Determinants of action potential duration in neonatal rat ventricle cells.

STUDY OBJECTIVE: The aim was to study the currents that determine the action potential duration in ventricular cells from neonatal rats. DESIGN: Microelectrode measurements of action potentials from ventricle strips were compared with action potentials obtained from isolated myocytes with the whole cell patch clamp method in current clamp mode. Ionic currents were studied in myocytes in voltage clamp mode using recognised modulators of channel activity. EXPERIMENTAL MATERIAL: Neonatal rats (2 d old) were decapitated and myocytes were prepared from the apical third of collagenase treated hearts. MEASUREMENTS AND MAIN RESULTS: Modification of the action potential by 1.8-5.0 mM Ca, 2.0 mM Co, 8 mM 4-aminopyridine, 1.8 mM Sr, and 20 mM tetraethylammonium suggested the presence of the slowly inactivating Ca current ICa,L, an early outward current Ieo, and at least one other K current. Action potentials from myocytes and ventricular strips were comparable. Voltage clamp experiments were confirmatory and revealed currents with the following properties: (1) ICa,L: a Ca current with a current density of 21.7 microA.cm-2, activated between -30 and -20 mV, saturated at 1.8 Cao, inactivated faster at 5 than at 1.8 mM Cao, more permeable to Ba and Sr than to Ca, and with Sr as charge carrier blocked by Ca; (2) Ieo: the peak current had a linear I/V relation between 0 and 70 mV and was abolished by 4 mM 4-aminopyridine; (3) IK1: the current was an inward rectifier that showed a relaxation at potentials negative to -90 mV. CONCLUSIONS: Action potentials obtained from neonatal rat ventricle with microelectrodes are comparable with those measured in myocytes in current clamp mode. The action potential duration is mainly determined by ICa,L, Ieo, and IK1, and there is no evidence for the presence of a delayed rectifier.

Taurine depresses INa and depolarises the membrane but does not affect membrane surface charges in perfused rabbit hearts.

STUDY OBJECTIVE: The aim was to determine whether taurine influences the membrane surface charges in cardiac muscle. DESIGN: Screening of the negative charges at the outside surface of the membrane results in a shift of the steady state inactivation of the sodium system towards less negative potentials. This feature was used to study eventual effects of taurine on surface charges and the data were compared to the known influence of varying extracellular calcium. EXPERIMENTAL MATERIAL: New Zealand rabbits (6-7 weeks, 1.25-1.75 kg) were anesthetised and the hearts were rapidly excised and perfused with the Langendorff technique. MEASUREMENTS AND MAIN RESULTS: Standard microelectrodes were used to determine the effects of 20 mM taurine and varying Ca concentrations (from 0.3 to 5.0 mM) on action potential parameters. The resting potential was varied by changing extracellular K between 2.5 and 10 mM. Taurine significantly depolarised the membrane by about 3 mV between 5 and 10 mM Ko but not at 2.5 mM; the maximum rate of depolarisation (dV/dTmax) decreased significantly at all Ko except at 10 mM where taurine caused arrhythmias or cardiac arrest. The dV/dTmax upsilon resting potential relationship (a measure for the steady state sodium current inactivation) was not changed by taurine, but the current was depressed as a function of membrane potential, the depression being more pronounced at more positive membrane potentials. An increase in Cao from 0.3 to 5.0 mM displaced the half maximal value of the dV/dtmax upsilon resting potential relationship from -79 to -67 mV, showing that the screening effect of Ca on the negative charges at the outside surface of the membrane could be detected with this experimental approach. CONCLUSIONS: The decrease of the fast Na current by taurine can explain the arrhythmias observed at 10 mM external potassium, whereas the surface charges of the glycocalix were not affected.

Effects of K+ channel blockers on the action potential of hypoxic rabbit myocardium.

