Differences in the effect of metabolic inhibition on action potentials and calcium currents in endocardial and epicardial cells

BACKGROUND. Ischemia-induced electrophysiological changes are more prominent in epicardial cells than in endocardial cells. Epicardial action potentials shorten more than endocardial action potentials during ischemia. Since the L-type Ca2+ current plays an important role in the maintenance of action potential duration, we hypothesized that the Ca2+ current is affected more in epicardial cells than in endocardial cells during ischemia. METHODS AND RESULTS. To test this hypothesis, we examined the effect of metabolic inhibition, a major component of ischemia, on action potentials and the Ca2+ current in single cells isolated from the endocardial and epicardial layers of the feline left ventricle. The membrane voltage and current were measured by using the whole-cell mode of the patch-clamp technique. During control periods, action potentials recorded from epicardial myocytes had lower amplitude, a prominent notch between phases 1 and 2, and shorter action potential duration compared with those recorded from endocardial myocytes. However, the amplitude and current-voltage relation of the Ca2+ current were similar in endocardial and epicardial cells at test potentials of -30 to 60 mV elicited from a holding potential of -40 mV. The time course of inactivation of the Ca2+ current also was identical in the two cell types. After 15 minutes of superfusion with glucose-free Tyrode's solution containing 1 mM CN-, action potential duration was reduced by 13 +/- 7% in endocardial cells and by 80 +/- 9% in epicardial cells (p less than 0.01). The peak Ca2+ current was reduced by 21 +/- 9% in endocardial cells and by 37 +/- 6% in epicardial cells (p less than 0.01). CONCLUSIONS. We conclude that enhanced depression of the Ca2+ current may account in part for the greater action potential shortening in epicardial cells during ischemia and metabolic inhibition.     

Verapamil diminishes action potential changes during metabolic inhibition by blocking ATP-regulated potassium currents.

Verapamil has beneficial effects on ischemic myocardium, including reduction in electrophysiological derangements, prevention of intracellular K+ loss, and preservation of high-energy phosphates, but the mechanisms underlying these actions are not clear. Recent studies have demonstrated a role of ATP-regulated K+ (KATP) current in action potential shortening and K+ loss during ischemia and metabolic inhibition. Therefore, we studied the effects of verapamil on KATP current in feline ventricular myocytes to test the hypothesis that the drug prevents ischemic electrophysiological disturbances by affecting the KATP channel. Membrane potentials and currents were recorded using standard patch-clamp techniques. During 15-minute superfusion with 1 mM CN-, action potential duration measured at 90% repolarization was reduced from 259 +/- 12 to 98 +/- 15 msec (62% reduction) in the absence of verapamil and from 266 +/- 11 to 183 +/- 16 msec (31% reduction) in the presence of 2 microM verapamil (p less than 0.01). In inside-out membrane patches, the KATP current, activated in the absence of ATP, was significantly suppressed by intracellular application of 2 microM verapamil, but the single-channel conductance was not changed. Verapamil did not change the mean open and closed times of the channel within bursts (e.g., the mean open time was 1.92 +/- 0.18 and 1.82 +/- 0.21 msec in the absence and presence of 2 microM verapamil, respectively), but it shortened the mean lifetime of bursts from 41.1 +/- 3.5 to 24.9 +/- 2.8 msec (p less than 0.01) and prolonged the closed time between bursts from 39.4 +/- 4.6 to 78.5 +/- 5.1 msec (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological properties and responses to simulated ischemia in cat ventricular myocytes of endocardial and epicardial origin

In multicellular preparations, there are differences in action potential configuration between endocardium and epicardium, and electrophysiological alterations induced by ischemia are more drastic in epicardium than in endocardium. The present study was designed to examine electrophysiological properties of single cardiac myocytes enzymatically isolated from the endocardial and epicardial surfaces of the cat left ventricle and to determine whether the differential responses to ischemia of intact tissue occur in single cells. Action potentials recorded from the isolated single cells of epicardial surface had lower action potential amplitude and a prominent notch between phase 1 and phase 2, compared with those of the cells isolated from the endocardial surface; these findings are similar to those in intact endocardial and epicardial preparations. Resting membrane potentials recorded from both endocardial and epicardial single cells were sensitive to the change in extracellular K+ concentration and had properties of a K+ electrode. Action potential duration was frequency dependent in both cell types and was shorter in epicardial cells than in endocardial cells at a stimulation rate of 3 Hz. When the cells were superfused with Tyrode's solution that was altered to mimic an ischemic environment in vivo (PO2, 30-40 mm Hg; pH 6.8; [K+], 10 mM; and glucose free), resting membrane potential, action potential amplitude, and action potential duration were reduced, and the refractory period was shortened in both endocardial and epicardial single cells, but there were no differences in the degree of changes in action potentials and refractory periods induced between the two cell types. Action potential changes induced by L-alpha-lysophosphatidylcholine (5-40 mg/l) were also similar in endocardial and epicardial single cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulfonylureas, ATP-sensitive K+ channels, and cellular K+ loss during hypoxia, ischemia, and metabolic inhibition in mammalian ventricle

