Monophasic action potential recordings from intact mouse heart: validation, regional heterogeneity, and relation to refractoriness.

INTRODUCTION: The monophasic action potential (MAP) technique has been validated in humans and larger animals, but, in mice, MAP recordings available to date show little resemblance to the murine ventricular transmembrane action potential (TAP) measured by conventional microelectrodes. We developed a miniaturized MAP contact electrode technique to establish in isolated mouse hearts: (1) optimal electrode size; (2) validation against TAP; (3) relationship between repolarization and refractoriness; and (4) regional repolarization differences. METHODS AND RESULTS: In 30 Langendorff-perfused mouse hearts, MAP electrodes of tip diameter 1.5, 1.0, and 0.25 mm were tested by comparing MAPs and TAPs from epicardial and endocardial surfaces of both ventricles. Only the MAP contact electrode of 0.25-mm tip diameter consistently produced MAP recordings that had wave shapes nearly identical to TAP recordings. MAP durations measured at 30%, 50%, 70%, and 90% repolarization (APD30, APD50, APD70, APD90) highly correlated with TAP measurements (r = 0.97, P < 0.00001). APD50 was significantly longer in endocardial than in epicardial recordings (right ventricle: 9.3+/-1.1 msec vs 3.9+/-1.1 msec; left ventricle: 9.9+/-2.1 msec vs 6.2+/-1.9 msec; both P < 0.001), demonstrating transmural repolarization differences. Effective refractory period (ERP) determined at basic cycle lengths from 70 to 200 msec correlated with 80%+/-6% of total repolarization, with an ERP/APD90 ratio of 0.85+/-0.14. CONCLUSION: Murine myocardial repolarization, regional repolarization heterogeneity, and relation to refractoriness can be assessed reliably by this miniaturized MAP contact electrode technique, which renders action potential wave shapes similar to that of intracellular microelectrodes. This technique may be useful for exploring repolarization abnormalities in genetically altered mice.

Gadolinium decreases stretch-induced vulnerability to atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is frequently associated with atrial dilatation caused by pressure or volume overload. Stretch-activated channels (SACs) have been found in myocardial cells and may promote AF in dilated atria. To prove this hypothesis, we investigated the effect of the SAC blocker gadolinium (Gd(3+)) on AF propensity in the isolated rabbit heart during atrial stretch. METHODS AND RESULTS: In 16 isolated Langendorff-perfused rabbit hearts, the interatrial septum was perforated to equalize biatrial pressures. Caval and pulmonary veins were occluded. Intra-atrial pressure (IAP) was increased in steps of 2 to 3 cm H(2)O by increasing the pulmonary outflow fluid column. Vulnerability to AF was evaluated by 15-second burst pacing at each IAP level. At baseline, IAP needed to be raised to 8.8+/-0.2 cm H(2)O (mean+/-SEM) to induce AF. A dose-dependent decrease in AF vulnerability was observed after Gd(3+) 12.5, 25, and 50 micromol/L was added. AF threshold increased to 19.0+/-0.5 cm H(2)O with Gd(3+) 50 micromol/L (P<0.001 versus baseline). Spontaneous runs of AF occurred in 5 hearts on a rise of IAP to 13.8+/-3.3 cm H(2)O at baseline but never during Gd(3+). Atrial effective refractory period shortened progressively from 78+/-3 ms at 0.5 cm H(2)O to 52+/-3 ms at 20 cm H(2)O (P<0.05). Gd(3+) 50 micromol/L had no significant effect on effective refractory period. CONCLUSIONS: Acute atrial stretch significantly enhances the vulnerability to AF. Gd(3+) reduces the stretch-induced vulnerability to AF in a dose-dependent manner. Block of SAC might represent a novel antiarrhythmic approach to AF under conditions of elevated atrial pressure or volume.

Female gender is a risk factor for torsades de pointes in an in vitro animal model.

