DIFFERENCES IN IONIC CURRENTS BETWEEN CANINE MYOCARDIAL AND PURKINJE CELLS.

An electrophysiological analysis of canine single ventricular myocardial (VM) and Purkinje (P) cells was carried out by means of whole cell voltage clamp method. The following results in VM versus P cells were obtained. INa3 was present, had a threshold negative to the fast activating-inactivating INa1, its slow inactivation was cut off by INa1, and contributed to Na+ influx at INa1 threshold. INa1 was smaller and had a less negative threshold. There was no comparable slowly inactivating INa2, accounting for the shorter action potential. Slope conductance at resting potential was about double and decreased to a minimum value at the larger and less negative IK1 peak. The negative slope region of I-V relation was smaller during fast ramps and larger during slow ramps than in P cells, occurred in the voltage range of IK1 block by Mg2+, was not affected by a lower Vh and TTX and was eliminated by Ba2+, in contrast to P cells. ICa was larger, peaked at positive potentials and was eliminated by Ni2+. Ito was much smaller, began at more positive values, was abolished by less negative Vh and by 4-aminopyridine, included a sustained current that 4-aminopyridine decreased but did not eliminate. Steeper ramps increased IK1 peak as well as the fall in outward current during repolarization, consistent with a time-dependent block and unblock of IK1 by polyamines. During repolarization, the positive slope region was consistently present and was similar in amplitude to IK1 peak, whereas it was small or altogether missing in P cells. The total outward current at positive potentials comprised a larger IK1 component whereas it included a larger Ito and sustained current in P cells. These and other results provide a better understanding of the mechanisms underlying the action potential of VM and P cells under normal and some abnormal (arrhythmias) conditions.

Characterization of the slowly inactivating sodium current INa2 in canine cardiac single Purkinje cells.

The aim of our experiments was to investigate by means of a whole cell patch-clamp technique the characteristics of the slowly inactivating sodium current (I(Na2)) found in the plateau range in canine cardiac Purkinje single cells. The I(Na2) was separated from the fast-activating and -inactivating I(Na) (labelled here I(Na1)) by applying a two-step protocol. The first step, from a holding potential (V(h)) of -90 or -80 mV to -50 mV, led to the quick activation and inactivation of I(Na1). The second step consisted of depolarizations of increasing amplitude from -50 mV to less negative values, which led to the quick activation and slow inactivation of I(Na2). The I(Na2) was fitted with a double exponential function with time constants of tens and hundreds milliseconds, respectively. After the activation and inactivation of I(Na1) at -50 mV, the slope conductance was very small and did not change with time. Instead, during I(Na2), the slope conductance was larger and decreased as a function of time. Progressively longer conditioning steps at -50 mV resulted in a progressive decrease in amplitude of I(Na2) during the subsequent test steps. Gradually longer hyperpolarizing steps (increments of 100 ms up to 600 ms) from V(h) -30 mV to -100 mV were followed on return to -30 mV by a progressively larger I(Na2), as were gradually more negative 500 ms steps from V(h) -30 mV to -90 mV. At the end of a ramp to -20 mV, a sudden repolarization to approximately -35 mV fully deactivated I(Na2). The I(Na2) was markedly reduced by lignocaine (lidocaine) and by low extracellular [Na(+)], but it was little affected by low and high extracellular [Ca(2+)]. At negative potentials, the results indicate that there was little overlap between I(Na2) and the transient outward current, I(to), as well as the calcium current, I(Ca). In the absence of I(to) and I(Ca) (blocked by means of 4-aminopyridine and nickel, respectively), I(Na2) reversed at 60 mV. In conclusion, I(Na2) is a sodium current that can be initiated after the inactivation of I(Na1) and has characteristics that are quite distinct from those of I(Na1). The results have a bearing on the mechanisms underlying the long plateau of Purkinje cell action potential and its modifications in different physiological and pathological conditions.

On the mechanisms of cholinergic control of the sinoatrial node discharge.

