Heterogeneity of action potential waveforms and potassium currents in rat ventricle.

OBJECTIVE: The ionic mechanisms for differences in action potential waveforms in rat left ventricle were studied by recording L-type Ca2+ current, transient outward K+ current, and inwardly rectifying background K+ current in single myocytes. METHODS: Single cells were obtained from adult rat hearts by enzymatic dispersion of tissue segments from the epicardium at the apex and the endocardium at the base of the left ventricle. Whole cell voltage clamp methods together with cell shortening measurements were used to identify the K+ currents involved in early and late repolarisation and to correlate changes in action potential shape with inotropic responses. 4-Aminopyridine was used to block the transient outward K+ current, I(t), to evaluate the contribution of this current to repolarisation. RESULTS: Action potential recordings demonstrated that cells from endocardial tissue at the base of the left ventricle have a considerably longer action potential than those from epicardial tissue at the apex. 4-Aminopyridine had a much more pronounced action potential lengthening and inotropic effects on cells from epicardium than on myocytes from endocardium suggesting that I(t) is larger in the epicardium. Voltage clamp measurements confirmed this. In contrast, the L-type Ca2+ current, the resting membrane potential, and the inwardly rectifying background K+ current were very similar in these two regions of left ventricle. CONCLUSIONS: One significant factor contributing to the heterogeneity of action potential waveforms in rat left ventricle is a differential distribution of a Ca+ independent transient outward K+ current, I(t). Regional differences in action potential duration have important implications for the gradient of repolarisation in rat left ventricle, for the genesis of the T wave of the electrocardiogram, and for both electrical and mechanical restitution (refractoriness).

Increase in action potential duration and inhibition of the delayed rectifier outward current IK by berberine in cat ventricular myocytes.

1. In the present work, the effects of the antiarrhythmic drug, berberine, on action potential and ionic currents of cat ventricular myocytes were studied. 2. Berberine prolonged action potential duration in cat ventricular myocytes without altering other variables of the action potential. 3. The drug at concentrations of 0.3-30 microM blocked only the delayed rectifier (IK) current with an IC50 = 4.1 microM. Berberine produced a tonic block and a phasic block that was increased with the duration of the depolarizing pulse. The blocking effect on IK was use-dependent, but not frequency-dependent. 4. In cardiac preparations two delayed rectifier currents have been found: a rapid (IKr) current and a slow (IKs) current. In the present work it has been found that berberine at the concentrations used, selectively blocked IKr. 5. At concentrations higher than 10 microM it also decreased the transient outward (Ito1) current. The drug did not have effects on the inward rectifier (IK1) or the high threshold calcium current (Ica-L). 6. These results show that berberine is a specific potassium channel blocker. The increase in action potential duration induced by berberine can be explained mainly by its blocking effects on IK.

Effects of imipramine on the transient outward current in rabbit atrial single cells.

1. The effects of imipramine on action potential characteristics and transient outward potassium current (It) of rabbit isolated atrial myocytes were studied using the whole-cell configuration of the patch-clamp technique. 2. Imipramine, 3 microM, decreased action potential amplitude and lengthened the action potential duration measured at 50% of repolarization, whereas it did not modify the final phase of repolarization or the resting membrane potential. These results are similar to those reported in multicellular rabbit atrial preparations. 3. Imipramine, 0.1-100 microM, induced a concentration-dependent inhibition of the peak amplitude of It, a shortening of the time to peak current and an increase in the inactivation rate. The acceleration of the current inactivation is to a major extent responsible for the decrease in the integral of the outward current measured at 50 ms after the start of the pulse. 4. The drug-induced block of It was not associated with changes in the voltage-dependence of the steady-state inactivation curve or in the process of recovery from inactivation of the current. Extrapolation to zero block shows that imipramine did not block It before its activation at the onset of the depolarization. These results suggested that imipramine does not affect the inactivated or the resting state of It channels. 5. It is concluded that in rabbit isolated atrial cells, imipramine inhibits It and that this effect is responsible for the lengthening of the action potential duration produced by this drug.

Quinidine-induced open channel block of K+ current in rat ventricle.