Sulfonylurea derivatives glibenclamide and tolbutamide are selective blockers of ATP-sensitive K+ (KATP) channels. However, their ability to prevent cellular K+ loss and shortening of action potential duration during ischemia or hypoxia in the intact heart is modest compared with their efficacy at blocking KATP channels in excised membrane patches. In the isolated arterially perfused rabbit interventricular septum, the increase in unidirectional K+ efflux and shortening of action potential duration during substrate-free hypoxia were effectively blocked by glibenclamide, but only by very high concentrations (100 microM); during hypoxia with glucose present, glibenclamide was only partially effective at reducing K+ loss. During total global ischemia (10 minutes), up to 100 microM glibenclamide or 1 mM tolbutamide attenuated shortening of action potential duration but only reduced [K+]0 accumulation by a maximum of 32 +/- 6%. In isolated patch-clamped guinea pig ventricular myocytes in which the whole-cell ATP-sensitive K+ current was activated by exposure to the metabolic inhibitors, glibenclamide (up to 100 microM) and tolbutamide (10 mM) were only partially effective at blocking the whole-cell ATP-sensitive K+ current (maximum block, 51 +/- 10% and 50 +/- 9%, respectively), especially when ADP was included in the patch electrode solution. In inside-out membrane patches excised from these myocytes, glibenclamide blocked unitary currents through KATP channels with a Kd of 0.5 microM and a Hill coefficient of 0.5 in the absence of ADP at the cytosolic membrane surface, but block was incomplete when 100 microM ADP (+2 mM free Mg2+) was present. ADP had a similar effect on block of KATP channels by tolbutamide. These findings suggest that free cytosolic [ADP], which rises rapidly to the 100 microM range during early myocardial ischemia and hypoxia, may account for the limited efficacy of sulfonylureas at blocking ischemic and hypoxic cellular K+ loss under these conditions.     

Activation of ATP-sensitive K+ channels by cromakalim. Effects on cellular K+ loss and cardiac function in ischemic and reperfused mammalian ventricle.

Pharmacological modulation of [K+]o accumulation and action potential changes during acute myocardial ischemia is under evaluation as a promising new antiarrhythmic and cardioprotective strategy during myocardial ischemia and reperfusion. We studied the effects of cromakalim, a K+ channel opener that activates ATP-sensitive K+ channels, in isolated arterially perfused rabbit interventricular septa subjected to ischemia and reperfusion and, through use of the patch clamp technique, in inside-out membrane patches excised from guinea pig ventricular myocytes. During aerobic perfusion, 5 microM cromakalim shortened action potential duration (APD) from 217 +/- 7 to 201 +/- 10 msec, had no effect on [K+]o, and reduced tension by 17 +/- 3% (n = 11). During ischemia, pretreatment with 5 microM cromakalim resulted in 1) more rapid APD shortening (71 +/- 9 versus 166 +/- 7 msec at 10 minutes and 63 +/- 12 versus 122 +/- 8 msec at 30 minutes), 2) similar [K+]o accumulation after 10 minutes (8.9 +/- 0.3 versus 9.6 +/- 0.5 mM) but a trend toward increased [K+]o accumulation after 30 minutes (11.0 +/- 1.7 versus 9.6 +/- 1.0 mM), and 3) similar times for tension to decline to 50% of control (2.14 +/- 0.16 versus 2.14 +/- 0.19 minutes) but shorter time to fall to 20% of control (4.34 +/- 0.33 versus 4.90 +/- 0.22 minutes; p = 0.003). After 60 minutes of reperfusion following 30 minutes of ischemia, recovery of function was similar, with a trend toward better recovery of developed tension (to 58 +/- 9% versus 39 +/- 10% of control; p = 0.18) and tissue ATP levels in cromakalim-treated hearts but no differences in APD or rest tension. Thus, 5 microM cromakalim had mild effects in normal heart but greatly accelerated APD shortening during ischemia without markedly increasing [K+]o accumulation, possibly because the more rapid APD shortening reduced the time-averaged driving force for K+ efflux through ATP-sensitive K+ channels. A significant cardioprotective effect during 30 minutes of ischemia plus 60 minutes of reperfusion could not be demonstrated in this model. In excised membrane patches studied at room temperature, the ability of cromakalim to activate ATP-sensitive K+ channels was significantly potentiated by 100 microM but not 15 microM cytosolic ADP, suggesting that in addition to the modest fall in cytosolic ATP during early ischemia, the rapid increases in cytosolic ADP may further sensitize cardiac ATP-sensitive K+ channels to activation by cromakalim.(ABSTRACT TRUNCATED AT 400 WORDS)     

Potassium rectifier currents differ in myocytes of endocardial and epicardial origin.