It has been proposed that cholinergic agonists inhibit the sinoatrial node discharge by shifting the activation range of the hyperpolarization-activated inward current If to more negative values or by increasing potassium conductance. In the former instance, cesium will potentiate the cholinergic inhibition by blocking any residual If; in the latter instance, Cs+ and Ba2+ will antagonize the inhibitory action by blocking K+ channels. The changes in discharge induced by high and low concentrations of carbachol were studied using an electrophysiologic technique in isolated guinea pig sinoatrial node perfused in vitro in the absence and presence of different concentrations of Cs+ and Ba2+. In Tyrode solution, high carbachol concentrations (0.5-2 microM) slowed the sinoatrial node by hyperpolarizing the membrane and by reducing the amplitude of diastolic depolarization; and stopped the sinoatrial node by preventing the attainment of threshold potential. Adding Cs+ (10 mM) to carbachol increased the rate in slowly discharging sinoatrial node and induced spontaneous discharge in quiescent sinoatrial node. In high [K+]o (approximately 12 mM), carbachol slowed or stopped the slow responses and adding Cs+ accelerated or induced discharge. Both in Tyode and in high [K+]o, in the presence of Cs+, carbachol did stop the sinoatrial node. In the presence of carbachol, Ba2+ (0.1 mM) accelerated or induced discharge, as Cs+ did. Atropine (1 microM) prevented both the slowing or suppression by carbachol and the acceleration of sinoatrial node by Cs+ in the presence of carbachol. Low carbachol concentrations (0.05-0.1 microM) decreased the rate to a similar extent in the absence and the presence of a low concentration of Cs+ (2 mM, which blocks If but not K+ channels), but markedly less in the presence of 0.5-0.75 mM Ba2+ (which block K+ channels but not If). We conclude that cholinergic agonists slow or stop the sinoatrial node by a shifting the membrane potential toward the more negative subsidiary pacemaker range and away from the threshold. The results with Cs+ and Ba2+ indicate that both high and low concentrations of carbachol decrease sinoatrial node discharge by activating the I(K,ACh) channels rather than by decreasing If.

Role of I(K) and I(f) in the pacemaker mechanisms of sino-atrial node myocytes.

The role of I(K) (delayed rectifier current) and I(f) (hyperpolarization-activated current) in dominant and subsidiary pacemaker ranges was studied in single myocytes isolated from the guinea pig sino-atrial node by means of a perforated patch-clamp technique. In the dominant pacemaker range (approx. -55 to -40 mV), I(K) tails are present whereas I(f) is not activated. In the subsidiary pacemaker range (approx. -80 to -70 mV), I(f) is large whereas I(K) is minimal and reversing. The threshold for I(f) activation is more negative at short time intervals. Larger or longer depolarizations to -40 mV and +20 mV deactivate I(f) more and are followed by faster reactivation of I(f). Steps of 200-300 ms duration to +20 mV completely deactivate I(f). The slope conductance decreases during depolarizations at -40 and +20 mV and quickly re-increases after the steps. The I(f) deactivation range is between -70 and +10 mV, with a V(1/2) of -35 mV. Depolarizations from -80 to +20 mV at a rate of 120/min limit the subsequent I(f) reactivation owing to the short diastole. We conclude that I(K) plays a predominant role in the dominant pacemaker range and I(f) does so in the subsidiary pacemaker range. Either pacemaker mechanism is used by sino-atrial node cells depending on the diastolic potential range. A previous depolarization markedly increases the amplitude and rate of I(f) reactivation.

Oscillatory zones and their role in normal and abnormal sheep Purkinje fiber automaticity.

The mechanisms by which low [K(+)](o) induces spontaneous activity was studied in sheep Purkinje fibers. Purkinje strands were superfused in vitro and membrane potentials were recorded by means of a microelectrode technique. The results show that low [K(+)](o) increases the slope and amplitude of early diastolic depolarization, sharpens the transition between early and late diastolic depolarizations, induces an after-potential and large pre-potentials through a negative shift of an oscillatory zone. Pre-potentials occur progressively sooner during diastole and merge with the after-potential to induce uninterrupted spontaneous discharge. During recovery, when the rate slows, after- and pre-potentials separate once more, the slower discharge decreasing the after-potentials but not the pre-potentials. Low [K(+)](o) has little effect on the plateau, but markedly slows phase 3 repolarization and may altogether prevent it. At depolarized levels, voltage oscillations, slow responses, sinusoidal fluctuations or quiescence may be present depending on voltage. During the recovery, a train of either sub-threshold oscillations or spontaneous action potentials appear towards the end of phase 3 repolarization. The cessation of the action potentials unmasks large sub-threshold oscillations, that occur in the oscillatory zone. Drive, high [Ca(2+)](o) and norepinephrine increase slope and amplitude of early diastolic depolarization as low [K(+)](o) does. In low [K(+)](o), Cs(+) prevents spontaneous discharge at polarized levels, but not the decrease in resting potential nor the onset of slow responses at depolarized levels. Cs(+) blocks the train of oscillations and of action potentials occurring during recovery. We conclude that low [K(+)](o) steepens early diastolic depolarization and increases its amplitude through an after-potential that results from an increased Ca(2+) load; allows the attainment of the threshold through Cs(+)-sensitive voltage oscillations which develop when the oscillatory zone is entered either by diastolic depolarization or by phase 3 repolarization; and causes voltage oscillations also at depolarized levels, but through a Cs(+)-insensitive different mechanism.