1. The effects of quinidine on calcium-independent outward K+ currents in rat ventricular myocytes were studied using whole-cell patch clamp techniques. 2. Quinidine sulphate (6 microM) significantly prolonged repolarization of the ventricular action potential. This effect was larger during early repolarization (25% level) than at later times (90% level). 3. Quinidine reduced the amplitude of a transient outward current, and accelerated its rate of decay by approximately 4 fold at membrane potentials between 0 to +50 mV. Quinidine also reduced the amplitude of a slowly inactivating, tetraethylammonium-sensitive 'pedestal' component of the outward current. 4. The quinidine-induced block of the transient outward current was dependent on time and membrane potential. Maximal block occurred with depolarizations of about 100 ms duration, and longer depolarizations (up to 1.5 s) produced little additional block. The membrane potential dependence of quinidine-induced block was very similar to the membrane potential dependence of activation of the transient outward current. The membrane potential dependence of steady-state inactivation of the transient outward current was not significantly affected by quinidine. 5. These results show that quinidine blocks outward K+ currents in rat ventricular cells. The time and potential dependence of this block suggests that quinidine blocks the transient outward K+ current by acting primarily on the open state of these channels.

Effects of bupivacaine on membrane currents of guinea-pig ventricular myocytes.

The effects of bupivacaine on the membrane currents of single guinea-pig ventricular myocytes were investigated using the whole-cell patch-clamp technique. Bupivacaine decreased the inward calcium current in a concentration-dependent way at concentrations of 10 microM and higher. Bupivacaine also decreased the delayed outward current without modifying the inward-rectifying potassium current. From these results it can be concluded that bupivacaine, at concentrations lower than 10 microM, does not exert its negative inotropic effect by decreasing the calcium current. This mechanism may play a role at higher concentrations of the drug.

Caffeine inhibits depolarization-activated outward currents in rat ventricular myocytes.

The effects of caffeine (10 mM) on depolarization-activated, calcium-independent outward K+ currents were investigated in isolated rat ventricular myocytes, using whole-cell clamping. The external solution contained CoCl2 2 mM and the internal solution contained ethylene glycol-bis(-aminoethyl ether) N,N,N',N'-tetraacetic acid 10 mM. Caffeine decreased the peak amplitude of the total current and the sustained plateau current. Caffeine did not modify the steady state inactivation curve, which was fitted by two Boltzmann functions. Caffeine blocked the tetraethylammonium-sensitive slowly activating and inactivating outward current by 32% and the 4-aminopyridine-sensitive rapidly activating and inactivating transient outward current by 19%. Caffeine did not modify the inactivation rate or the time course of the recovery from inactivation of the transient current. Ryanodine 10 microM did not modify any of the current components and the effect of caffeine was not modified by ryanodine pretreatment. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine 100 microM, did not modify the depolarization-activated calcium-independent outward currents.

Differential effects of histamine on left and righ guinea-pig atria.

In electrically stimulated strip from right guinea-pig atria histamine produced a positive inotropic effect and it increased the amplitude and duration of calcium-dependent slow action potentials, which suggests an increase in the slow inward current. These effects were apparently mediated by H2-receptors. On strips from left atria histamine produced an H1-receptor mediated positive inotropic effect, without alteration of slow action potentials. The mechanism of this positive inotropic effect remains unknown.

Effects of metoprolol on action potential and membrane currents in guinea-pig ventricular myocytes.

The effects of the beta-adrenoceptor antagonist metoprolol on action potentials and membrane currents were studied in single guinea-pig ventricular myocytes. The experiments were carried out using the nystatin-method of whole-cell technique. This method was used in order to prevent the run-down of the calcium current. Metoprolol at concentrations of 10-100 mumol/l shortened action potential in a dose-dependent way. The drug only decreased resting membrane potential at a concentration of 100 mumol/l in two out of five cells. Under voltage-clamp conditions, metoprolol blocked the high threshold calcium current at concentrations of 30 and 100 mumol/l to 82 +/- 4% and 73 +/- 5% from control, respectively. The drug decreased the inward rectifying potassium current in a concentration-dependent manner. This effect was evident for inward current at voltages negative to the apparent reversal potential and for outward current at voltages between -30 and -80 mV. This blocking effect on the inward rectifying potassium current can explain the effect on resting membrane potential. At voltages positive to -30 mV metoprolol increased a time-independent outward current. This metoprolol-enhanced outward current was blocked by barium and cesium. This result suggests that the metoprolol-enhanced current is carried by potassium. The current component enhanced by metoprolol was not sensitive to glibenclamide and tetraethylammonium applied externally, which suggests that the adenosine triphosphate-sensitive channel is not the target of metoprolol. The activation of this time-independent outward current by metoprolol and the blocking effects on the calcium current seem to explain the shortening in action potential induced by the drug.