Recent clinical observations indicate that female gender is associated with a higher risk of developing torsades de pointes (TdP) cardiac arrhythmia. In this study, we used the Langendorff technique in isolated perfused rabbit hearts and the whole-cell patch-clamp technique in ventricular myocytes to examine the gender difference in TdP incidence and gain insight into the underlying mechanisms. Isolated rabbit hearts were perfused by using the Langendorff technique. TdP was induced by abrupt changes of cycle length (deltaCL) in the presence of Tyrode's solution containing 1 mM 4-aminopyridine (4AP) and 50% reduced Mg2+ and K+ (low K/Mg). The effects of 1 mM 4AP on cardiac potassium currents were characterized by using the patch-clamp technique. Results demonstrated that (a) no significant gender difference was observed in the absolute QT interval before or after 4AP perfusion in the presence of low K/Mg; (b) 4AP caused marked QT prolongation in the ECG; (c) a significantly higher TdP incidence (nine of 12) was found in female hearts compared with male hearts (three of 12; p < 0.05); (d) 1 mM 4AP primarily inhibited Ito, although a slight inhibition of IKr also occurred in low-K/Mg Tyrode's solution. (e) No inhibition of IK1 was observed. (f) No gender difference was found in the potassium current block produced by 4AP. Female gender is associated with a higher incidence of TdP in an experimental isolated heart model and mechanisms subsequent to QT prolongation may contribute to such a gender difference.

The antiestrogen tamoxifen blocks the delayed rectifier potassium current, IKr, in rabbit ventricular myocytes.

Tamoxifen is an antiestrogen drug commonly used to treat breast cancer and has been shown to cause prolongation of the electrocardiographic QT interval in humans. Because QT prolongation could influence cardiac arrhythmias, we sought to determine the electrophysiologic mechanism(s) underlying the tamoxifen action. The whole-cell patch-clamp technique was used to study the effect of tamoxifen on the delayed rectifier (IKr), the inward rectifier (IK1), the transient outward current (Ito), and the inward L-type calcium current (ICa) in rabbit ventricular myocytes. By switching to the current-clamp mode, the effect of tamoxifen on action potential duration (APD) was also studied. Tamoxifen blocked IKr in a time-, concentration- and voltage-dependent fashion. IKr tail currents were completely blocked by 10 micromol/l tamoxifen with no recovery after 15 min of washout. At +50 mV, tamoxifen 1 and 3.3 micromol/l blocked IKr by 39.5 +/- 1.7% (P <.01) and 84.8 +/- 1.3% (P <.01) respectively, while no significant block of IK1 or Ito was observed. Significant block of ICa by tamoxifen was also observed at concentrations greater than 1 micromol/l, with almost complete inhibition at 10 micromol/l. Tamoxifen showed no significant effect on APD at concentrations up to 3.3 micromol/l. We conclude that tamoxifen potently blocks both IKr and ICa at clinically relevant concentrations. The observed QT prolongation by tamoxifen in humans may be a result of its predominant effect on IKr. Inhibition of IKr, in conjunction with other QT-prolonging factors in patients could increase their risk of developing torsades de pointes-type cardiac arrhythmias.

Gender difference in the cycle length-dependent QT and potassium currents in rabbits.

Women are known to have a longer electrocardiographic Q-T than men, which may contribute to their being at greater risk of developing drug-induced polymorphic ventricular arrhythmias. However, little is known about the underlying mechanisms. In the present study, we evaluated potential gender differences in Q-T interval in isolated perfused rabbit hearts using the Langendorff technique and evaluated the density of outward potassium currents in single ventricular myocytes using the whole-cell patch-clamp technique. We found that female hearts demonstrated a greater Q-T lengthening (delta Q-T%) upon an increase in cycle length (CL), resulting in a significantly longer Q-T (301 +/- 4.8 ms, CL = 2.3 s) at a long CL in female hearts compared with male hearts (267 +/- 4.0 ms, P < .01). Ventricular myocytes isolated from female hearts showed a smaller IK(tail) and peak IKI outward current density. A 50% reduction in extracellular K+ and Mg++ shifted the I-V relationship of IKI and Ito and reduced their amplitude. However, neither the I-V relationship of IKr nor the gender difference in the Q-T-CL relationship was significantly altered. We conclude that 1) female rabbit ventricular myocytes have significantly lower IKr and IKl outward current densities than do male cells, which may contribute to the gender difference in Q-T, and 2) a lower base-line IKr density may contribute to the steeper Q-T-CL relationship in female hearts.