Role of dual pacemaker mechanisms in sinoatrial node discharge.

We investigated whether in the sinoatrial node (SAN) there are two different pacemaker mechanisms and whether either one can maintain spontaneous discharge. These questions were studied by means of an electrophysiological technique and of blockers of different diastolic currents in rabbit and guinea pig isolated SAN. In SAN subsidiary pacemakers of both species, Cs(+) (5-10 mM) or high [K(+)](o) (10-12 mM) decreased the maximum diastolic potential, abolished diastolic depolarization (DD) at polarized levels (subsidiary DD), unmasked a U-shaped dominant DD at depolarized levels, but did not stop the SAN. In rabbit SAN, E4031 (1 microM) and d-sotalol (100 microM) did not stop discharge, but did so after block of subsidiary DD by high [K(+)](o) or Cs(+). In guinea pig SAN, in Tyrode solution E4031, d-sotalol or indapamide (100 microM) did not stop SAN discharge. In the presence of Cs(+) or high [K(+)](o) indapamide (but not E4031 or d-sotalol) stopped the SAN. Ba(2+) (1-5 mM) led to stoppage of discharge both in Tyrode solution and in high [K(+)](o) or Cs(+). Depolarization by blockers of DD unmasked sinusoidal fluctuations, which during recovery were responsible for resumption of discharge. We conclude that in rabbit and guinea pig SAN, two different pacemaker mechanisms (Cs(+)- and K(+)-sensitive subsidiary DD, and Cs(+)- and K(+)-insensitive dominant DD) can independently sustain discharge, but block of both mechanisms leads to quiescence. Abolition of dominant DD by blockers of I(K) is consistent with a decay of I(K) as the dominant pacemaking mechanism, I(Kr) being more important in rabbit and I(Ks) in guinea pig. Sinusoidal fluctuations appear to be an essential component of the pacemaking process.

Role of Na-Ca exchange in the action potential changes caused by drive in cardiac myocytes exposed to different Ca2+ loads.

The role of Na-Ca exchange in the membrane potential changes caused by repetitive activity ("drive") was studied in guinea pig single ventricular myocytes exposed to different [Ca2+]o. The following results were obtained. (i) In 5.4 mM [Ca2+]o, the action potentials (APs) gradually shortened during drive, and the outward current during a train of depolarizing voltage clamp steps gradually increased. (ii) The APs shortened more and were followed by a decaying voltage tail during drive in the presence of 5 mM caffeine; the outward current became larger and there was an inward tail current on repolarization during a train of depolarizing steps. (iii) These effects outlasted drive so that immediately after a train of APs, currents were already bigger and, after a train of steps, APs were already shorter. (iv) In 0.54 mM [Ca2+]o, the above effects were much smaller. (v) In high [Ca2+]o APs were shorter and outward currents larger than in low [Ca2+]o. (vi) In 10.8 mM [Ca2+]o, both outward and inward currents during long steps were exaggerated by prior drive, even with steps (+80 and +120 mV) at which there was no apparent inward current identifiable as I(Ca). (vii) In 0.54 mM [Ca2+]o, the time-dependent outward current was small and prior drive slightly increased it. (viii) During long steps, caffeine markedly increased outward and inward tail currents, and these effects were greatly decreased by low [Ca2+]o. (ix) After drive in the presence of caffeine, Ni2+ decreased the outward and inward tail currents. It is concluded that in the presence of high [Ca2+]o drive activates outward and inward Na-Ca exchange currents. During drive, the outward current participates in the plateau shortening and the inward tail current in the voltage tail after the action potential.

