The effects of adriamycin on normal and ouabain-toxic canine Purkinje and ventricular muscle fibers

In the "Na+ lag hypothesis" of cardiac glycoside action, [Ca++]i increases through sodium-calcium exchange following block of the sodium-calcium ATPase. This accumulation of [Ca++]i has been suggested to be responsible for digitalis-induced delayed after depolarizations and arrhythmias. We used standard microelectrode techniques to study the effects of adriamycin, 10-200 microM, on the canine Purkinje fiber transmembrane potential. Adriamycin, 10 and 50 microM, had no effect on the action potential other than inducing a 28% increase in duration at 50 microM (p less than 0.01). Adriamycin, 100 and 200 microM, further prolonged action potential duration and decreased amplitude and Vmax. We then studied the effects of adriamycin, 50 microM (a concentration that purportedly blocks sodium-calcium exchange), on ouabain-induced delayed afterdepolarizations and aftercontractions in Purkinje fiber and ventricular muscle. After initially increasing delayed afterdepolarization amplitude in five of nine Purkinje fibers, adriamycin reversibly reduced their amplitude in all nine, by 78%, at a drive cycle length of 500 msec (P less than 0.01). Adriamycin, 50 microM, had no effect on calcium ion-induced slow response action potentials, suggesting this concentration does not significantly reduce the slow inward current. In ventricular muscle, adriamycin, 50 microM, did not significantly reduce contraction but did depress aftercontractions (P less than 0.025). In sum: in concentrations that have no effect on the AP other than prolonging duration, adriamycin, 50 microM, reversibly depresses ouabain-induced delayed afterdepolarizations and aftercontractions; however, adriamycin, 50 microM, does not depress calcium ion-dependent action potentials. Although the action of adriamycin on delayed afterdepolarizations and aftercontractions is consistent with direct inhibition of the transient inward current and/or an indirect reduction via reduced activity of sodium-calcium exchange, whether either or both of these mechanisms is involved must be defined by further experimentation.     

Nonlinear relation between Vmax and INa in canine cardiac Purkinje cells

We studied the relation of the maximal upstroke velocity (Vmax) of action potentials to the peak sodium current (INa) under voltage clamp in single, internally perfused, canine cardiac Purkinje cells under conditions that ensured membrane action potentials due only to INa. Three different methods of altering sodium channel availability were investigated: voltage-dependent inactivation, tetrodotoxin (TTX) block, and use-dependent block by quinidine. Under all three conditions, the relation of Vmax to INa was nonlinear, and no relation was found that would allow prediction of INa results from Vmax measurements. With voltage-dependent inactivation or TTX block, sodium channel availability measured by Vmax was reduced less than availability measured by peak INa, so that Vmax overestimated sodium channel availability. This overestimation of sodium channel availability by Vmax could be attributed to greater sodium channel mobilization during the slowed action potential upstrokes. The overestimation varied with experimental temperature as a consequence of changes in sodium channel kinetics. Vmax also overestimated sodium channel availability during TTX exposure so that the Kd for TTX block was 4.5 micron from Vmax measurements but only 1.6 microM from INa measurements. Use-dependent block of INa by quinidine had a striking voltage-dependent component under voltage clamp that could not be appreciated from action potentials. Consequently, block could be underestimated or overestimated by Vmax measurements. We conclude that Vmax measurements represent a convenient index for INa, but Vmax is not a reliable method for quantitative studies of sodium channel behavior.     

A quantitative comparison of the relation between the shape of the action potential and the pattern of stimulation in canine ventricular muscle and Purkinje fibers

The purpose of these experiments was to obtain a quantitative comparison of the relation between the shape of the action potential and the pattern of stimulation in canine ventricular muscle and Purkinje fibers. Papillary muscles with attached false tendons were removed from dogs and studied, utilizing standard microelectrode techniques. The area beneath the action potential was obtained by electronic integration and used as an index of changes in shape. With abrupt increases or decreases in the frequency of stimulation, there were rapid early changes in shape followed by slow changes in shape which required approximately 1 min before a new steady state was achieved. In ventricular muscle, the rapid and slow changes of shape were opposite in direction. With brief pauses, the area of Purkinje potentials increased while that of ventricular muscle decreased, thus markedly enhancing nonuniformity of recovery. With premature stimuli, the area of Purkinje potentials decreased while that of muscle fibers increased. These observations suggest that there are fundamental differences in factors which control repolarization in Purkinje fibers and muscle. The observed phenomena could not have been predicted by previously published work on the steady state relationship between the shape of the action potential and frequency of stimulation. Furthermore, none of the currently proposed models for the generation of the cardiac action potential will account for all of these observations. The discordant behavior of action potential shapes in Purkinje and muscle during changes in the pattern of stimulation may contribute to the genesis of cardiac arrhythmias.     

