duration.     

Effects of ritanserin on transmembrane action potentials in canine Purkinje fibers.

Ritanserin has been reported to be a potential antiarrhythmic. We studied the cellular electrophysiologic effects of ritanserin in canine Purkinje fibers. Ritanserin produced significant depressant effects on transmembrane action potentials elicited in canine Purkinje fibers. At concentrations of 10 and 40 mg/liter, ritanserin decreased Vmax (the upstroke velocity) of action potential in a dose-dependent fashion and shortened the duration of fast response action potential. These concentrations of ritanserin also reduced the amplitude and duration of the slow response action potentials induced in Purkinje fibers treated with isoproterenol (10(-5) M) and high K+ (22 mM). These in vitro results suggest that the cellular electrophysiologic actions of ritanserin may be due to its direct actions on cardiac sodium and calcium channels, which, in turn, may account for its antiarrhythmic effects.     

The electrophysiological effects of disopyramide phosphate on canine ventricular muscle and Purkinje fibers in normal and low potassium

We studied the effect of lowering the extracellular potassium concentration ([K+]o) on the electrophysiological actions of disopyramide phosphate, a new antiarrhythmic drug. At low [K+]o, therapeutic concentrations of disopyramide phosphate caused significantly less depression of action potential amplitude and maximum upstroke velocity of both Purkinje fiber and ventricular muscle action potentials. The drug shifted the membrane responsiveness curve along the voltage axis to more negative membrane potentials regardless of [K+]o. However, a greater shift occurred when [K+]o was normal. Disopyramide phosphate prolonged both action potential duration and effective refractory period in all fibers but there was consistently greater prolongation of these parameters at low [K+]o. More importantly, disopyramide phosphate altered repolarization time course of action potentials in such a way that action potentials with dissimilar durations throughout the ventricular conducting system became more equal. The drug was less effective in decreasing this disparity in action potential durations throughout the ventricles in the presence of low [K+]o. These modifications of the electrophysiological actions of disopyramide by low [K+]o suggest that a therapeutic concentration of disopyramide might have less of an antiarrhythmic effect in the presence of hypokalemia.     

Extracellular [K+] fluctuations in voltage-clamped canine cardiac Purkinje fibers.

Membrane currents and extracellular [K+] were measured in canine Purkinje strands during voltage-clamp steps to plateau or diastolic potentials. Extracellular [K+] increased during step depolarizations and decreased during step hyperpolarizations. On hyperpolarization, the largest fraction of the K+ depletion occurred during the initial 500 ms of the voltage-clamp step and was correlated with a potassium depletion current, the id. A slower component of the depletion also occurred on hyperpolarization and had a time constant consistent with cylindrical diffusion of potassium within the Purkinje strands. On depolarization, there is an accumulation of K+ that is correlated with the plateau current ix. On termination of depolarizing test pulses, the K+ accumulation decays with a time course similar to the ix tail current. Surprisingly, no accumulation of K+ occurred during the arrhythmogenic transient inward current, TI, suggesting that the selectivity of this current should be reevaluated.     

Carbon dioxide effects on the ventilatory response to sustained hypoxia

We examined the interrelation between CO2 and the ventilatory response to moderate (80% arterial saturation) sustained hypoxia in normal young adults. On a background of continuous CO2-stimulated hyperventilation, hypoxia was introduced and sustained for 25 min. Initially, with the introduction of hypoxia onto hypercapnia, there was a brisk additional increase in inspiratory minute ventilation (VI) to 284% of resting VI, but the response was not sustained and hypoxic VI declined by 36% to a level intermediate between the initial increase and the preexisting hypercapnic hyperventilation. Through the continuous hypercapnia, the changes in hypoxic ventilation resulted from significant alterations in tidal volume (VT) and mean inspiratory flow (VT/TI) without changes in respiratory timing. In another experiment, sustained hypoxia was introduced on the usual background of room air, either with isocapnia or without maintenance of end-tidal CO2 (ETCO2) (poikilocapnic hypoxia). Regardless of the degree of maintenance of ETCO2, during 25 min of sustained hypoxia, VI showed an initial brisk increase and then declined by 35-40% of resting VI to a level intermediate between the initial response and resting room air VI. For both isocapnia and poikilocapnic conditions, the attenuation of VI was an expression of a diminished VT. Thus the decline in ventilation with sustained hypoxia occurred regardless of the background ETCO2, suggesting that the mechanism underlying the hypoxic decline is independent of CO2.     

Multiple effects of calcium antagonists on plateau currents in cardiac Purkinje fibers

We studied the influence of Mn, La, and D600 on action potentials and plateau currents in cardiac Purkinje fibers. The Ca antagonists each abolished the second inward current, but they failed to act selectively. Voltage clamp experiments revealed two additional effects: decrease of slow outward current (iotachi) activation, and increase of net outward time-independent plateau current. These effects occurred at inhibitor concentrations used in earlier studies, and were essential to the reconstruction of observed Ca antagonist effects on electrical activity. The inhibitory influence of Mn, La, and D600 on iotachi suggested that iotachi activation might depend upon prior Ca entry. This hypothesis was not supported, however, when [Ca]omicron was varied: elevating [Ca]omicron enhanced Ca entry, but iotachi was nevertheless depressed. Thus, the results suggested instead that Ca antagonists and Ca ions have rather similar effects on iotachi, possibly mediated by changes in membrane surface charge.     

Oxidized low density lipoproteins exert arrhythmogenic effects in rabbit Purkinje fibers.