[Connection between electrophysiology and cardiac protection]. / Rapporti tra elettrofisiologia e cardioprotezione.

A diseased myocardium may require protection from further damage (anoxia, ischemia) due to its own mechanical activity and this process involves a complex number of adaptations. The aim of cardioprotection is to decrease either the work of the heart or improve its ability to deliver a normal cardiac output without further myocardial damage. From an electrophysiological point of view, cardioprotection may involve the protection either from an abnormal electrophysiology (e.g., arrhythmias) or from the damage of an abnormal mechanical activity of the myocardium through electrophysiological modifications. Since the action potential is the signal that regulates both duration and frequency of myocardial contraction, its modifications affect the mechanical performance of the heart. In addition, some of ion fluxes across the cell membrane require energy and therefore add to energy requirements of the myocardium. The mechanisms of cardioprotection involve removal of the disease process (e.g., tachycardia, coronary stenosis), reduction in damage (e.g., preconditioning and reperfusion, coronary vasodilators), decrease in demand (e.g., decrease in cardiac output, rate, sympathetic discharge, calcium load, blood pressure), reduced performance (e.g., decreased contraction through shortening of the action potential by the opening of KATP channels in anoxic cells, administration of KATP channel openers, calcium antagonists, cardioplegic solution in bypassed hearts, removal of calcium overload), and remodeling. However, cardioprotection is not a process limited to the heart, in the sense that the work of the heart cannot be reduced independently of the needs of the circulation. In addition, cardioprotection (physiological and pharmacological) includes advantages and disadvantages and may be more or less specific in addressing myocardial dysfunction. Nevertheless, cardioprotection may avoid serious complications (e.g., arrhythmias), slow down the disease process, and improve the heart function enough to allow more demanding treatments (e.g., surgical interventions).

On the mechanism of cesium-induced voltage and current tails in single ventricular myocytes.

The mechanisms by which different concentrations of cesium modify membrane potentials and currents were investigated in guinea pig single ventricular myocytes. In a dose-dependent manner, cesium reversibly decreases the resting potential and action potential amplitude and duration, and induces a diastolic decaying voltage tail (Vex), which increases at more negative and reverses at less negative potentials. In voltage-clamped myocytes, Cs+ increases the holding current, increases the outward current at plateau levels while decreasing it at potentials closer to resting potential, induces an inward tail current (Iex) on return to resting potential and causes a negative shift of the threshold for the inward current. During depolarizing ramps, Cs+ decreases the outward current negative to inward rectification range, whereas it increases the current past that range. During repolarizing ramps, Cs+ shifts the threshold for removal of inward rectification negative slope to less negative values. Cs+-induced voltage and current tails are increased by repetitive activity, caffeine (5 mM) and high [Ca2+]O (8.1 mM), and are reduced by low Ca2+ (0.45 mM), Cd2+ (0.2 mM) and Ni2+ (2 mM). Ni2+ also abolishes the tail current that follows steps more positive than ECa. We conclude that Cs+ (1) decreases the resting potential by decreasing the outward current at more negative potentials, (2) shortens the action potential by increasing the outward current at potentials positive to the negative slope of inward rectification, and (3) induces diastolic tails through a Ca2+-dependent mechanism, which apparently is an enhanced electrogenic Na-Ca exchange.

Cesium effects on i(f) and i(K) in rabbit sinoatrial node myocytes: implications for SA node automaticity.

Cesium blocks the hyperpolarization-activated current i(f) but blocks neither the delayed-rectifier current i(K) nor the sinoatrial (SA) node discharge. It has been proposed that the failure of Cs+ to block SA discharge is either an incomplete block or a negative shift of i(f). However, an alternative possibility is that i(K) (rather than i(f)) has a predominant role in the SA-pacemaker potential. To investigate this point, the effects of Cs+ on both i(f) and i(K) in the pacemaker range of potentials were studied in the same single SA node cell at the same time by means of the perforated patch-clamp technique. Hyperpolarizing steps from a holding potential (Vh) of -35 mV into and past the pacemaker-potential range resulted in a progressively larger i(f) associated with an increasing slope conductance. Cs+ (2 mM) reversibly blocked both i(f) and the slope conductance increase, suggesting that the current activated was indeed predominantly i(f). Subsequently, hyperpolarizing steps to -50, -60, and -70 mV were applied in the absence (to activate only i(f)) and in the presence of a prior depolarizing step to +10 mV (to activate i(K) as well, as the action potential normally does). Cs+ almost abolished i(f) but only slightly decreased i(K). It is concluded that the failure of Cs+ to block the SA- node spontaneous discharge is not due to a shift of i(f) out of the pacemaker range (due to run-down) or an incomplete block of i(f). Instead, the resistance of i(K) to block by Cs+ is consistent with a predominant role of i(K) for the discharge of the SA node, although i(f) can contribute under normal or special circumstances. The reduction of i(K) by Cs+ raises the question whether the Cs+ slows the SA-node discharge not only by suppressing I(f), but also by reducing i(K).