犬三层心室肌细胞的分离和鉴别

目的探讨有效分离和鉴别犬三层心室肌细胞的方法。方法用带有左室前降支的犬心肌组织块灌流分离心室肌细胞,得到的心肌细胞先根据解剖部位大致分成三层,再采用膜片钳技术,在电流钳模式下,随机选择各层15个细胞记录动作电位(AP),根据AP的形态、时程、频率依赖性进一步判断是否为相应层的心室肌细胞。结果经左室前降支插管灌流心肌组织块,可以得到数量多、状态好的心肌细胞。在15个心室肌细胞中,能准确判断其层次的有:外层7个、中层5个、内层8个。结论经冠状动脉插管灌流分离犬心室肌细胞是可行的,结合解剖部位和动作电位特点,能有效鉴别不同层的心室肌细胞。     

Morphology of electrophysiologically identified junctions between Purkinje fibers and ventricular muscle in rabbit and pig hearts

Purkinje fiber-ventricular muscle (PV) junctions were identified by extracellular recording in isolated, superfused preparations from rabbit and pig hearts. Microelectrode recordings from different cell types at the PV junctions were obtained, and the cells recorded from were retrieved microscopically. To this end 26 tissue blocks were serially sectioned at 4 microns. Microscopic identification of the very cell recorded from was obtained in five of seven Purkinje, five of 16 transitional, and two of two ventricular muscle cell recordings. In addition, some tissue blocks from both junctional and nonjunctional sites identified only by extracellular recording were examined in serial sections. Transitional cells in the rabbit heart are thin, broad bandlike cells (30-35 by 3-5 microns) arranged in one or two sheets in the subendocardium between the Purkinje layer and ventricular mass. Transitional cells are coupled via short, thin strands to both Purkinje and ventricular muscle cells. A second type of PV coupling was observed frequently in the pig, but in only one of 21 cases in the rabbit. Here, a short, linear segment of small transitional cells connected large-diameter Purkinje cells to ventricular muscle cells. Distances found between Purkinje-transitional cell coupling sites and transitional cell-ventricular muscle coupling sites varied from 100 to 1,000 microns in the rabbit heart and from 50 to several hundred micrometers in the pig heart. Action potentials from transitional cells typically showed multiple components in their upstroke. Both our morphological and electrophysiological findings are compatible with the existence of a relatively high-resistance barrier between Purkinje and transitional cells and between transitional and ventricular muscle cells.     

Variations in the functional electrical coupling between the subendocardial Purkinje and ventricular layers of the canine left ventricle

Action potential propagation from the subendocardial Purkinje network into the ventricular muscle is an essential link in cardiac activation. Studies of papillary muscles have indicated that ventricular muscle activation by the Purkinje network occurs only at discrete, localized regions near the papillary muscle base. Over the rest of the endocardial surface, however, the spatial distribution of these subendocardial Purkinje to ventricular muscle connections has been less well defined. We therefore studied in vitro 12 canine left ventricular preparations (eight from the septum, four from the lateral wall), using a high-density (1-mm spacings), high-resolution extracellular mapping technique to determine the subendocardial Purkinje and ventricular muscle activation sequences. These studies show that the distribution of subendocardial Purkinje to ventricular muscle electrical coupling is spatially inhomogeneous, and that the junctional regions themselves have variable degrees of electrical coupling. We also attempted to determine whether ventricular muscle coupling to the Purkinje network might influence Purkinje network conduction velocity. We found that on the papillary muscle apex, a region without direct Purkinje to ventricular muscle propagation, Purkinje network conduction velocity was slowed, suggesting that the Purkinje network might be electrically loaded by the underlying ventricular muscle. Finally, we performed numerical simulations using a model consisting of two layers of excitable cells to evaluate the effects that different electrical coupling patterns and/or different coupling resistivities between the two layers might have on activation of each layer. These simulation studies suggest that a coupling pattern having discrete junctional sites between the two layers (similar to our findings for subendocardial Purkinje to ventricular muscle coupling) is beneficial, as this arrangement allows more rapid activation of both layers by minimizing electrical loading of the thin Purkinje layer by the thicker ventricular muscle layer.     