Nitric oxide modulates synaptic glutamate release during anoxia.

The ability of nitric oxide to enhance vesicular glutamate release during anoxia was examined in the present study. Whole-cell patch clamp recordings were obtained from CA1 pyramidal neurons in rat hippocampal slices perfused in media containing tetrodotoxin. These cells exhibit spontaneous inward currents previously identified as glutamatergic miniature excitatory postsynaptic currents (mEPSCs). The frequency of these mEPSCs increases during exposure to anoxia. The anoxia-induced increase in frequency is reduced when experiments are performed in the presence of the competitive nitric oxide (NO)-synthase inhibitors N(G)-nitro-L-arginine methyl ester and N(G)-nitro-L-arginine, as well as reduced hemoglobin. Arginine reversed the suppression by the NO-synthase inhibitors. The N-methyl-D-aspartate (NMDA) receptor antagonists 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid and MK-801 also suppressed the anoxia-induced increase in mEPSC frequency. These data indicate that NMDA receptor-activated NO production may enhance vesicular synaptic glutamate release, which would in turn contribute to excitotoxicity during hypometabolic states.

Adenosine A1 antagonism increases specific synaptic forms of glutamate release during anoxia, revealing a unique source of excitation.

The role of the adenosine A1 receptor in the modulation of anoxia-induced synaptic glutamate release was examined in CA1 pyramidal neurons by whole-cell voltage-clamp recording in the rat hippocampal slice preparation. Anoxia leads to an increased action potential-independent synaptic glutamate release in the form of a higher frequency of miniature excitatory postsynaptic currents (mEPSCs). This increase is not significantly affected when slices are preincubated in the adenosine A1 receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX). A second population of spontaneous inward currents, however, occurs in DPCPX-treated slices during a well-defined period following the onset of anoxia. Their suppression by glutamate antagonists, tetrodotoxin, or by the cutting of the Schaffer collateral pathway indicates that they represent action potential-dependent, glutamatergic excitatory postsynaptic currents (ap-EPSCs) originating from CA3 pyramidal neurons. CA3 neurons were examined in current-clamp whole-cell patch mode to determine the origin of this increased orthodromic excitation. After the onset of anoxia, CA3 cells initially exhibit a small depolarization or hyperpolarization associated with a decrease in input resistance. This is followed by transient depolarization (the depolarizing "nub"), which is associated with an increase in input resistance. The nub evoked single as well as bursts of action potentials in CA3 neurons. The occurrence of these CA3 nub-elicited action potentials coincides with that of ap-EPSCs recorded in the CA1 cells. Recording with cesium- rather than standard potassium-containing electrodes results in the suppression of the nub and its associated increase in input resistance. In conclusion we have shown that adenosine tone plays an important role in suppressing anoxia-induced spontaneous ap-EPSCs but not action potential-independent mEPSCs in CA1 neurons. These EPSCs originate from a depolarization in CA3 pyramidal neurons, which is associated with an increase in resistance. This previously undescribed phenomenon likely results from a decrease in the conductance of an unidentified potassium channel.

Mechanism of early anoxia-induced suppression of the GABAA-mediated inhibitory postsynaptic current.