Whole-cell voltage-clamp experiments and single-channel current recordings in cell-attached patch mode were performed on enzymatically dissociated single ventricular myocytes harvested from feline endocardial and epicardial surfaces. The studies were designed to compare the characteristics of inward rectifier K+ current (IK1) and delayed rectifier K+ current (IK) between endocardial and epicardial cells and to test the hypothesis that the differential characteristics of IK1 and/or IK are responsible for the differences in action potential configuration between the two cell types. IK1 in endocardial cells displayed a distinct N-shaped current-voltage (I-V) relation, with a prominent outward current at potentials between -80 and -30 mV. In epicardial cells, an outward current region was much smaller, and the I-V relation demonstrated a blunted N-shaped I-V relation. In single-channel current recordings in cell-attached patch mode, neither unitary current amplitude of IK1 nor probability of channel opening was different between endocardial and epicardial cells, suggesting that the difference in the number of functional channels might be responsible for the differential IK1 I-V relations. The characteristics of IK also differed between endocardial and epicardial cells. The time course of growth of tail current of IK (IK,tail) (activation of IK) was significantly enhanced and that of IK,tail deactivation was delayed in epicardial cells compared with endocardial cells. The time constant of the slow component of IK activation at +20 mV was 3,950 +/- 787 msec in endocardial cells and 2,746 +/- 689 msec in epicardial cells (p less than 0.05); the corresponding values for IK deactivation at -50 mV were 1,041 +/- 387 msec and 1,959 +/- 551 msec, respectively (p less than 0.01). The voltage dependence of steady-state activation of IK,tail was similar between endocardial and epicardial cells, suggesting that the probability of channel opening at any potential was not different in the two cell types. The amplitude and density of fully activated IK (IK,full) were significantly greater in epicardial cells than in endocardial cells. At repolarization to -20 mV, IK,full amplitude was 452 +/- 113 pA in endocardial cells and 578 +/- 135 pA in epicardial cells (p less than 0.05), and the corresponding values for IK,full density were 2.86 +/- 0.73 and 4.21 +/- 0.83 microA/cm2, respectively (p less than 0.05). A nonstationary fluctuation analysis revealed that the amplitude of IK unitary current was similar between endocardial and epicardial cells (0.23 +/- 0.07 versus 0.22 +/- 0.03 pA, p = NS).(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and properties of ATP-sensitive potassium channels in myocytes from rabbit Purkinje fibres

OBJECTIVE: Our goal was to identify the ATP-sensitive potassium (KATP) channels in cardiac Purkinje cells and to document the functional properties that might distinguish them from KATP channels in other parts of the heart. METHODS: Single Purkinje cells and ventricular myocytes were isolated from rabbit heart. Standard patch-clamp techniques were used to record action potential waveforms. and whole-cell and single-channel currents. RESULTS: The KATP channel opener levcromakalim (10 microM) caused marked shortening of the Purkinje cell action potential. Under whole-cell voltage-clamp, levcromakalim induced an outward current, which was blocked by glibenclamide (5 microM), in both Purkinje cells and ventricular myocytes. Metabolic poisoning of Purkinje cells with NaCN and 2-deoxyglucose caused a significant shortening of the action potential (control 376 +/- 51 ms; 6 min NaCN/2-deoxyglucose 153 +/- 21 ms). This effect was reversed with the application of glibenclamide. Inside-out membrane patches from Purkinje cells showed unitary current fluctuations which were inhibited by cytoplasmic ATP with an IC50 of 119 microM and a Hill coefficient of 2.1. This reflects approximately five-fold lower sensitivity to ATP inhibition than for KATP channels from ventricular myocytes under the same conditions. The slope conductance of Purkinje cell KATP channels, with symmetric, 140 mM K+, was 60.1 +/- 2.0 pS (mean +/- SEM). Single-channel fluctuations showed mean open and closed times of 3.6 +/- 1.5 ms and 0.41 +/- 0.2 ms, respectively, at -60 mV and approximately 21 degrees C. At positive potentials. KATP channels exhibited weak inward rectification that was dependent on the concentration of internal Mg2+. Computer simulations, based on the above results, predict significant shortening of the Purkinje cell action potential via activation of KATP channels in the range 1-5 mM cytoplasmic ATP. CONCLUSIONS: Purkinje cell KATP channels may represent a molecular isoform distinct from that present in ventricular myocytes. The presence of KATP channels in the Purkinje network suggests that they may have an important influence on cardiac rhythm and conduction during periods of ischemia.     