Multiple effects of putative alpha-adrenoceptor agonists on the electrical and mechanical activity of guinea-pig papillary muscle.

The effects of the putative alpha-adrenoceptor agonists phenylephrine, methoxamine and clonidine on force of contraction and on calcium-dependent action potentials were studied in guinea-pig papillary muscles. Phenylephrine increased the force of contraction by stimulating alpha-adrenoceptors as well as beta-adrenoceptors. It increased the amplitude and duration of slow action potentials, but this effect was exclusively due to stimulation of beta-adrenoceptors. The positive inotropic effect mediated by alpha-adrenoceptors can presumably not be explained by an increase in calcium influx during the action potential via the slow inward current. Methoxamine had no effect on the force of contraction at 10(-5) and 10(-4) mol/l, but at 10(-4) mol/l it slightly decreased amplitude and duration of slow action potentials. Clonidine produced a large increase in force of contraction and in amplitude and duration of slow action potentials. These effects were due to stimulation of H2-histamine receptors. It is concluded that in guinea-pig papillary muscle the tested putative alpha-adrenoceptor agonists do not share a common alpha-adrenoceptor effect, but produce prominent effects which are mediated through either beta-adrenoceptors (phenylephrine), or H2-histamine-receptors (clonidine) or are non-specific (methoxamine) in nature.

Electrophysiological interactions between quinidine-lidocaine and quinidine-phenytoin in guinea-pig papillary muscle.

The interactions between quinidine and lidocaine or phenytoin at the sodium channel level have been studied in the present work. The maximum upstroke velocity (Vmax) of the guinea-pig papillary muscle action potential has been used as a measure of the sodium current. Lidocaine interfered with the use-dependent blocking effects on Vmax of quinidine, by decreasing the fraction of sodium channels blocked by quinidine during the conditioning action potential, in an apparently competitive way. These results strongly suggest that quinidine and lidocaine bind to a common receptor site. Alternatively, it has been suggested that lidocaine and quinidine bind to different but related receptor sites, since lidocaine may induce allosteric changes in quinidine's receptor. Phenytoin increased the use-dependent blocking effects on Vmax of quinidine by slowing the time course of the slow component of reactivation of Vmax induced by quinidine. Phenytoin did not change the fraction of sodium channels blocked by quinidine during the conditioning action potential. These results suggest that phenytoin binds to a different receptor site than quinidine.

Characterization of the effects of histamine on the transmembrane electrical activity of guinea-pig and rabbit SA- and AV-node cells.

The effects of histamine on the transmembrane electrical activity of cells of small preparations (0.5 X 0.5 mm) of guinea-pig and rabbit sinoatrial- and atrioventricular-nodes were studied. Histamine at concentrations above 10(-7) mol/l increased the firing rate, the rate of diastolic depolarization, the maximum diastolic potential, the amplitude and the maximum rate of depolarization of the action potential of pacemaker cells of rabbit and guinea-pig sinoatrial cells and rabbit atrioventricular cells. These effects were antagonized by the H2-receptor blocker cimetidine (2.5 X 10(-6)mol/l) but they were not modified by the H1-receptor blocker chlorphenamine (2.5 and 5 X 10(-6)mol/l). Small preparations of guinea-pig atrioventricular node did not exhibit spontaneous activity, but it was induced by histamine and blocked by cimetidine. Histamine increased the maximum upstroke velocity of propagated action potential of cells of the central part of complete atrioventricular node in both species studied. These effects were blocked by cimetidine, but not by chlorphenamine. It is concluded that the increase in automaticity induced by histamine in guinea-pig and rabbit sinoatrial and atrioventricular nodes was due to stimulation of H2-receptors. Histamine did not depress electrical activity of atrioventricular node cells, but rather increased it. This effect was due to H2-receptor stimulation.