Overdrive Excitation in the Guinea Pig Sinoatrial Node Superfused in High [K(+)](o).

The aim of the present experiments was to study the characteristics and mechanisms of the rhythm induced by overdrive ('overdrive excitation', ODE) in the sinoatrial node (SAN) superfused in high [K(+)](o) (8-14 mM). It was found that: (1) overdrive may induce excitation in quiescent SAN and during a slow drive; (2) in spontaneously active SAN, overdrive may accelerate the spontaneous discharge; (3) immediately after the end of overdrive, a pause generally precedes the onset of the induced rhythm; (4) during the pause, an oscillatory potential (V(os)) may be superimposed on the early diastolic depolarization (DD); (5) during the subsequent late DD, a different kind of oscillatory potential appears near the threshold for the upstroke (ThV(os)) which is responsible for the initiation of spontaneous activity; (6) once started, the induced rhythm is fastest soon after overdrive; (7) faster drives induce longer and faster spontaneous rhythms; (8) the induced action potentials are slow responses followed by DD with a superimposed V(os), but ThV(os) is responsible for ODE; (9) the induced rhythm subsides when ThV(os) miss the threshold and gradually decay; (10) low [Ca(2+)](o) abolishes ODE; (11) in quiescent SAN, high [Ca(2+)](o) induces spontaneous discharge through ThV(os) and increases its rate by enhancing V(os) and shifting the threshold to more negative values, and (12) tetrodotoxin abolishes ODE as welll as the spontaneous discharge induced by high [Ca(2+)](o). In conclusion, in K(+)-depolarized SAN, ODE may be present in the apparent absence of calcium overload, is Ca(2+)- and Na(+)-dependent and is mediated by ThV(os) and not by V(os). Copyright 1997 S. Karger AG, Basel

Mechanisms of suppression and initiation of pacemaker activity in guinea-pig sino-atrial node superfused in high [K+]o.

The electrophysiological mechanisms by which changes in [K+]o suppress and initiate pacemaker activity were studied in guinea-pig isolated sino-atrial node (SAN) superfused in vitro. High [K+]o (10 mM or higher) gradually decreases maximum diastolic potential and action potential amplitude, until only subthreshold responses and eventually quiescence follow. When the threshold potential is missed, an oscillatory afterpotential (Vos) is often superimposed on early diastolic depolarization (DD). During the subsequent late DD, gradually increasing oscillatory prepotentials (ThVos) appear, whose depolarizing phase may initiate an action potential. If ThVos miss the threshold, they gradually decrease in size. In quiescent SAN, on decreasing high [K+]o, the resumption of spontaneous activity is caused by ThVos. In high [K+]o, Cs+ and Ba2+ may induce spontaneous activity in quiescent SAN and accelerate spontaneously active SAN. A low [Ni2+]o does not suppress SAN, whereas nifedipine blocks excitation (but not DD); and high [Ca2+]o induces spontaneous discharge in quiescent SAN. Tetrodotoxin and low [Na+]o often cause block of conduction. In conclusion, high [K+]o suppresses SAN discharge not by abolishing DD, but by preventing the attainment of the threshold. A slower rhythm may be maintained by ThVos arising during the late DD. After arrest, resumption of activity is due to gradually increasing ThVos. The effects of current blockers suggest that in high [K+]o the mechanism underlying DD may involve IK, but not I(f) or ICa. Initiation of discharge by high [Ca2+]o and induction of quiescence by nifedipine suggest a role of Ca2+ in excitation (but not in DD). The effects of tetrodotoxin and low [Na+]o suggest a role of Na+ in conduction within SAN superfused in high [K+]o.