1. In order to assess the role of different ionic currents in hypoxia-induced action potential shortening, we investigated the effects of blockers of voltage-dependent and ATP-sensitive K(+)-channel on the membrane potential of hypoxic rabbit hearts and papillary muscles. The response to blocking of the inward rectifier was studied at three external K+ concentration: 2.5, 5, and 7.5 mM. 2. Hypoxia produced a progressive decline in action potential duration (APD) that levelled off after 15 to 20 min. Steady state APD values at 25% and 95% repolarization (APD25 and APD95) were 26.0 +/- 1.9% and 42.2 +/- 2.4% of controls respectively. 3. Tetraethylammonium (TEA, 10 mM) delayed but did not reduce APD shortening at the steady state. 4. Blocking of IK1 with a mixture of 0.2 mM Ba2+ and 4 mM Cs+ lengthened APD in normoxia and prevented APD95 shortening in hypoxia. The APD25 shortening was significantly attenuated at all [K]o. 5. Glibenclamide (Glib, 30 microM) did not prevent APD shortening, but produced a progressive action potential (AP) lengthening after 15 min of hypoxia. Steady levels of 48 +/- 3.5% and 62 +/- 5.0% of controls for APD25 and APD95 respectively were reached after 45 min. 6. The relation between APD25 and pacing rate was determined in normoxic and hypoxic papillary muscles and the effects of 2 mM 4-aminopyridine (4-AP) were examined. Hypoxia attenuated the APD25 shortening currently observed when the stimulation rate was lowered from 1 to 0.1 Hz without altering the plateau reduction occurring at frequencies above 2 Hz. These effects were potentiated by 4-AP.7. Our data suggest that the accelerated AP repolarization in hypoxic rabbit myocardium represents a delicate balance of several outward currents: IKI, IK-ATP. and at least one yet unidentified current component rather insensitive to changes in [K]o and to K+ channel blockers.

Explanted cardiac cells: a model to study drug actions?

Results from drug action studies are reviewed together with techniques to produce preparations of explanted cardiac cells and parameters that can be measured in these preparations. Results from drug action studies on explanted cells are poorly integrated into our present knowledge of drug effects derived from investigations of more conventional models. Recent reports indicate that most explanted cardiac cells change their electrophysiological properties after explantation and this may be responsible for the difficulties in interpreting results obtained from this preparation. To exploit the potential of explanted cardiac cells as a model to study drug actions it is recommended that (a) the preparation be characterized with electrophysiological methods and (b) the preparation of explanted cardiac cells be selected so that it facilitates the solution of a well-defined pharmacological problem.

Interaction of taurine with the fast Na-current in isolated rabbit myocytes.

We studied in whole cell configuration with the patch clamp method the effect of taurine on the macroscopic Na current in adult ventricular rabbit myocytes. Because these cells have a large surface [13,750 +/- 704 microns2 (19), mean +/- S.E.M. (n)], we reduced [Na]o to 45 mM and worked at room temperature to obtain acceptable voltage control. When the cells were held at -80 mV, taurine (20 mM) had the following effects: 1) The current voltage relationships crossed over so that taurine increased INa at potentials negative to -45 mV, and at more positive potentials it depressed the current; 2) taurine reduced the maximal Na conductance from 536.3 +/- 72.2 to 253.6 +/- 33.6 microS.cm-2; 3) the crossing over of the I/V curves was mainly caused by a hyperpolarizing shift of V1/2 of the steady-state activation by 6.3 mV; 4) the crossing over was independent of a -4.6 mV shift of V1/2 of the steady-state inactivation and 5) taurine increased significantly the time constant of reactivation between -90 and -70 mV, but we did not find evidence that taurine changed the time constant of inactivation between -40 and +20 mV. We conclude that positive to -45 mV taurine causes a block of INa channels that resembles that of local anesthetic antiarrhythmic drugs. Negative to -45 mV taurine counteracts the local anesthetic effect causing increased excitability and improved conduction in the range of the threshold potential and -45 mV.

Diffusion and electrogenic components of the resting potential in explanted neonatal rat ventricle cells.