Electrophysiological responses of canine atrial endocardium and epicardium to acetylcholine and 4-aminopyridine.

OBJECTIVES: Prior studies demonstrated marked electrophysiological and pharmacological differences between canine ventricular epicardium and endocardium. For atrium, however, it has been assumed that, because of the thin wall, electrical properties of epicardium and endocardium are similar. The aim of the present study was to compare the action potential (AP) characteristics in epicardial and endocardial atrial cells before and following addition of acetylcholine (ACh) and 4-aminopyridine (4-AP). METHODS AND RESULTS: Microelectrode techniques were used to study the effects of ACh (10(-7)-10(-5) M) and 4-AP (0.5 mM) on epicardial and endocardial AP of canine right atrial free wall at cycle lengths (CL) of 250 to 2000 ms. ACh hyperpolarized epicardial and endocardial cells (by 5-8 mV at 10(-5) M). In control, AP duration to 90% repolarization (APD90) was longer in endocardium at all CL. ACh shortened APD90 in either tissue with more prominent effect in endocardium (at 10(-5) M and CL = 2000 ms, from 179 +/- 10 to 90 +/- 11 ms in epicardium and from 209 +/- 10 to 65 +/- 6 ms in endocardium, P < 0.05). As a result, at 10(-5) M, APD90 in endocardium was shorter than in epicardium at all CL 4-AP effects on AP duration were similar in both tissue types. No effects of 4-AP was seen at CL = 250 ms and at long CL, the compound shortened APD90 and prolonged AP duration to 50% repolarization. CONCLUSIONS: (1) ACh exerts direct effects on atrial epicardial and endocardial AP; (2) 4-AP-sensitive transient outward current (Itol) is expressed both in canine atrial epicardial and endocardial cells; (3) differential response of epicardial and endocardial APD to ACh may alter the gradient of repolarization across the atrial wall and contribute to vagally induced atrial flutter and fibrillation.     

Interrelation between pinacidil and intracellular ATP concentrations on activation of the ATP-sensitive K+ current in guinea pig ventricular myocytes

The patch-clamp technique was used to study the relation between pinacidil and intracellular ATP concentration [( ATP]i) on the activation of the outward K+ current in guinea pig ventricular myocytes. Pinacidil shortened the action potential duration, exhibiting stronger effect at 2 mM [ATP]i than at 5 mM [ATP]i. Pinacidil at 5 microM or higher concentrations activated the time-independent outward current at potentials positive to -80 mV, and the pinacidil-activated current was suppressed by increasing [ATP]i from 2 to 5 mM. The dose-response curve of pinacidil at different [ATP]i showed a shift to the right and a depression of the maximum response at increased [ATP]i. The pinacidil-induced shortening of the action potential duration and outward current were inhibited by application of 0.3-1.0 microM glibenclamide. In single-channel current recordings, pinacidil activated the intracellular ATP-sensitive K+ channel current without changing the unitary amplitude, and increased open probability of the channel, an effect dependent on [ATP]i. The pinacidil-activated single-channel current was blocked by glibenclamide. These results prove the notion that pinacidil activates the ATP-sensitive K+ channel current, which explains the action potential shortening in cardiac cells after application of pinacidil.     

Mechanisms for the positive inotropic effect of alpha 1-adrenoceptor stimulation in rat cardiac myocytes.

alpha 1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+ sensitivity and action potential prolongation, which can increase net Ca2+ entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2-0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+ entry and differentiate between the two mechanisms. At 22-23 degrees C the specific alpha 1-adrenoceptor agonist methoxamine (100 microM) prolonged action potential duration at 50% repolarization from 55 +/- 2 to 81 +/- 5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3 +/- 0.6 to 7.8 +/- 1 microns (n = 18). Reduction of the transient outward current and other K+ currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52 +/- 6 to 98 +/- 12 msec and increased unloaded cell shortening from 2.9 +/- 0.4 to 6.6 +/- 0.6 microns (n = 4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70-500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3 +/- 0.7 to 4.97 +/- 0.8 microns at pulse potentials positive to -40 mV. Similar alpha 1-adrenoceptor responses were observed at 35 degrees C during action potential and voltage-clamp experiments, which could be blocked by 10 microM prazosin. In myocytes loaded with the Ca2+ indicator indo-1, alpha 1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+ transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+ transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to alpha 1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+ current reduction. In addition, modulation of K+ channels could occur independent of changes in contractility and/or [Ca2+]i.     