The electrophysiological effects of oxidized low density lipoproteins (ox-LDLs) have been studied in rabbit Purkinje fibers using standard microelectrode techniques, in comparison with native LDLs (n-LDLs) and lysophosphatidylcholine (LPC). At the concentration of 100 micrograms protein/ml, ox-LDL but never n-LDL induced the abrupt occurrence of abnormal electrical activities during the basic stimulation of 1 Hz (6/13 fibers) and the development of either early afterdepolarizations (6/13 fibers) or abnormal automaticity (4/13 fibers) at low frequencies (0.1 and 0.03 Hz). Short trains of rapid stimulation (2, 3, 4 and 5 Hz) did not trigger delayed afterdepolarizations. However, early afterhyperpolarizations were commonly seen after each action potential. 30 microM LPC caused quite similar electrophysiological derangements. The results suggest that ox-LDLs may exert arrhythmogenic effects partly explained by their LPC content.     

The effects of adrenergic blockade on fetal response to hypoxia

The actions of the adrenergic blocking agents propranolol and phentolamine upon the responses of 124-135 days fetal sheep to hypoxia induced by causing pregnant ewes to breathe 9% O2 and 3% CO2 in N2 have been studied. During hypoxia fetal heart rate fell and any tendency for this to return was prevented by propranolol and stimulated by phentolamine. The ability of the fetal heart rate to return during hypoxia appears to be related to the rise in plasma catecholamines. Hypoxia induced increases in plasma ACTH and cortisol and in plasma metabolites appear to have the same characteristics as those changes induced by catecholamine infusion; the former being largely an alpha-receptor effect and the latter being beta-receptor mediated. The results indicate but do not prove that many of the fetal responses to hypoxia could be caused by the rise in plasma catecholamines.     

An analysis of calcium effects on diastolic depolarization in sheep cardiac Purkinje fibers

The events by which [Ca]O modifies diastolic depolarization (DD) were analyzed in sheep cardiac Purkinje fibers perfused in vitro. Cs (2 mM) reduced diastolic depolarization (DD) at different [Ca]O and in 10.8 mM [Ca]O revealed an oscillatory potential (VOS) and the decay of a prolonged depolarization (Vex). In the presence of Cs, procedures that reduce Cai (a slower driving rate, lower [Ca]O or tetrodotoxin) abolished VOS and Vex and partially restored DD. In 10.8 mM [Ca]O and at all driving rates, Cs reduced DD slope, DD amplitude and VOS amplitude but had little effect on the VOS time to peak. In 10.8 mM [Ca]O, decreasing calcium overload by different means (2.6 microM TTX, 0.2 mM Cd) abolished VOS and decreased DD slope and amplitude. Substituting Na with Li induced marked aftercontractions but small VOS. In 10.8 mM [Ca]O, Li increased the amplitude of the aftercontractions and decreased that of VOS. Li also depolarized slightly the resting membrane and abolished the voltage undershoot (Emax) at the end of the action potential. In low [K]O, Li repolarized the resting membrane but the repolarization was maintained only in the presence of Ca. It is concluded that Ca overload causes both VOS and Vex which can either be masked by or can mask DD depending on the magnitude of DD and of Ca overload. VOS is apparently caused by an electrogenic Na-Ca exchange since Li-induced Ca overload increases the aftercontraction but decreases VOS.     

The relationship among intracellular sodium activity, calcium, and strophanthidin inotropy in canine cardiac Purkinje fibers

The role of sodium and calcium ions in strophanthidin inotropy was studied by measuring simultaneously the electrical, mechanical, and intracellular sodium ion activities in electrically driven cardiac Purkinje fibers under conditions that change the intracellular sodium or calcium level (tetrodotoxin, strophanthidin, high calcium, and norepinephrine). Tetrodotoxin (TTX; 1-5 X 10(-6)M) shifted the action potential plateau to more negative values, shortened the action potential duration, and decreased the contractile tension and the intracellular sodium ion activity (aiNa). The changes in tension and in aiNa caused by TTX appear to be related since they had similar time courses. Strophanthidin (2-5 X 10(-7)M) increased tension and aiNa less in the presence of TTX, and, for any given value of aiNa, tension was less than in the absence of TTX. Increasing extracellular calcium (from 1.8 to 3.3-3.6 mM) or adding norepinephrine (0.5-1 X 10(-6)M) increased tension and decreased aiNa less in the presence than in the absence of TTX. When two of the above procedures were combined, the results were different. Thus, during the increase in aiNa and tension caused by strophanthidin in the presence of TTX, increasing calcium or adding norepinephrine increased tension markedly but did not increase aiNa further. In a TTX-high calcium or TTX-norepinephrine solution, adding strophanthidin increased both tension and aiNa, and the increase in tension was far greater than in the presence of TTX alone. The results indicate that: (a) the contractile force in Purkinje fibers is affected by a change in aiNa; (b) a decrease in aiNa by TTX markedly reduces the inotropic effect of strophanthidin, possibly as a consequence of depletion of intracellular calcium; (c) increasing calcium influx with norepinephrine or high calcium in the TTX-strophanthidin solution produces a potentiation of tension development, even if aiNa does not increase further; and (d) when the calcium influx is already increased by high calcium or norepinephrine, strophanthidin has its usual inotropic effect even in the presence of TTX. In conclusion, the positive inotropic effect of strophanthidin requires that an increase in aiNa be associated with suitable calcium availability.