Spontaneously beating explanted neonatal rat ventricle cells stop beating and show a steady potential (the mean resting potential, -46.2 mV at 6.0 mM Ko) when exposed to 10 mM Cao or 4 mM Mn. When Ko was increased, resting potential changed only slightly between 3 and 15 mM, but the resting potential versus Ko characteristically approached the slope of a K electrode at high Ko. Elimination of Cl from the medium did not alter the K dependence of the resting potential. However, a hyperpolarization of 9 mV per 10-fold change was observed when Nao was decreased from 50 to 4 mM. Ouabain (10(-4) M) depolarized the membrane within 2 min to a stable level of about -30 mV in spontaneously beating cells and in those treated with Ca channel blockers. This potential was considered as the diffusion component of the membrane potential, Vdiff. Consequently the difference between resting potential and Vdiff represents the ouabain-sensitive or the electrogenic component of the resting potential. Using linearized versions of the Mullins and Noda as well as the Goldman - Hodgkin - Katz equations, we calculated that a PNa/PK between 0.25 and 0.35, a Na/K exchange ratio of 2.0, and a Ki of 160 mM adequately described the K dependence of the resting potential. We demonstrated the contribution of electrogenic Na extrusion to the resting potential of mammalian cardiac cells in culture. Therefore the existence of a composite resting potential precludes the direct comparison of potential measurements obtained under conditions liable to independently modify either the diffusion or the electrogenic component.

Comparative effects of metabolic inhibition on the action potential from three experimental models of cardiac cells.

Because of conflicting reports on the basic mechanisms responsible for the action potential changes produced by hypoxia or metabolic inhibitors, we investigated the effect of 2,4-dinitrophenol, substrate deprivation, and 2-deoxy-D-glucose on three preparations of rat myocardium: adult and newborn ventricle and cultured cells derived from neonatal rats. The latter exhibit slow action potentials in contrast to the other two, which show fast action potentials. Cultured cells were insensitive to 2,4-dinitrophenol and substrate deprivation but were markedly inhibited by blocking of glycolysis. The action potential of adult cells was shortened in the three conditions tested but was most sensitive to blocking of oxidative phosphorylation, which abolished propagated electrical activity after 15 min of exposure. The response of newborn ventricle was intermediate between the other two. Our data indicated that as far as maintenance of the membrane electrical properties is concerned, the relative importance of different metabolic pathways varies with development after birth or with time in culture.

Voltage control during inward current flow in rat ventricular muscle using a double sucrose gap technique.

In rat ventricular muscle, measurements of the membrane potential with microelectrodes during depolarizing voltage steps showed that deviation of the membrane potential from the command signal were never larger than 15 mV during flow of the fast inward current and that voltage control was regained within 15 ms after the beginning of the voltage step. During the flow of the slow inward current, tail currents elicited by interrupting the time course of the slow current at different time intervals returned exponentially to the steady-state level, thus indicating acceptable voltage control. It is concluded that rat ventricular muscle is a rather favorable preparation for voltage-clapm experiments and this is attributed mainly to the geometry of the preparation.

Early action potential shortening in hypoxic hearts: role of chloride current(s) mediated by catecholamine release.

We tested the hypothesis that the early action potential shortening induced by hypoxia in perfused hearts is attributable to chloride currents activated or modulated by endogenous catecholamine release. Rabbit hearts perfused at 33 degrees C and paced at 2.5-2.8 Hz were used for membrane potential recordings with microelectrodes. Catecholamine depletion was induced with reserpine treatment. The effects of nadolol (10 microM), the stilbenedisulfonic acid derivatives DIDS (10 microM) and SITS (1 mM), and diphenylamine-2 carboxylate (DPC, 100 microM) on action potential characteristics were determined at different times during hypoxia. The effect of chloride transport blockers on the outward currents induced by 200 nM carbonyl cyanide (CCCP) or by 1 microM isoproterenol in isolated cells was also tested. In control hearts, action potential duration (APD) at 25 and 95% repolarization decreased by 50 +/- 9% and 32 +/- 7% respectively after 5 min of hypoxia. This effect was fully antagonized by reserpine pretreatment, by respiratory acidosis, and by nadolol when present from the beginning of hypoxia. None of these agents affected action potential characteristics in normoxia and nadolol had no effect when added after 15 min of hypoxia. Lowering the chloride concentration to 17.5 mM reproduced the effects of nadolol and reserpine. DIDS and SITS lengthened APD in normoxia and attenuated the early APD shortening in hypoxia. DPC had no effect in normoxia but fully counteracted APD shortening produced by isoproterenol or early hypoxia. In isolated cells, DIDS did not affect the glibenclamide sensitive outward current induced by CCCP and DPC blocked the isoproterenol induced current. The data suggest that in whole hearts, chloride currents mediated by endogenous catecholamine release are involved in the early action potential shortening induced by hypoxia with preservation of glycolysis.