Simultaneous recording of action potentials from endocardium and epicardium during ischemia in the isolated cat ventricle: relation of temporal electrophysiologic heterogeneities to arrhythmias

We studied the effects of ischemia on transmembrane action potentials, conduction time, and refractory periods of both endocardial and epicardial muscle cells of coronary-perfused cat left ventricles. Oxygenated Tyrode's solution was perfused through the left anterior descending coronary artery, while the preparation was superfused with Tyrode's solution gassed with 95% N2 and 5% CO2. Transmembrane action potentials recorded simultaneously from endocardial and epicardial cells were normal during coronary perfusion. When perfusion was discontinued ("ischemia"), rapid deterioration of action potentials and prolongation of conduction time were observed in both endocardial and epicardial cells. The magnitude of the reduction of action potential amplitude and action potential duration (APD), and of prolongation of conduction time, was greater in epicardial cells than in endocardial cells, although the change in resting membrane potential was almost the same. However, APD of endocardial cells decreased progressively during 30 min of ischemia, whereas APD of epicardial cells was reduced maximally at 10 min and then partially recovered. Shortening of refractory periods of endocardial cells paralleled APD shortening, whereas refractory periods of epicardial cells decreased for the first 10 min and then increased. At 10 min of ischemia, APD and refractory periods of epicardial cells were significantly shorter than those of endocardial cells. At 30 min of ischemia, refractory periods of epicardial cells exceeded those of endocardial cells because of development of greater postrepolarization refractoriness in epicardial cells. Accompanying these different changes in APD and refractory periods of endocardial and epicardial cells, spontaneous extrasystolic impulses increased and rapid runs of extrasystolic impulses could be induced by extrastimuli.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-regulated K+ channels protect the myocardium against ischemia/reperfusion damage

The role of ATP-regulated K+ channels in protecting the myocardium against ischemia/reperfusion damage was explored using glibenclamide and pinacidil to block and activate the channels, respectively. Electrical and mechanical activity of arterially perfused guinea pig right ventricular walls was recorded simultaneously via an intracellular microelectrode and a force transducer. The preparations were subjected to either 1) 20 minutes of no-flow ischemia with or without glibenclamide (1 and 10 microM) followed by reperfusion, or 2) 30 minutes of no-flow ischemia with or without pinacidil (1 and 10 microM) followed by reperfusion. No-flow ischemia for 20 minutes produced changes in electrical and mechanical activity that were completely reversed on reperfusion; resting membrane potential declined by 13 +/- 1.2 mV, action potential duration at 90% repolarization (APD90) decreased by 62%, and developed tension fell by greater than 95%, but resting tension did not change significantly. Glibenclamide (10 microM) had no effect on activity during normal perfusion, but during ischemia, resting membrane potential fell slightly further (17 +/- 1.8 mV) and APD90 declined by only 24%. Developed tension declined more slowly and to a lesser extent, but resting tension rose significantly between 10 and 20 minutes of ischemia. Reperfusion of glibenclamide-treated tissues elicited arrhythmias (extrasystoles and tachycardia), and the preparations failed to recover mechanical function. Glibenclamide at 1 microM produced qualitatively similar effects, albeit less severe. After 30 minutes of no-flow ischemia in untreated tissues, resting tension increased by approximately 130% during the no-flow period. Reperfusion caused arrhythmias (extrasystoles, tachyarrhythmias, and fibrillation) and failed to restore resting or developed tension to preischemic levels. Pinacidil at 1 microM did not affect electrical or contractile function, but at 10 microM it had a negative inotropic effect, decreasing APD90 and developed tension by 5% and 18%, respectively. Both concentrations of the drug caused a faster and greater decline in APD90 during the no-flow period. Resting tension did not change during 30 minutes of no-flow ischemia in the presence of pinacidil, and reperfusion led to 85% and complete recovery of electrical and mechanical activity at 1 and 10 microM, respectively. The data indicate that glibenclamide enhances whereas pinacidil reduces myocardial damage caused by ischemia/reperfusion. The results are consistent with the hypothesis that activation of ATP-regulated K+ channels during ischemia is an important adaptive mechanism for protecting the myocardium when blood flow to the tissue is compromised.     

Differences in transient outward currents of feline endocardial and epicardial myocytes

Whole-cell voltage-clamp experiments were performed on enzymatically dissociated single ventricular myocytes harvested from feline endocardial and epicardial surfaces. The studies were designed to test the hypothesis that the differences in the amplitude of transient outward current (Ito) contribute to the difference in action potential configuration between endocardial and epicardial myocytes. In the control state, action potentials recorded from epicardial cells demonstrated a prominent notch between phases 1 and 2, and membrane current recordings displayed a prominent Ito, whereas in endocardial cells the notch in action potentials and Ito were small. External application of 4-aminopyridine (2 mM) reduced the amplitudes of notch and Ito in epicardial cells but not in endocardial cells. After application of 4-aminopyridine (2 mM) and caffeine (5 mM), the notch and Ito were abolished completely in both endocardial and epicardial cells. The first component of Ito (Ito1) was present in all epicardial cells studied (n = 20); it was absent in 12 of the 20 endocardial cells, and a small Ito1 was present in the remaining eight endocardial cells. The mean amplitude of Ito1 was significantly greater in epicardial than in endocardial cells. At a test voltage of +80 mV, the amplitude of Ito1 was 102.0 +/- 47.7 pA/pF in epicardial cells and 3.3 +/- 3.3 pA/pF in endocardial cells (p less than 0.01). The second component of Ito (Ito2) was present in all endocardial (n = 30) and epicardial (n = 30) cells studied. The amplitude of Ito2 was significantly greater in epicardial than in endocardial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early afterdepolarizations and triggered activity induced by cocaine. A possible mechanism of cocaine arrhythmogenesis.