1. We investigated the mechanism of hypoxia-induced depression of gamma-aminobutyric acid-A (GABAA)-mediated inhibitory postsynaptic currents (IPSCs) in CA1 neurons of hippocampal slices from 21- to 28-day-old rats. Cells were examined by whole-cell patch-clamp recording and hypoxia was induced by switching perfusion of the slice from oxygenated artificial cerebral spinal fluid (ACSF) to ACSF saturated with 95% N2-5% CO2. 2. Synaptic responses evoked by stimulation of the Schaffer collateral-commissural projection at a fixed holding potential (VH = -60 mV) during anoxia revealed that the IPSC appeared more sensitive than the excitatory postsynaptic current to anoxia-induced depression. All subsequent studies examined the GABAA-mediated IPSC synaptic responses in isolation by direct stimulation of GABA interneurons in the stratum radiatum in the presence of extracellular 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP) (20 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (50 microM) to block glutamatergic currents and intracellular QX-314 (lidocaine N-ethyl bromide, 1 mM) to block GABAB-mediated currents. When studied in this manner (VH = -60 mV) the GABAA-mediated IPSC appeared to change from an outward to inward current after exposure to anoxia. 3. The current-voltage relationship of GABAA-mediated IPSCs revealed that these changes resulted from a positive shift in the IPSC reversal potential without a significant change in the conductance. Thus under patch clamp apparent IPSC inhibition may result from a decrease in the extracellular concentration of chloride ions. Similar findings were observed with micropipettes that contained high intracellular chloride concentrations. 4. Miniature spontaneous IPSCs were examined in the presence of tetrodotoxin (1 microM) with micropipettes containing high intracellular chloride concentrations. The miniature IPSCs (mIPSCs) appeared as spontaneous transient inward currents. Consistent with an anoxia-induced decrease in extracellular chloride, the mean amplitude of the mIPSCs increased after the onset of anoxia. A significant decrease in rise and decay time was also noted during anoxia. The frequency of the mIPSCs also increased by approximately 300%. 5. The resting input resistance of the cells was examined by measuring the current resulting from a 20-mV hyperpolarizing pulse. A significant reduction in resistance was observed 2 min after the onset of anoxia. This still occurred, although to a lesser degree, in the presence of glutamatergic blockers (20 microM CPP plus 50 microM CNQX). In the presence of both GABAergic (picrotoxin, 100 microM) and glutamatergic blockers no significant reduction in resting input resistance was apparent after 2 min of anoxia.(ABSTRACT TRUNCATED AT 400 WORDS)

Arachidonic acid participates in the anoxia-induced increase in mEPSC frequency in CA1 neurons of the rat hippocampus.

Patch clamp in the whole cell configuration was used to examine the effects of a variety of agents that influence arachidonic acid metabolism on vesicular glutamate release in CA1 neurons of rat hippocampal slices. As previously demonstrated, anoxia induced a significant increase in the frequency of spontaneous glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) during the first 5 min following anoxia. This increase in frequency was almost completely abolished if slices were preincubated in artificial cerebral spinal fluid (ACSF) containing the phospholipase C/A2 inhibitor, bromophenacyl-bromide (BPB; 20 microM) or the cyclooxygenase inhibitors, indomethacin (20 microM) and piroxicam (10 microM). This observation may be important to our understanding of the neuroprotective action of these agents. These data suggest that arachidonic acid (AA) and its cyclooxygenase products or by-products (oxygen free radicals) contribute to vesicular glutamate release during the early phase of anoxia.

Adenosine antagonists prevent hypoxia-induced depression of excitatory but not inhibitory synaptic currents.

Hypoxia induces depression of excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) in CA1 neurons of the hippocampus. The effect of antagonists that act at the A1 adenosine receptor on hypoxia-induced depression of EPSCs and IPSCs were examined in hippocampal slices with the patch clamp technique (whole-cell configuration). The A1 receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (200 nM) and 8-phenyltheophilline (8-PT) (10 microM) significantly prevented depression of EPSCs by hypoxia but failed to protect IPSCs. This result suggests that the hypoxia-induced depression of the EPSC involves the activation of adenosine receptors (possibly of the A1 subtype), whereas depression of the IPSC results from a different mechanism.

Activation of a common potassium channel in molluscan neurones by glutamate, dopamine and muscarinic agonist.