Effects of the novel antiarrhythmic compound TR 2985 (ropitoin) on action potentials of different mammalian cardiac tissues.

Ropitoin (TR 2985) is a novel antiarrhythmic drug. In the present work we have found that the main effect of the compound on the normal action potential of different cardiac tissues, guinea-pig atrial and ventricular muscle (1-3 mumol/l), and dog Purkinje fibres (0.5-1.0 mumol/l) was a depression of the maximum upstroke velocity. This effect was dependent on the frequency of stimulation, being stronger at higher frequencies. In the presence of the drug (3 mumol/l), we observed a shift of 9 mV of the resting membrane potential-maximum upstroke velocity relationship to more negative potentials. Under control conditions recovery from inactivation of maximum upstroke velocity was complete in less than 200 ms. Ropitoin induced a very slow component of recovery of the maximum upstroke velocity, explaining the frequency-dependent effects of the drug. The slow recovery of the maximum upstroke velocity induced by ropitoin was dependent on the membrane potential being faster at a more hyperpolarized membrane potential. The time course of the recovery was also dependent on pH; acidosis slowed it considerably. Ropitoin increased action potential duration of atrial muscle at 20% and 90% of repolarization. In contrast, the compound shortened action potential duration of ventricular muscle at 20% and 90% of repolarization, and dog Purkinje fibres at 50% and 90% of repolarization. In addition, ropitoin (1-3 mumol/l) depressed guinea-pig ventricular slow action potentials. This effect was the stronger the higher the stimulation frequency.

Chloroquine blocks the background potassium current in guinea pig atrial myocytes.

In this study we examined the effects of chloroquine on the muscarinic potassium current, I(K-ACh), and the inward rectifying potassium current, I(K1). We utilized three ways to induce I(K-ACh): activating the M2-muscarinic receptors with carbachol, activating the purinergic A1-receptors with adenosine and directly activating the G(K)-protein coupled with these receptors in an irreversible way with GTPgammaS. In experiments using the whole-cell configuration of the patch-clamp technique, we found that chloroquine, independently from the manner of activation of I(K-ACh), was able to block this current with similar potency. These results strongly suggest that chloroquine may be acting directly on the muscarinic potassium channel. Chloroquine also blocked I(K1) with similar potency, in both guinea pig atrial and ventricular myocytes.

Further characterization of the effects of imipramine on plateau membrane currents in guinea-pig ventricular myocytes.

The effects of imipramine, a tricyclic antidepressant, on action potential characteristics and plateau membrane currents were studied in isolated guinea-pig ventricular myocytes, using the whole cell configuration of the patch-clamp technique. Imipramine (1, 5 and 15 mumols/l) decreased in a concentration-dependent manner the amplitude and shortened the duration of the action potential, but it had no effect on resting membrane potential. At all three concentrations tested, imipramine decreased the delayed outward potassium current, this effect being apparently voltage-independent since it did not modify the activation curve. Imipramine, 5 and 15 mumols/l, also produced an inhibition of the peak high threshold calcium current, but did not change the shape of the current-voltage relationship or the apparent reversal potential of this current. Therefore, imipramine probably decreased the maximum available calcium conductance. However, the inward rectifying potassium current was not affected by any concentration of imipramine tested. Imipramine, 1 and 5 mumols/l, shortened the duration of the action potentials elicited in the presence of the inorganic calcium channel blocker cobalt chloride, and at 5, but not at 1 mumol/l, also shortened the action potentials obtained in the presence of the sodium channel blocker tetrodotoxin. Washout of imipramine completely reversed all its effects within 15 minutes. All these results suggest that imipramine at a concentration of 1 mumol/l produced a shortening in action potential duration by inhibiting the late sodium current flowing during the plateau phase of the action potential. At concentrations of 5 and 15 mumols/l the effect of imipramine on action potential duration can also be explained by a blocking effect on the high threshold calcium current.