Differences in the electrophysiological response of canine ventricular subendocardium and subepicardium to acetylcholine and isoproterenol. A direct effect of acetylcholine in ventricular myocardium

A prolongation of the ventricular effective refractory period in response to cholinergic agonists or vagal stimulation has been demonstrated in a number of in vivo animal models. However, exposure of isolated myocardial tissues obtained from these hearts to as much as 10(-4) M acetylcholine has been shown to produce essentially no change in action potential duration or effective refractory period. The discrepancy between the in vivo and in vitro findings generally has been explained on the basis of accentuated antagonism, whereby parasympathetic agonists exert their influence through antagonism of the effects of beta-adrenergic tone in vivo. The fact that acetylcholine exerts little if any direct effect on the electrical activity of ventricular myocardium, although well accepted, is based exclusively on studies performed using endocardial preparations. Our recent demonstration of major electrophysiological differences between canine ventricular endocardium and epicardium prompted us to examine the effects of acetylcholine and the role of accentuated antagonism in these two tissue types. Using standard microelectrode techniques, we show that acetylcholine (10(-7)-10(-5) M) has little if any effect in canine ventricular endocardium but a pronounced effect to either prolong or markedly abbreviate action potential duration and effective refractory period in epicardium. These effects of acetylcholine on epicardium are attended by an accentuation of the spike and dome morphology of the action potential, are readily reversed with atropine, fail to appear when epicardium is pretreated with the transient outward current blocker 4-aminopyridine, are accentuated in the presence of isoproterenol (10(-7) to 5 x 10(-6) M), and persist in the presence of propranolol. Isoproterenol-induced abbreviation of action potential duration and effective refractory period is also shown to be more pronounced in epicardium than in endocardium; equimolar concentrations of acetylcholine completely antagonize the effects of isoproterenol in endocardium and epicardium. We conclude that acetylcholine exerts important direct effects on the electrical response of canine ventricular myocardium, which are accentuated in the presence of beta-adrenergic agonists. Our findings suggest the differential response of epicardium and endocardium to acetylcholine is due to the presence of a transient outward current-mediated spike and dome morphology in the epicardial action potential. Finally, the data suggest that acetylcholine may exert antiarrhythmic as well as arrhythmogenic effects through its actions to alter conduction and refractoriness.     

Microvascular pressures and resistances in the left ventricular subepicardium and subendocardium

These experiments tested the hypothesis that differences in the distribution of subepicardial and subendocardial microvascular resistances may alter the transmural distributions of microvascular pressures. Isolated blood- and physiological saline-perfused porcine hearts were surgically incised to enable exposure of the subendocardial and subepicardial microcirculations. Microvascular pressures were measured during cardiac arrest and maximal vasodilation at various perfusion pressures to formulate relations between perfusion pressure and microvascular pressure in the different subendocardial (both free wall and papillary muscle) and subepicardial segments. Measurements of arteriolar and venular pressures in both myocardial regions were performed in comparably sized vessels (80-120 microns in diameter). At a coronary perfusion pressure of 100 mm Hg, subendocardial arteriolar and venular pressures were 60 +/- 4 and 33 +/- 3 mm Hg, respectively. In contrast, at the same coronary perfusion pressure, arteriolar and venular pressures in the subepicardial microcirculation averaged 80 +/- 6 and 22 +/- 3 mm Hg, respectively (p less than 0.05 versus subendocardium). At all levels of coronary perfusion pressure, arteriolar pressures were significantly lower in the subendocardium than in the subepicardium (p less than 0.05). Venular pressures were also higher in the subendocardial microcirculation than in the subepicardial microcirculation at all but the lowest perfusion pressure (p less than 0.05). The relative distribution of resistances in arteries, microvessels, and veins was also different between the subepicardium and subendocardium. Specifically, in the subendocardium, arterial and venous resistances were higher, percentage-wise, but microvascular resistance was proportionately lower than that in the subepicardium (p less than 0.05). From these data, it is concluded that the distribution of microvascular resistances and pressures is different during maximal vasodilation in the subepicardial and subendocardial microcirculations of the left ventricle. It is also speculated that differences in autoregulatory capacity and vulnerability to ischemia may be partially related to unequal distribution of microvascular resistances across the wall of the left ventricle.     