BACKGROUND. Cocaine may produce life-threatening cardiac arrhythmias, but it is not clear whether this is an indirect effect of coronary vasoconstriction and ischemia or a direct myocardial effect of the substance. Except for its effects on the Na+ current as a local anesthetic, little is known about the direct electrophysiological actions on cardiac cells. Therefore, we studied the effects of cocaine on action potentials and membrane currents in isolated feline ventricular myocytes to test the hypothesis that cocaine-induced arrhythmogenesis may be based on cellular and ionic mechanisms. METHODS AND RESULTS. Action potentials and membrane currents were recorded using the patch clamp technique. Single cells were isolated from feline left ventricles by enzymatic digestion. Exposure to cocaine (10 or 50 microM) depressed the plateau phase of the action potential and prolonged action potential duration. Action potential duration measured at 90% repolarization (APD90) was increased from 280 +/- 12 msec to 325 +/- 17 msec (p less than 0.01) by 5-minute exposure to 10 mumol cocaine, when the cells were stimulated at 1 Hz. During exposure to 50 mumol cocaine, APD90 was markedly increased from 298 +/- 13 msec to 437 +/- 35 msec (p less than 0.01) in seven of 16 cells, and early afterdepolarizations (EADs) developed in these cells. The take-off potential and the amplitude of EADs were -28.3 +/- 2.3 mV and 16.8 +/- 1.2 mV, respectively. Triggered activity arising from EADs was induced in four of the seven cells. Addition of 1 nmol isoproterenol augmented EADs and induced sustained triggered activity, whereas they were suppressed by exposure to 2 microM verapamil. Whole-cell voltage clamp experiments revealed that cocaine (50 microM) reduced the peak L-type Ca2+ current from 1.03 +/- 0.13 nA to 0.79 +/- 0.11 nA (23% reduction, p less than 0.05). Cocaine also reduced the peak delayed rectifier K+ current from 362 +/- 51 pA to 113 +/- 32 pA (69% reduction, p less than 0.01). However, cocaine did not affect activation and inactivation kinetics of these channels. Cocaine had no effect on the inward rectifier K+ current. CONCLUSIONS. We conclude that cocaine can prolong action potential duration and induce EADs and triggered activity by blocking the delayed rectifier K+ current, and that cocaine-induced abnormalities of repolarization, modulated by its inhibitory effects on catecholamine reuptake, may play a role in the potential of cocaine for induction of acute fatal arrhythmias.     

ATP-sensitive potassium channel modulation of the guinea pig ventricular action potential and contraction

The role of ATP-sensitive potassium (KATP) channels in modulating the action potential and contraction of guinea pig ventricular myocytes was investigated. Under voltage clamp, the maximum whole-cell KATP channel conductance was estimated (195 +/- 10 nS, n = 6) by exposing the cells to complete metabolic blockade (2 mM cyanide in the presence of 10 mM 2-deoxy-glucose). In isolated inside-out membrane patches, the ATP dependence of KATP channel activity under relevant conditions was measured (half-maximal inhibition at 114 microM). Under current clamp (with intracellular ATP concentration = 5 mM), the effect of graded KATP channel activation on the action potential and the twitch was estimated by injection of a current (proportional to voltage) that simulated the KATP conductance. As this "conductance" was increased, the action potential was shortened, and contractile amplitude declined, as expected. From the results of these experiments, the quantitative dependence of the action potential duration on intracellular ATP concentration was estimated, without relying on a mathematical model of the cell membrane. The results imply that KATP-dependent action potential shortening is likely to occur if ATP concentration falls below normal levels (approximately 5 mM), as may happen regionally, or globally, during myocardial ischemia.     