Differences in regional distribution of K+ current densities in rat ventricle.

The objective of the present work is to study the ionic mechanisms for the regional differences in action potential duration in rat ventricle. This regional diversity has been related to differences in the regional distribution of some potassium currents in several species. Single cells were obtained by enzymatic dispersion of tissue segments from rat ventricular muscle. Whole cell voltage-clamp methods were used to identify the K+ currents involved in action potential repolarisation in the different regions. 4-Aminopiridine, TEA and voltage protocols were used to isolate the following potassium currents: transient outward, Ito, delayed rectifier, Ik, and sustained current, Iss. In the present work, we have studied the distribution of these three repolarising currents, and that of the inward rectifier, Ikl, in the free wall of the right ventricle, the subepicardium of the apex of the left ventricle and in the subendocardium of the base of the left ventricle. Action potential duration was longer in the left than in the right ventricle, and in the former it was longer in the subendocardium of the base than in the subepicardium of the apex. The main difference was in the phase 1, suggesting the implication of Ito. This was confirmed with voltage-clamp experiments. In conclusion, this work shows that Ito current density is higher in the regions with the shorter action potential, whereas there are no differences in the regional distribution of Ik, Iss or Ikl.

Inhibition of cardiac inward rectifier currents by cationic amphiphilic drugs.

Cardiac inward rectifier channels belong to three different classes of the KIR channel protein family. The KIR2.x proteins generate the classical inward rectifier current, IK1, while KIR3 and KIR6 members are responsible for the acetylcholine responsive and ATP sensitive inward rectifier currents IKAch and IKATP, respectively. Aberrant function of these channels has been correlated with severe cardiac arrhythmias, indicating their significant contribution to normal cardiac electrophysiology. A common feature of inward rectifier channels is their dependence on the lipid phosphatidyl-4,5-bisphospate (PIP2) interaction for functional activity. Cationic amphiphilic drugs (CADs) are one of the largest classes of pharmaceutical compounds. Several widely used CADs have been associated with inward rectifier current disturbances, and recent evidence points to interference of the channel-PIP2 interaction as the underlying mechanism of action. Here, we will review how six of these well known drugs, used for treatment in various different conditions, interfere in cardiac inward rectifier functioning. In contrast, KIR channel inhibition by the anionic anesthetic thiopental is achieved by a different mechanism of channel-PIP2 interference. We will discuss the latest basic science insights of functional inward rectifier current characteristics, recently derived KIR channel structures and specific PIP2-receptor interactions at the molecular level and provide insight in how these drugs interfere in the structure-function relationships.

Improvement of the metabolic status recovers cardiac potassium channel synthesis in experimental diabetes.

AIMS: The fast transient outward current, I(to,fast) , is the most extensively studied cardiac K(+) current in diabetic animals. Two hypotheses have been proposed to explain how type-1 diabetes reduces this current in cardiac muscle. The first one is a deficiency in channel expression due to a defect in the trophic effect of insulin. The second one proposes flawed glucose metabolism as the cause of the reduced I(to,fast) . Moreover, little information exists about the effects and possible mechanisms of diabetes on the other repolarizing currents of the human heart: I(to,slow) , I(Kr) , I(Ks) , I(Kur) , I(Kslow) and I(K1) . METHODS: We recorded cardiac action potentials and K(+) currents in ventricular cells isolated from control and streptozotocin- or alloxan-induced diabetic mice and rabbits. Channel protein expression was determined by immunofluorescence. RESULTS: Diabetes reduces the amplitude of I(to,fast) , I(to,slow) and I(Kslow) , in ventricular myocytes from mouse and rabbit, with no effect on I(ss) , I(Kr) or I(K1) . The absence of changes in the biophysical properties of the currents and the immunofluorescence experiments confirmed the reduction in channel protein synthesis. Six-hour incubation of myocytes with insulin or pyruvate recovered current amplitudes and fluorescent staining. The activation of AMP-K reduced the same K(+) currents in healthy myocytes and prevented the pyruvate-induced current recovery. CONCLUSION: Diabetes reduces K(+) current densities in ventricular myocytes due to a defect in channel protein synthesis. Activation of AMP-K secondary to deterioration in the metabolic status of the cells is responsible for K(+) channel reductions.