Ionic determinants of spontaneous activity in clusters of cultured cardiac cells from newborn rats.

The spontaneous activity of cell clusters derived from ventricle cells of newborn rats was studied using a recording television microscope. The influence of varying concentrations of sodium, potassium, calcium, tetrodotoxin (TTX), and that of 2 mM MnCl2 was tested. The spontaneous activity of the cell clusters persisted in TTX but it was abolished by Mn. The beating rate increased when [Ca]0 and [Na]0 were changed from 0.3 mM to 3.0 mM and from 30 mM to 75 mM; it decreased with a change of [Na]0 from 75 mM to 142 mM. It is concluded that electrogenesis in their behavior to very young embryonic rat heart cells or cells of the rabbit sinoauricular node.

Electrophysiologic effects and electrolyte changes in total myocardial ischemia.

The ventricular membrane potential was measured in the perfused rabbit heart under control conditions and during total cardiac ischemia produced by an arrest of the coronary perfusion. The steady-state inactivation characteristic of the sodium system and the sodium dependence of the upstroke velocity were determined by measurements of the maximum rate of rise (Vmax) as a function of the resting potential (RP) and the [Na]0 to [Na]i ratio. The intracellular concentrations of sodium and potassium ([Na]i, [K]i) were estimated from measurements of the cellular electrolyte content, the total water content, and the volume of the extracellular space. Ischemia produced a net sodium gain of 58 mmol/kg dry weight and a slight loss of potassium. As a consequence, [Na]i increased and [K]i decreased. Under ischemia the resting potential was closer to the potassium equilibrium potential than in the controls. The changes in action potential configuration and plateau level suggested that ischemia inhibited the slow inward current. The Vmax decreased within 10 min of ischemia. Neither membrane depolarization nor a reduction in the sodium gradient could entirely explain the low Vmax of ischemic cells. It is concluded that the electrophysiologic effects of ischemia result from changes in ionic gradients and membrane electrical properties.

Intracellular chloride activity in rabbit papillary muscle: effect of ouabain.

Intracellular chloride activity (aiCl) and membrane potential (Vm) were measured in rabbit papillary muscle under in vitro conditions. The cellular chloride concentration (Cli) was estimated from measurements of total water content, extracellular space, and total chloride concentration. The effects of therapeutic (10(-8) M) and toxic (10(-6) M) concentrations of ouabain on these parameters were tested. The chloride-sensitive microelectrodes were of the liquid-ion exchanger type. Selectivity for HCO-3 was taken into account in the calculation of aiCl. In 11 control experiments made with two different protocols aiCl was determined in subendocardial and in deeper cells. The mean membrane potentials were -78.7 and -78.0 mV and the mean cytoplasmic chloride activities were 17.5 and 17.7 mM, respectively. The chloride equilibrium potentials were -43.5 and -43.2 mV. These results indicated that chloride is not passively distributed in rabbit papillary muscle. Ouabain (10(-8) M) did not change Vm or aiCl. At a toxic concentration of ouabain, Vm fell to -68.0 mV in superficial cells and to -67.8 mV in deeper cells, but aiCl remained unchanged . These results suggested that under in vitro conditions intracellular chloride is distributed within more than one cellular compartment.

Disopyramide phosphate effects on slow and depressed fast responses.

We studied the effects of disopyramide phosphate on explanted neonatal rat ventricle cells exhibiting depressed fast responses or naturally occurring slow response action potentials together with automatic activity. Disopyramide suppressed the spontaneous activity at a concentration of 2.5 micrograms/mL with a half-maximal value of 10 micrograms/mL. Before spontaneous activity was lost, there was an increase in beating rate possibly related to membrane depolarization. In depressed fast and slow response action potentials there was an increase in action potential duration (APD) which was consistently found both at the level of the plateau and at 90% repolarization. Comparison of the APD increase observed after disopyramide treatment and that after exposure to 20 mM tetraethylammonium suggested a block of a potassium conductance as a possible cause underlying the change in APD. The Vmax values of the depressed fast response decreased at constant membrane potential and this was attributed to the local anesthetic effect of the drug. In addition, we report two novel findings: (i) a decrease of Vmax of the slow response action potentials which may be secondary to membrane depolarization, and (ii) an increase in the duration of slow action potentials, possibly caused by inhibition of a potassium conductance.