Barium-induced diastolic depolarization and controlling mechanisms in guinea pig ventricular muscle.

Barium-induced diastolic depolarization (Ba(2+)-DD) and its modulation were studied in guinea pig papillary muscle and single ventricular myocytes. In papillary muscles, Cs+ (4 mM) abolished the DD induced by Ba2+ (0.05-0.2 mM) by abolishing the undershoot at the end of the action potential (AP; consistent with a block of an outward current). Acetylcholine (ACh 1 microM) had little effect on Ba(2+)-DD, whereas norepinephrine (NE 1 microM) enhanced it by increasing the undershoot and by inducing an oscillatory potential. Low [Ca2+]o (0.54 mM) decreased the resting potential and increased Ba(2+)-DD amplitude. High [Ca2+]o (8.1 mM) had opposite effects. Cs+ also reduced Ba(2+)-DD in low [Ca2+]o. In isolated myocytes, Ba(2+)-DD and the pacemaker current induced by Ba2+ (Ba(2+)-IKdd) increased on depolarization and reversed on hyperpolarization. Although not significantly, high [Ca2+]o slightly decreased and low [Ca2+]o slightly increased Ba(2+)-IKdd. Cd2+ markedly reduced the slow inward current ICa and the AP duration (APD), but did not affect Ba(2+)-IKdd. We conclude that Ba(2+)-DD (a) is entirely due to a voltage- and time-dependent decrease in gK1, since it is abolished by Cs+ (no contribution of a nonblocked decaying IK) by eliminating the undershoot (no If), (b) is potentiated by NE through an increased undershoot and an oscillatory potential, (c) is modified by high and low [Ca2+]o mostly through changes in the resting potential, and (d) is not affected by the block of the slow channel by Cd2+.

On the antiarrhythmic actions of magnesium in single guinea-pig ventricular myocytes.

1. The hypotheses that magnesium quickly abolishes arrhythmias by acting as a calcium antagonist or by increasing outward potassium currents were tested in guinea-pig isolated ventricular myocytes by recording membrane potentials and currents by means of a single microelectrode discontinuous voltage clamp method. 2. High [Mg2+]o (4-16 mmol/L) slightly increased the amplitude and duration of the action potential (AP) in some myocytes, but overall the changes were not significant. 3. High [Mg2+]o did not decrease the slow inward current (ICa) and had little effect on voltage- and time-dependent outward potassium currents whether or not ICa was allowed to flow. 4. Zero [Mg2+]o decreased the duration, but not amplitude, of the AP. Zero [Mg2+]o had little effect on ICa and on outward currents except for a small increase in outward current in the region of the negative slope of the inward rectifier current-voltage relationship. 5. In our myocytes, in contrast to [Mg2+]o, high [Ca2+]o significantly increased the amplitude and decreased the duration of the AP; at the same time, high [Ca2+]o increases ICa and the outward potassium current. 6. High [Mg2+]o decreased the amplitude of the oscillatory potentials (Vos)induced by various Ca(2+)-overloading procedures (high [Ca2+]o, noradrenaline, strophanthidin and barium). 7. It is concluded that the mechanisms by which high [Mg2+]o quickly suppresses cardiac arrhythmias are related to an extracellular action of Mg2+ and do not include a block of ICa or an increase in outward current. Mg2+ can be antiarrhythmic by decreasing Vos amplitude and possibly by screening the fixed negative charges at the external surface of the sarcolemma.

Ca(++)-dependence of caffeine modulation of the rate-force relation in canine cardiac Purkinje fibers.

The effects of caffeine on rate-force relation and their Ca++ dependence were studied in canine cardiac Purkinje fibers. At constant rate, caffeine increased and then decreased force. During drives at different rates (15, 60 and 120/min), 1 mM caffeine caused the largest positive inotropic effect at the slowest rate, but in 8.1 mM [Ca++]o caffeine no longer increased force even at 15/min. Interruptions of drive were followed by a positive and then a negative staircase: caffeine (1-4 mM) blunted these effects. A sudden decrease in rate caused a positive staircase: caffeine decreased and caffeine plus 8.1 mM [Ca++]o abolished it. A sudden increase in rate caused a negative staircase and on recovery a positive staircase: caffeine reduced and caffeine plus 8.1 mM [Ca++]o could reverse them. In low [Ca++]o (0.54 mM), caffeine caused only a positive inotropic effect and did not modify the rate-force relation patterns. High [Ca++]o ( > 8.1 mM) reversed the staircase patterns induced by a rate increase; adding caffeine shifted the reversal to lower [Ca++]o. Low [Na]o altered the rate-force relation similarly to high [Ca++]o, and caffeine exaggerated its effects. Decreasing [Ca++]i by means of tetrodotoxin or high [K+]o antagonized the caffeine effects on the rate-force relation. We conclude that caffeine markedly modulates the rate-force relation through a Ca(++)-dependent mechanism. This mechanism appears to involve a supraoptimal increase in [Ca++]i rather than a decrease in Ca++ released during the action potential.