1. The potassium currents evoked in isolated and identified neurones of molluscan pedal ganglia by either glutamate, dopamine or the muscarinic agonist F-2268 were investigated using voltage and patch clamp techniques. 2. Potassium currents induced by either dopamine or F-2268 could be blocked by pertussis toxin, as well as by a prolonged intracellular injection of the G protein inhibitor, GDP-beta-S. Loading the neurones with the G protein activator, GppNHp, on the other hand, induced a potassium current. This current was not additive to the currents evoked by agonist application. 3. Intracellular injection of the calcium buffer BAPTA failed to affect any of the agonist-induced currents, although it effectively blocked the after-hyperpolarization following directly evoked action potentials. 4. The activity of the potassium channels seen in cell-attached patches was greatly enhanced by application to the bath of either glutamate, dopamine, or F-2268. 5. The only effect of an addition of agonists to the bath was to increase the open probability (Po) of the K+ channel already active in the control conditions. The identity of the spontaneously active and agonist-activated channels was concluded from the identity of their channel conductances, rectification properties and current amplitudes. 6. Phorbol-12,13-dibutyrate, when applied to the bath, induced an increase in open time and caused an increase in Po, as did the agonists. Staurosporine completely prevented changes of Po induced by the phorbol ester but not those induced by the agonists. 7. The same inwardly rectifying potassium channel may be opened by both the receptor-linked G protein (with glutamate, dopamine, F-2268) and by protein kinase C (with phorbol ester) activation. 8. Strong evidence was obtained against the involvement of any known secondary messenger systems (formation of nucleotides, phosphoinositide turnover and subsequent activation of protein kinase C, formation of nitric oxide, metabolism of arachidonic acid) in the transduction mechanism of F-2268-, dopamine- and glutamate-induced responses. 9. Since none of the known secondary messenger systems seems to affect the activation by agonists applied to receptors outside the patch of channels located under the patch electrode, it appears that some as yet undescribed linking system must exist that could connect the spatially separated receptor-G protein complex and the potassium channel.

Early anoxia-induced vesicular glutamate release results from mobilization of calcium from intracellular stores.

1. The cause of the increased frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) resulting from anoxia was investigated in CA1 neurons of the in vitro rat hippocampal slice. These neurons were examined by whole-cell patch-clamp recording, and hypoxia was induced by switching the perfusion of the slice from oxygenated artificial cerebral spinal fluid (ACSF) to ACSF saturated with 95% N2-5% O2. Except where noted, experiments were carried out in ACSF containing 1 microM tetrodotoxin (TTX). 2. Although anoxia resulted in a significant increase in the frequency of mEPSCs, the amplitude, rise time, and half-decay time of the mEPSCs were unchanged. This increase in frequency indicates that there is a change in presynaptic neurotransmitter release mechanisms, probably an increase in calcium concentration, soon after the onset of anoxia. The unchanged kinetics and amplitude of the mEPSCs indicate that anoxic-induced synaptic changes are not a result of changes in the postsynaptic glutamate receptor. 3. When hippocampal slices were exposed to anoxic conditions in ACSF with calcium excluded, an increase in mEPSC frequency equal to that in normal ACSF was observed. When 0.2 mM of CdCl2 was added to the zero-calcium ACSF, anoxia still resulted in increases in mEPSC frequency equal to those of normal ACSF. It is therefore concluded that the anoxia-induced increase in mEPSC frequency does not result from an increase in a transmembrane calcium influx. The zero-calcium plus 0.2 mM CdCl2 ACSF solution completely abolished orthodromically elicited synaptic potential (in the absence of TTX), indicating that calcium currents that mediate normal orthodromic transmitter release were completely abolished in the latter experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Site of synaptic depression during hypoxia: a patch-clamp analysis.