Influence of explantation procedure on the electrical and morphological properties of cultured neonatal rat ventricle cells.

The ultrastructure and electrophysiological properties of ventricle cells from newborn rats were studied before and after explantation. The cultured cells were dissociated either with trypsin or with collagenase, the latter enzyme being used with and without stirring with a magnetic bar. The explanted cells were studied 10 hr and 48 hr or more after explantation. At 10 hr after explantation, the cells exhibited fast-rising action potentials, but their myofibrils were disorganized, except for stirred collagenase-dispersed cells, which were also depolarized and inexcitable. At 2 days and later after explantation, all preparations had well-defined sarcomeres and myofibrils oriented in parallel similar to the ventricle before explantation, but the cells showed slow-response action potentials together with spontaneous activity. These findings suggest that the disorganization of myofibrils does not reflect damage to the surface membrane. Moreover, collagenase seems more damaging to the cells than trypsin under similar conditions (comparable periods of mechanical stirring), especially 10 hr after explantation.

Effects of K-channel blockers, calcium, and verapamil suggest different pacemaker mechanisms in cultured neonatal rat and embryonic chick ventricle cells.

We compared the determinants of spontaneous activity in explanted neonatal (2-day-old) rat ventricle cells and in reaggregates derived from 15-day-old chick embryos. We studied the beating rate with an optical recording method and the underlying electrical activity with glass microelectrodes using the K current blockers cesium (Cs) and tetraethylammonium, varied Ca concentrations, and the Ca antagonist verapamil. In the rat (i) Cs increased the beating rate that was mediated by an increase in the slope of the diastolic potential. (ii) Ca increased the beating rate dramatically at low and medium concentrations to decrease it again at 8 mM Cao. This increase in the beating rate was mediated by an increase of the slope of the diastolic depolarization. (iii) The beating rate decreased with verapamil at concentrations between 0.5 and 2.0 microM. The effects of Cs and Ca suggest that an increase in net inward current (block of IK1) underlies the positive chronotropic effect of Cs and that the pacemaker mechanism is determined by a Ca inward current or an IT1 type current modulated by variations of Cai. In the chick reaggregates (i) Cs and tetraethylammonium decreased the beating rate that was mainly brought about by a decrease in the slope of diastolic depolarization. (ii) Ca increased the beating rate but to a lesser degree than in the rat and there was no decrease of the beating rate at higher concentrations. (iii) The increase in the beating rate was not mediated by an increase in the slope of the diastolic potential but mainly by a depolarization of the maximum diastolic potential. (iv) Verapamil inhibited electrogenesis before any change in the diastolic potential was evident. The negative chronotropic effect of Cs and tetraethylammonium is compatible with the notion that a voltage- and time-dependent K current was inhibited and that this current determines the pacemaker. Moreover, the Ca component of the pacemaker mechanism in explanted rat ventricle cells resembles either that of the sinoatrial node or represents triggered activity.

Electrogenesis of freshly explanted spontaneously active clusters of neonatal rat ventricle cells.

Spontaneously active explanted rat ventricle cells show 4 to 7 h after explantation fast rising TTX sensitive action potentials but 24 h after explantation, electrogenesis is determined by slow rising. Mn sensitive action potentials. Because 4 to 7 h after explantation the electrical properties of the cells resemble more those of the tissue they were derived from than of cells 24 h after explantation, we conclude that the occurrence of slow rising action potentials is not directly related to the method of tissue disintegration but develops gradually after explantation.

Background K+ currents and response to metabolic inhibition during early development in rat cardiocytes.