奎尼丁对吡那地尔诱导的犬右心室跨壁复极离散的影响

目的　由吡那地尔诱导犬右心室肌细胞产生“全或无”复极,观察奎尼丁对这种跨壁复极离散的影响。方法　应用标准玻璃微电极技术在1000ms刺激周长下,记录犬右心室肌细胞不同部位(外膜下、M区、内膜下)在不同情况[正常对照、吡那地尔(2 5μmol/L)、吡那地尔( 2 5μmol/L) +奎尼丁(5μmol/L) ]的动作电位。结果　吡那地尔( 2 5μmol/L)在3层细胞产生“全或无”复极,使跨壁复极离散增大,动作电位时程跨壁复极离散由(48 .5±9 .2)ms升为(128. 7±13. 5)ms(P<0. 01),进一步灌注奎尼丁(5μmol/L)后,减为(54 .3±10 .8)ms(P<0. 01)。奎尼丁部分恢复动作电位2相平台,延长了被吡那地尔缩短的动作电位时程。结论　在犬右心室肌组织,奎尼丁(5μmol/L)减小了由吡那地尔造成的跨壁复极离散,维持了跨壁电稳定性。     

Sodium channel block produces opposite electrophysiological effects in canine ventricular epicardium and endocardium.

Using microelectrode techniques we compared the effects of tetrodotoxin (TTX, 2-3 microM), DL-propranolol (1-3 micrograms/ml), and flecainide acetate (10-15 microM) on isolated canine ventricular epicardial (epicardium) and endocardial (endocardium) tissues. Propranolol, TTX, and flecainide decreased Vmax and phase 0 amplitude in a use-dependent manner in both tissues. The effects of propranolol were slow to develop and wash out. TTX and propranolol always abbreviated action potential duration in endocardium. Action potential duration was abbreviated by 23.8 +/- 5.6 msec after propranolol (1 microgram/ml, basic cycle length [BCL] = 1,000 msec) and 10.8 +/- 12.9 msec after TTX (2 microM, BCL = 1,000 msec). In epicardium, the reduction of phase 0 and 1 amplitudes led to a slowing of the second action potential upstroke and an increase in the amplitude of phase 2. This accentuation of the notch resulted in a paradoxical prolongation of the epicardial action potential. Action potential duration was prolonged 34.4 +/- 11.3 msec after 4 hours of exposure to propranolol (1 microgram/ml, BCL = 1,000 msec), 11.1 +/- 6.3 msec after 15 minutes of exposure to TTX (2 microM, BCL = 1,000 msec), and 19.9 +/- 8.2 msec after 25-45 minutes of exposure to flecainide (15 microM, BCL = 500 msec). With stronger sodium block, phase 1 terminated at more negative potentials, the second upstroke often failed to appear, and an all-or-none repolarization ensued causing a marked abbreviation of the epicardial action potential. In some epicardial preparations, we observed marked abbreviation at some sites but prolongation at other sites after sodium blockade with flecainide. The dispersion of repolarization was often attended by reentrant activity. The differential response of epicardium and endocardium to sodium blockade was not observed when the preparations were pretreated with 4-aminopyridine or ryanodine, agents known to diminish the transient outward current and epicardial notch. Acceleration-induced prolongation of refractoriness was observed after sodium blockade in epicardium but not in endocardium. Postrepolarization refractoriness also occurred in epicardium but not in endocardium after TTX, propranolol, or flecainide exposure. The data indicate that propranolol, TTX, and flecainide, via their action to block sodium current, may exert opposite effects on action potential duration and refractoriness in cells spanning the ventricular wall. The presence of the transient outward current in epicardium but not in endocardium appears to contribute importantly to these differences.(ABSTRACT TRUNCATED AT 400 WORDS)     

A role for a glibenclamide-sensitive, relatively ATP-insensitive K+ current in regulating membrane potential and current in rat aorta

OBJECTIVE: ATP-sensitive K+ channels have been classified based on their inhibition by cytoplasmic ATP. Recent evidence in vascular smooth muscle has suggested that these channels show weak sensitivity to intracellular ATP. However, it is not known whether these channels regulate the resting K+ conductance in vascular smooth muscles. Therefore, the aim of the present investigation was to characterize this current in rat aorta myocytes and to examine whether it contributes to setting the membrane potential. METHODS: The conventional and nystatin-permeablised whole cell patch clamp techniques were used to characterize the effect of glibenclamide on membrane potential and K+ current in enzymatically dispersed rat aorta myocytes. RESULTS: The mean resting potential measured in current clamp mode using the permeabilized patch approach was -54 +/- 5 mV (n = 8). Glibenclamide (10 microM) caused a reversible 24-mV depolarization in these cells. In symmetrical K+ (135 mM) solution an inward glibenclamide-sensitive (10 microM) current (-4.1 +/- 0.7 pA/pF; n = 5), hereafter termed Iglib, was observed at a membrane potential of -80 mV when cells held at -60 mV were ramped from -80 to +80 mV. In the absence of any nucleotide in the pipette solution, Iglib measured by the conventional whole-cell method was -23.69 +/- 4.65 pA/pF (n = 9). With 1 and 3 mM ATP in the pipette, the average current density was -25 +/- 6.3 pA/pF (n = 8), and -9.4 +/- 2.7 pA/pF (n = 9), respectively. In the absence of ATP, 1 mM GDP significantly (P < 0.01) increased Iglib (-44.8 +/- 8.4 pA/pF; n = 13). Inclusion of 1 mM ATP in the GDP-containing pipette solution had no significant effect on the current amplitude (-56.4 +/- 10.7 pA/pF; n = 7). Iglib fell to -11.0 +/- 2.9 pA/pF (n = 10) if 1 mM GDP and 3 mM ATP were present. In symmetrical K+, the Iglib observed in the presence of 1 mM ATP in the pipette was increased by more than two-fold in the presence of 10 microM levcromakalim. In PSS containing 5 mM K+, a significant glibenclamide-sensitive current was observed at -45 mV membrane potential when cells dialyzed with 1 mM ATP were ramped between -80 to 30 mV. CONCLUSION: These results demonstrate that Iglib channels in rat aorta myocytes differ from classical KATP channels, being relatively insensitive to intracellular ATP. Iglib therefore appears to have an important role in contributing to the maintenance of the resting potential in rat aortic smooth muscle.     