Cesium, Na(+)-K(+) Pump and Pacemaker Potential in Cardiac Purkinje Fibers.

The mechanisms of the hyperpolarizing and depolarizing actions of cesium were studied in cardiac Purkinje fibers perfused in vitro by means of a microelectrode technique under conditions that modify either the Na(+)-K(+) pump activity or I(f). Cs(+) (2 mM) inconsistently increased and then decreased the maximum diastolic potential (MDP); and markedly decreased diastolic depolarization (DD). Increase and decrease in MDP persisted in fibers driven at fast rate (no diastolic interval and no activation of I(f)). In quiescent fibers, Cs(+) caused a transient hyperpolarization during which elicited action potentials were followed by a markedly decreased undershoot and a much reduced DD. In fibers depolarized at the plateau in zero [K(+)](o) (no I(f)), Cs(+) induced a persistent hyperpolarization. In 2 mM [K(+)](o), Cs(+) reduced the undershoot and suppressed spontaneous activity by hyperpolarizing and thus preventing the attainment of the threshold. In 7 mM [K(+)](o), DD and undershoot were smaller and Cs(+) reduced them. In 7 and 10 mM [K(+)](o), Cs(+) caused a small inconsistent hyperpolarization and a net depolarization in quiescent fibers; and decreased MDP in driven fibers. In the presence of strophanthidin, Cs(+) hyperpolarized less. Increasing [Cs(+)](o) to 4, 8 and 16 mM gradually hyperpolarized less, depolarized more and abolished the undershoot. We conclude that in Purkinje fibers Cs(+) hyperpolarizes the membrane by stimulating the activity of the electrogenic Na(+)-K(+) pump (and not by suppressing I(f)), and blocks the pacemaker potential by blocking the undershoot, consistent with a Cs(+) block of a potassium pacemaker current. Copyright 1995 S. Karger AG, Basel

The pacemaker current in cardiac Purkinje myocytes.

It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward current i(f) underlies the pacemaker potential. Because some findings are at odds with this interpretation, we used the whole cell patch clamp method to study the currents in the voltage range of diastolic depolarization in single canine Purkinje myocytes, a preparation where many confounding limitations can be avoided. In Tyrode solution ([K+]o = 5.4 mM), hyperpolarizing steps from Vh = -50 mV resulted in a time-dependent inwardly increasing current in the voltage range of diastolic depolarization. This time-dependent current (iKdd) appeared around -60 mV and reversed near EK. Small superimposed hyperpolarizing steps (5 mV) applied during the voltage clamp step showed that the slope conductance decreases during the development of this time-dependent current. Decreasing [K+]o from 5.4 to 2.7 mM shifted the reversal potential to a more negative value, near the corresponding EK. Increasing [K+]o to 10.8 mM almost abolished iKdd. Cs+ (2 mM) markedly reduced or blocked the time-dependent current at potentials positive and negative to EK. Ba2+ (4 mM) abolished the time-dependent current in its usual range of potentials and unmasked another time-dependent current (presumably i(f)) with a threshold of approximately -90 mV (> 20 mV negative to that of the time-dependent current in Tyrode solution). During more negative steps, i(f) increased in size and did not reverse. During i(f) the slope conductance measured with small (8-10 mV) superimposed clamp steps increased. High [K+]o (10.8 mM) markedly increased and Cs+ (2 mM) blocked i(f). We conclude that: (a) in the absence of Ba2+, a time-dependent current does reverse near EK and its reversal is unrelated to K+ depletion; (b) the slope conductance of that time-dependent current decreases in the absence of K+ depletion at potentials positive to EK where inactivation of iK1 is unlikely to occur. (c) Ba2+ blocks this time-dependent current and unmasks another time-dependent current (i(f)) with a more negative (> 20 mV) threshold and no reversal at more negative values; (d) Cs+ blocks both time-dependent currents recorded in the absence and presence of Ba2+. The data suggest that in the diastolic range of potentials in Purkinje myocytes there is a voltage- and time-dependent K+ current (iKdd) that can be separated from the hyperpolarization-activated inward current i(f).