Interaction between overdrive excitation and overdrive suppression in canine Purkinje fibres

The interaction between overdrive excitation and overdrive suppression was studied in canine Purkinje fibres perfused in vitro with a solution containing noradrenaline (10(-6) mol . litre-1) and/or high calcium (8.1 mmol . litre-1). The following results were obtained: 1) in the presence of either high calcium or noradrenaline, a fast drive induced at times an acceleration of the spontaneous rhythm; 2) the simultaneous administration of high calcium and noradrenaline quite often allowed the onset of overdrive excitation that showed different patterns; 3) short (5 to 10 s) and fast (60 to 180 min) drives elicited a rhythm that was fastest immediately after overdrive, gradually slowed toward control and could be followed by suppression; 4) in other instances, overdrive caused little acceleration but failed to induce suppression; 5) drives longer than 15 s usually induced only suppression, although there could be a few beats before suppression; 6) intermittent drive led to an initial excitation and then to suppression; 7) driving at a rate slower than the spontaneous rate caused a temporary suppression of the spontaneous discharge which resumed even if the slow drive continued; 8) excitation was not induced by overdriving in low calcium, even in the presence of noradrenaline. It is concluded that many of the in vivo features of overdrive excitation can be reproduced in vitro in small strands of Purkinje fibres. The relationship between overdrive excitation and overdrive suppression involves the opposing actions of drive on the oscillatory potential and on diastolic depolarisation.     

Complex and rate-dependent beat-to-beat variations in Ca transients of canine Purkinje cells

Purkinje fibers play an essential role in transmitting electrical impulses through the heart, but they may also serve as triggers for arrhythmias linked to defective intracellular calcium (Ca²⁺) regulation. Although prior studies have extensively characterized spontaneous Ca²⁺ release in nondriven Purkinje cells, little attention has been paid to rate-dependent changes in Ca²⁺ transients. Therefore we explored the behaviors of Ca²⁺ transients at pacing rates ranging from 0.125 to 3 Hz in single canine Purkinje cells loaded with fluo3 and imaged with a confocal microscope. The experiments uncovered the following novel aspects of Ca²⁺ regulation in Purkinje cells: 1) the cells exhibit a negative Ca²⁺-frequency relationship (at 2.5 Hz, Ca²⁺ transient amplitude was 66 ± 6% smaller than that at 0.125 Hz); 2) sarcoplasmic reticulum (SR) Ca²⁺ release occurs as a propagating wave at very low rates but is localized near the cell membrane at higher rates; 3) SR Ca²⁺ load declines modestly (10 ± 5%) with an increase in pacing rate from 0.125 Hz to 2.5 Hz; 4) Ca²⁺ transients show considerable beat-to-beat variability, with greater variability occurring at higher pacing rates. Analysis of beat-to-beat variability suggests that it can be accounted for by stochastic triggering of local Ca²⁺ release events. Consistent with this hypothesis, an increase in triggering probability caused a decrease in the relative variability. These results offer new insight into how Ca²⁺ release is normally regulated in Purkinje cells and provide clues regarding how disruptions in this regulation may lead to deleterious consequences such as arrhythmias.     

Alternans of action potential duration after abrupt shortening of cycle length: differences between dog Purkinje and ventricular muscle fibers

The purpose of this study was to determine whether the alternans of action potential duration (APD) occurring in Purkinje and ventricular muscle fibers after an abrupt shortening of cycle length can be explained by the two factors controlling the cycle length-dependent APD changes (i.e., restitution and memory effect). Action potentials were recorded simultaneously from dog Purkinje fibers and ventricular muscle fibers using conventional microelectrode techniques. APD change during alternans was dependent on the preceding diastolic interval in the same manner as during restitution in Purkinje fibers but not in ventricular muscle fibers. The course of memory change was not affected by the presence of alternans in either fiber type. In Purkinje fibers, APD alternans was attenuated by a Ca2+ channel blocker, nisoldipine (2 X 10(-6) M), and augmented by a Ca2+ channel agonist, Bay K 8644 (3 X 10(-8) M). These effects were attributed to the changes in the kinetics and the amplitude of restitution. In ventricular muscle fibers, APD alternans was always preceded and accompanied by alternans of action potential shape. Alternans of both action potential shape and APD was suppressed by nisoldipine (2 X 10(-6) M) and attenuated by Bay K 8644 (3 X 10(-8) M). These results show that in Purkinje fibers, APD during alternans can be explained by restitution and memory effect. However, in ventricular muscle fibers, the mechanism of APD alternans is linked to factors controlling action potential shape. These findings are compatible with the hypothesis that APD alternans in Purkinje fibers depends on the differences in the recovery of membrane currents generated by the preceding action potential and in ventricular muscle fibers on the differences in the concentration and/or handling of intracellular calcium.     