1. The effect of hypoxia on synaptic physiology was investigated in hippocampal slices from 16- to 23-day-old rats. CA1 pyramidal cells were examined by whole cell patch-clamp recording, and hypoxia was induced by switching perfusion of the slice from oxygenated artificial cerebrospinal fluid (ACSF) to ACSF saturated with 95% N2-5%CO2. Synaptic responses were assessed by stimulating the Schaffer collateral-commissural projection with an electrode in the stratum radiatum every 20 s. 2. Within 100-200 s of the onset of hypoxia, the orthrodromically elicited synaptic response of the CA1 cells was largely inhibited. In addition, a slow inward current was observed after the onset of hypoxia. A transient outward current, preceding the inward current, was observed in only 2 of 17 cells examined. The slow inward current culminated in an irreversible rapid inward current at approximately 140 s after hypoxia. This rapid inward current occurred simultaneously with spreading depression as measured by field potentials. Tetrodotoxin (TTX) had no effect on the onset of this current, whereas kynurenic acid significantly delayed its occurrence. 3. Before the onset of hypoxia, spontaneous transient inward currents were apparent. The frequency of these events increased by three- to fourfold after hypoxia. The transient inward currents persisted in slices incubated in TTX, but were almost completely inhibited in slices incubated with the mixed N-methyl-D-aspartate (NMDA)/non-NMDA antagonist kynurenic acid. This identified the spontaneous events that were increased in frequency by hypoxia as glutamatergic miniature excitatory postsynaptic currents (mEPSCs). 4. The mean amplitude of the mEPSCs was not affected by hypoxia at a time at which the orthodromically elicited synaptic response was almost completely inhibited by hypoxia. In addition, the response of the postsynaptic cell to pressure ejection of glutamate was not inhibited under conditions of nearly complete blocked the synaptic response. Thus, by two measures, the postsynaptic response was not affected by hypoxia, indicating that the site of hypoxia-induced synaptic failure was at the presynaptic terminal. 5. The orthodromically elicited synaptic response consisted of an EPSC followed closely by an inhibitory postsynaptic current (IPSC). The IPSC portion of the elicited postsynaptic response was more sensitive to inhibition by hypoxia than was the EPSC. In some cells the EPSC exhibited a monophasic decline in amplitude during hypoxia. However, in a majority of cells, an initial decline in the amplitude of the EPSC was followed by a transient increase and subsequent depression.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of the time course of fast EPSCs and glutamate channel kinetics by aniracetam.

It is generally accepted that glutamate serves as the neurotransmitter at most excitatory synapses in the mammalian central nervous system (CNS). Synaptic release of glutamate may trigger a fast and a slow excitatory postsynaptic current (EPSC). The slow EPSC is mediated by N-methyl-D-aspartate (NMDA) receptor channels, whereas the fast EPSC is mediated by non-NMDA receptor channels. The nootropic agent aniracetam selectively and reversibly slows the desensitization kinetics of non-NMDA channels and lengthens their single-channel open times. Antiracetam also modulates the kinetics of the fast EPSC in a manner that mirrors its action on the kinetics of the non-NMDA channels. These results support the hypothesis that the properties of the non-NMDA glutamate channels rather than the rate of neurotransmitter clearance are the primary determinants of the kinetics of the fast EPSC in the mammalian CNS.

Ionic mechanisms of the rapid (nicotinic) phase of acetylcholine response in identified Planobarius corneus neurones.

Current responses to acetylcholine (ACh) and to suberyldicholine (D-6) applied from the double-barrelled ionophoretic micropipette were studied in two identified neurones (LPed-2 and LPed-3) isolated from the left ganglion of pulmonate mollusc, Planorbarius corneus. Experiments made with K2SO4-filled microelectrodes show that in LPed-2 neurone two kinds of cholinoreceptors are involved in the rapid phase of ACh response one of which induces chloride conductance and the other, sodium conductance. The Cl-dependent component can be separated from the cationic one by C-6 whereas the cationic component can be separated from the Cl--dependent one by furosemide. Cl- conductance can be induced selectively by D-6. In the LPed-3 neurone only Cl- conductance increases during rapid phase of ACh response. The reversal potential of Cl--dependent responses was found to be more negative than the resting potential in experiments made with K2SO4-filled microelectrodes but less negative than the resting potential in the case of KCl-filled microelectrodes. This difference seems to be due to the artificial increase of intracellular chloride concentration.