Role of voltage oscillations in the automaticity of sheep cardiac Purkinje fibers.

The role of oscillatory potentials occurring near the threshold for the fast sodium current (ThVos) in the induction of spontaneous and repetitive activity was studied in sheep Purkinje fibers superfused in vitro. In low extracellular potassium concentration, the steepness and amplitude of diastolic depolarization increased and ThVos appeared during quiescence. ThVos amplitude increased progressively until its depolarizing phase reached the threshold potential for the initiation of the action potential. Drive increased the amplitude of diastolic depolarization and of ThVos, and longer drives induced faster and longer-lasting repetitive activity ("overdrive excitation"). In quiescent fibers, barium depolarized the resting membrane and initiated spontaneous discharge through ThVos. Acetylcholine had similar actions. Cesium hyperpolarized the membrane, thereby suppressing ThVos and related spontaneous activity. Tetrodotoxin and lidocaine also suppressed ThVos, but not the driven action potentials. In low extracellular potassium plus high extracellular calcium concentrations, drive induced ThVos as well as the oscillatory potentials related to calcium overload (Vos), but caused overdrive excitation through ThVos, even when caffeine was present. We conclude from our results that in Purkinje "dominant" pacemaker fibers (i) diastolic depolarization initiates spontaneous activity by attaining the threshold for the upstroke of the action potential through the depolarizing phase of a ThVos; (ii) the depolarizing phase of ThVos is caused by a tetrodotoxin-sensitive Na+ component; (iii) ThVos is voltage dependent in that a small depolarization of the resting membrane induces it and a small hyperpolarization suppresses it; (iv) ThVos can induce overdrive excitation; and (v) ThVos occurs in the absence of calcium overload and has distinguishing characteristics from the Vos induced by calcium overload.

Cesium effects on dual pacemaker mechanisms in guinea pig sinoatrial node.

The aim of the present experiments was to study the effects of cesium (Cs) on dual pacemaker potentials and the underlying mechanisms in isolated sinoatrial (SA) node of the guinea pig superfused in vitro. Cs (1-20 mM) initially hyperpolarized the maximum diastolic potential (MDP) and then depolarized it in a concentration-dependent manner. In subsidiary pacemaker cells of the SA node, increasing [Cs]o abolished diastolic depolarization (DD), but then (by depolarizing the cells to less negative potentials) Cs allowed the appearance of a shallow DD that maintained spontaneous discharge even in 20 mM Cs. In 10 mM [K]o, the subsidiary pacemaker cells depolarized and exhibited action potentials similar to those in dominant pacemaker cells. In high [K]o, the Na/K pump activity is stimulated: adding Cs initially increased the MDP minimally, but the subsequent decrease in MDP persisted. In preparations quiescent in high [K]o, Cs only depolarized the membrane and could induce spontaneous discharge: the action potentials were followed by an undershoot and DD. In high [K]o plus norepinephrine, even 20 mM Cs did not suppress and might increase the rate of discharge. During quiescence in acetylcholine or carbachol (I(f) is blocked), Cs still transiently hyperpolarized the resting potential. In zero [K]o with or without carbachol (I(f) is absent or blocked), Cs hyperpolarized the quiescent membrane by stimulating the Na/K pump. Cs-induced hyperpolarization was reduced by 50-100 microM ouabain. We conclude that, in the SA node, dual pacemaker mechanisms are present in subsidiary cells. The pacemaker potential at more negative voltages is blocked by Cs, but the dominant type pacemaker potential is not blocked even by 20 mM Cs (which is known to completely block I(f). Cs initially hyperpolarizes apparently by stimulating the Na/K pump (not by blocking I(f)) and subsequently depolarizes the subsidiary pacemaker cells by blocking an outward current but not IK1 (absent in the SA node). Thus, in the SA node I(f) may play little role in pacemaker activity.