The effects of metabolic inhibition on K+ background currents and action potential duration were investigated in neonatal rat ventricle cells during early development. Action potentials and ionic currents were measured with the patch clamp technique in current and voltage clamp mode in cells isolated with collagenase from 1 day and 7 day old rats. During the first postnatal week, the cell surface increased from 1700 to 2210 microm2 and the membrane hyperpolarized from -66.1 to -72.0 mV. Concomitantly the action potential shortened and the plateau became more negative. Inhibition of oxidative phosphorylation (50 microM 2,4 DNP) or of glycolysis in 1 day old rats (5 mM 2-deoxyglucose, 2-DG) also shortened the action potential by about 50% after 5 min exposure. The background current measured in the absence of INa, ICa,L, and Ito included: (1) an inward rectifying component whose I/V curves crossed over when measured in 6, 15, or 30 mM [K]o and showed an increase in slope conductance when [K]o was raised. Inward rectification was abolished by 2.4 mM Ba2+ in 1 day old cells and by 0.2 mM one week after birth; (2) a glibenclamide (100 microM) sensitive component that developed with time after membrane rupture (5-10 min) showing a higher current density in 7 than in 1 day old animals (1.4 vs 0.2 microA x cm-2 at -50 mV); and (3) a small and almost linear leak component of comparable amplitude in both age groups. Inhibition of oxidative phosphorylation with 2.5 microM carbonylcyanide m-chlorophenylhydrazone induced the development of background currents with different properties in both age groups: An inwardly rectifying Ba2+ sensitive current in 1 day old cells and a glibenclamide sensitive outwardly rectifying current in the 7 day old group. In contrast, exposure to 5 mM 2-DG provoked in all cells the development of an outwardly rectifying current that was blocked by glibenclamide. We conclude that the electrophysiologic response to metabolic inhibition is determined by the relative importance of the metabolic pathways present which in turn depends on the developmental state of the cells.

Magnesium: effects on reperfusion arrhythmias and membrane potential in isolated rat hearts.

The effects of Mg2+ concentration (Mg2+o, 0, 1.2, 2.4, and 4.8 mM) on the incidence of reperfusion arrhythmias and on the cellular electrical activity were studied in spontaneously beating rat hearts. The surface electrogram and the membrane potential were recorded in control conditions, during 10 min of regional ischemia (ligature of the left anterior descending coronary artery), and on reflow. Changes in Mg2+o did not alter action potential morphology but the depolarization induced by ischemia decreased with increasing Mg2+o. In hearts perfused with Mg2+ free solution or 1.2 mM subthreshold delayed afterdepolarizations (DADs) were often detected during ischemia. Moreover, DADs could be identified as initial events in the production of extrabeats or tachycardia appearing on reperfusion under these conditions. Chaotic electrical activity during fibrillation precluded the observation of DADs. The overall incidence (100%) and severity of ventricular tachyarrhythmias (80% tachycardia and fibrillation) was similar in both groups. At high Mg2+o, subthreshold DADs were occasionally observed during ischemia and often on reperfusion where they did not lead to the development of overt arrhythmias. Consequently, the incidence, severity, and duration of arrhythmic episodes on reflow was markedly reduced. Raising Mg2+ only on reperfusion did not prevent the development of arrhythmias, whose morphology in the intracellular recordings was similar to that found in hearts perfused without Mg2+ or with 1.2 mM. The recovery of sinus rhythm after 10 min of reperfusion was linearly related to Mg2+o. Our data strengthen the view that reperfusion arrhythmias belong to the Ca2+ mediated non reentrant type and suggest that Mg2+ counteracts these arrhythmias by depressing cytosolic Ca2+ oscillations. Besides, it appears that raising Mg2+o reduces ischemic K+o accumulation. The resulting changes in resting potential could contribute to lower DADs amplitude and thus decrease the arrhythmogenic potential of the Ca2+i oscillations induced by reperfusion.

Influence of streptomycin on spontaneous activity of clusters of cultured cardiac cells from neonatal rats.

In clusters of trypsinized ventricle cells from neonatal rats which exhibit slow response action potentials, streptomycin in concentrations from 0.17-5.5 mM significantly inhibits the beating rate. Microelectrode experiments performed at a concentration of 5.5 mM revealed a reduction in the slope of diastolic depolarization from 149 to 53 mV/s whereas the maximum diastolic potential depolarized from -42.4 to -33.6 mV which entailed a decrease in overshoot and maximum rate of rise of the action potential. We conclude that the decrease of the slope of diastolic depolarization mainly determines the slowing of the beating rate and that streptomycin interferes with the pacemaker mechanism usually associated with the slow response.