Effect of higenamine on action potential of Purkinje and ventricular myocardial cells

Action potentials of isolated Purkinje cells of dogs and the electrophysiological actions of higenamine on the dog's Purkinje cells were studied by glass microelectrodes. The actions of higenamine on the action potential of Purkinje cells were: (1) increase of amplitude of the plateau phase, (2) enhancement of repolarization of phase 3 and shortening of action potential duration (APD), (3) lower concentration of higenamine (10(-7) g/ml) causing shortening of APD without any significant change of the effective refractory period (ERP), and higher concentration (10(-6) g/ml) of it causing shortening of both APD and ERP, (4) higher concentration (10(-6) g/ml) of higenamine increasing the slope of phase 4, decreasing the threshold of depolarization, and increasing the automaticity of the Purkinje cells, (5) action of higenamine on the Purkinje cells only partially blocked by tetrodotoxin (TTX) and verapamil. The above facts suggest that: (1) higher concentration of higenamine may induce tachyarrhythmias, while lower concentration may prevent arrhythmias; (2) in low concentration of higenamine, there is a relative increase of ERP. Thus, higenamine seems to be safer than isoproterenol in the treatment of bradyarrhythmias.     

Multiple types of Ca2+ currents in single canine Purkinje cells

Whole-cell Ca2+ channel currents were recorded from isolated single canine Purkinje and ventricular cells to determine whether there were multiple types of Ca2+ channels in these two cell types, as in many other excitable tissues. The experimental conditions were such that currents other than Ca2+ channel currents were largely suppressed. The charge carrier was either Ca2+ or Ba2+ (5mM). In every canine Purkinje cell studied (n = 36), we saw T and L Ca2+ channel currents that are similar to their counterparts in other tissues. Neither current was affected by tetrodotoxin (30 microM), but both were reduced by Mn2+ (5mM). Ni2+ (50 microM) blocked T more than L current. Nisoldipine (1 microM) apparently abolished the L current but also decreased the T current by 50%. Substitution of Ba2+ for Ca2+ augmented and prolonged L current but did not affect T current significantly. At 36 degrees C and with 5 mM [Ca2+]o, T current inactivated over a voltage range from -70 to -30 mV whereas L current inactivated between -30 and +20 mV. T current was detectable in only some of the ventricular cells studied (8 out of 12). In these cells the ratio of maximal T current to maximal L current (0.2 +/- 0.1, n = 8) was lower than the T/L ratio in Purkinje cells (0.6 +/- 0.2, n = 6). The density of peak L current in ventricular cells (7.5 +/- 1.7 pA/pF, n = 8) was higher than that in Purkinje cells (4.4 +/- 3.4 pA/pF, n = 6). Therefore, in ventricular cells the L current is the main Ca2+ current whereas in Purkinje cells, the T current also contributes significantly to membrane electrical activity. In Purkinje cells, beta-adrenoceptor stimulation by isoproterenol (1 microM) increased L current but did not affect T current. On the other hand, in 70% (7 out of 10) of the Purkinje cells, alpha-adrenoceptor stimulation by 10 microM norepinephrine (in the presence of 2 microM propranolol) increased the T current. Our observations show that the distribution of the two types of Ca2+ channels in canine ventricle is heterogeneous and that the two types of Ca2+ channels are modulated by catecholamines by different receptors.     

Adrenergic modulation of the transient outward current in isolated canine Purkinje cells

Single canine Purkinje cells were voltage clamped under Ca2+-free conditions using the patch pipette. Depolarizing pulses from a holding potential of -42 mV induced a time-dependent rapidly activating-slowly inactivating outward current, which was identified as the transient outward current. The current showed two exponential time constants of inactivation (48,352 msec at +58 mV and 53,325 msec at +78 mV). Norepinephrine in concentrations exceeding 10(-9) M modified the inactivation kinetics of this current without affecting the activation kinetics. The half-maximum dose for norepinephrine effect was 1.9 X 10(-8) M, and the effect was saturated at 10(-6) M. Norepinephrine reduced the amplitude of the fast time constant component of inactivation, while increasing the amplitude of the slow component, without changing their time constants. Norepinephrine also increased the amplitude of a time-independent current component. The beta-antagonist sotalol blocked the norepinephrine effect on the transient outward current. On the other hand, both activation of adenyl cyclase by forskolin and increase of intracellular cAMP concentration produced the same effect as exposure to norepinephrine. These results suggest a role for neurotransmitter regulation of the transient outward current in cardiac cells, perhaps by channel phosphorylation.