Ionic basis for action potential prolongation by phenylephrine in canine epicardial myocytes

INTRODUCTION: In canine ventricle, alpha-adrenergic agonists prolong action potential duration (APD) without any effect on the action potential notch, suggesting that, in this species, the effect on repolarization might be independent of inhibition of I(to). The present study investigated the action of the alpha-adrenergic agonist phenylephrine on the action potential and the repolarizing currents I(to) and I(K) in isolated canine epicardial myocytes. METHODS AND RESULTS: Isolated cells from canine epicardial tissue, and Purkinje fibers, were studied with the whole cell, voltage clamp method. Phenylephrine 0.1 microM increased APD by 13% +/- 4% at 90% repolarization without affecting the notch or amplitude. Under voltage clamp, concentrations of phenylephrine as high as 10 microM had no effect on I(to) in canine epicardial myocytes. However, I(to) of isolated canine Purkinje myocytes was reduced to 69% +/- 7% of control by 1 microM phenylephrine. Further studies in canine epicardial myocytes revealed an action of phenylephrine to inhibit I(K), and in particular I(Ks). Using a voltage protocol that included a two-step repolarization to separate I(Ks) and I(Kr) tail components, the largely I(Kr) component was not significantly affected by 1 microM phenylephrine, whereas the largely I(Ks) component was reduced to 81% +/- 5% of control value. CONCLUSION: Alpha-adrenergic prolongation of repolarization in canine epicardium does not result from inhibition of I(to). Rather, it appears that reduction of I(Ks) contributes to the action of phenylephrine. The unresponsiveness of epicardial I(to) is not a general characteristic of the canine heart, because Purkinje myocyte I(to) was inhibited, suggesting regional differences in the molecular basis of I(to) and/or alpha-adrenergic signaling in the canine heart.     

Amiodarone inhibits cardiac ATP-sensitive potassium channels

INTRODUCTION: ATP-sensitive K+ channels (K(ATP)) are expressed abundantly in cardiovascular tissues. Blocking this channel in experimental models of ischemia can reduce arrhythmias. We investigated the acute effects of amiodarone on the activity of cardiac sarcolemmal K(ATP) channels and their sensitivity to ATP. METHODS AND RESULTS: Single K(ATP) channel activity was recorded using inside-out patches from rat ventricular myocytes (symmetric 140 mM K+ solutions and a pipette potential of +40 mV). Amiodarone inhibited K(ATP) channel activity in a concentration-dependent manner. After 60 seconds of exposure to amiodarone, the fraction of mean patch current relative to baseline current was 1.0 +/- 0.05 (n = 4), 0.8 +/- 0.07 (n = 4), 0.6 +/- 0.07 (n = 5), and 0.2 +/- 0.05 (n = 7) with 0, 0.1, 1.0, or 10 microM amiodarone, respectively (IC50 = 2.3 microM). ATP sensitivity was greater in the presence of amiodarone (EC50 = 13 +/- 0.2 microM in the presence of 10 microM amiodarone vs 43 +/- 0.1 microM in controls, n = 5; P < 0.05). Kinetic analysis showed that open and short closed intervals (bursting activity) were unchanged by 1 microM amiodarone, whereas interburst closed intervals were prolonged. Amiodarone also inhibited whole cell K(ATP) channel current (activated by 100 microM bimakalim). After a 10-minute application of amiodarone (10 microM), relative current was 0.71 +/- 0.03 vs 0.92 +/- 0.09 in control (P < 0.03). CONCLUSION: Amiodarone rapidly inhibited K(ATP) channel activity by both promoting channel closure and increasing ATP sensitivity. These actions may contribute to the antiarrhythmic properties of amiodarone.