Mechanisms responsible for the altered cardiac repolarization dispersion in experimental hypothyroidism.

AIMS: To identify the causes for the inhomogeneity of ventricular repolarization and increased QT dispersion in hypothyroid mice. METHODS: We studied the effects of 5-propyl-2-thiouracil-induced hypothyroidism on the ECG, action potential (AP) and current density of the repolarizing potassium currents I(to,fast), I(to,slow), I(K,slow) and I(ss) in enzymatically isolated myocytes from three different regions of mouse heart: right ventricle (RV), epicardium of the left ventricle (Epi-LV) and interventricular septum. K(+) currents were recorded with the patch-clamp technique. Membranes from isolated ventricular myocytes were extracted by centrifugation. Kv4.2, Kv4.3, KChIP and Na/Ca exchanger proteins were visualized by Western blot. RESULTS: The frequency or conduction velocity was not changed by hypothyroidism, but QTc was prolonged. Neither resting membrane potential nor AP amplitude was modified. The action potential duration (APD)(90) increased in the RV and Epi-LV, but not in the septum. Hypothyroid status has no effect either on I(to,slow), I(k,slow) or I(ss) in any of the regions analysed. However, I(to,fast) was significantly reduced in the Epi-LV and in the RV, whereas it was not altered in cells from the septum. Western blot analysis reveals a reduction in Kv4.2 and Kv4.3 protein levels in both the Epi-LV and the RV and an increase in Na/Ca exchanger. CONCLUSION: From these results we suggest that the regional differences in APD lengthening, and thus in repolarization inhomogeneity, induced by experimental hypothyroidism are at least partially explained by the uneven decrease in I(to,fast) and the differences in the relative contribution of the depolarization-activated outward currents to the repolarization process.

Mechanism of transient outward K(+) channel block by disopyramide.

The block of the transient outward K(+) current (I(to)) by disopyramide was studied in isolated rat right ventricular myocytes using whole cell patch-clamp techniques. Disopyramide at a concentration of 10 to 1000 microM reduced peak I(to) and accelerated the apparent rate of current inactivation. The onset of block was assessed using a double pulse protocol with steps from -70 to +50 mV. As the duration of the first (conditioning) pulse was increased from 1 to 50 ms, block was increased. Further prolongation of the conditioning pulse resulted in relief of block, which was nearly complete with a 1-s conditioning pulse. In the absence of drug, the recovery from inactivation of I(to) at -70 mV was fast and best fit with a single exponential function having a time constant of 33 +/- 13 ms. In contrast, in the presence of 100 microM disopyramide, recovery from apparent inactivation was biexponential with time constants of 35 +/- 13 ms and 7.16 +/- 1.5 s. The time course of the slow component was used to estimate recovery of channels from block by disopyramide. Recovery from block was voltage-dependent, suggesting that disopyramide was trapped by the open channel. Taken together, these results suggest that disopyramide rapidly blocks channels in the open state and that unblock occurs from the inactivated state.

Electrophysiologic interactions between mexiletine-quinidine and mexiletine-ropitoin in guinea pig papillary muscle.

The effects of quinidine alone, ropitoin alone, and in combination with mexiletine at the sodium channel level were studied in isolated guinea pig papillary muscles using a sucrose gap technique. The maximum upstroke velocity (Vmax) was used as an indirect index of the magnitude of the sodium current. With quinidine (24 microM) and ropitoin (1 microM) added, trains of stimuli led to an exponential decline of Vmax to a new steady-state level (use-dependent block). The combination of mexiletine and quinidine decreased use-dependent Vmax block and magnitude of the slow component of reactivation induced by quinidine without modifying its time-constant of recovery. When we studied the interaction between mexiletine and ropitoin, we obtained similar results. Therefore, we conclude that mexiletine, quinidine, and ropitoin may bind to the same receptor site in the sodium channel. In addition, these antagonistic effects appeared only at frequencies greater than 0.5 Hz, whereas at very slow frequencies of stimulation we observed a synergistic effect in both interactions studied.