Antiarrhythmics--from cell to clinic: past, present, and future.
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Action Potentials↗
Biomedical subjects
J C Hancox
Publications and source records attributed to J C Hancox.
Inhibition by nickel of the L-type Ca channel in guinea pig ventricular myocytes and effect of internal cAMP.
The characteristics of nickel (Ni) block of L-type Ca current (I(Ca, L)) were studied in whole cell patch-clamped guinea pig cardiac myocytes at 37 degrees C in the absence and presence of 100 microM cAMP in the pipette solution. Ni block of peak I(Ca,L) had a dissociation constant (K(d)) of 0.33 +/- 0.03 mM in the absence of cAMP, whereas in the presence of cAMP, the K(d) was 0.53 +/- 0.05 mM (P = 0.006). Ni blocked Ca entry via Ca channels (measured as I(Ca, L) integral over 50 ms) with similar kinetics (K(d) of 0.35 +/- 0.03 mM in cAMP-free solution and 0.30 +/- 0.02 mM in solution with cAMP, P = not significant). Under both conditions, 5 mM Ni produced a maximal block that was complete for the first pulse after application. Ni block of I(Ca,L) was largely use independent. Ni (0. 5 mM) induced a positive shift (4 to 6 mV) in the activation curve of I(Ca,L). The block of I(Ca,L) by 0.5 mM Ni was independent of prepulse membrane potential (over the range of -120 to -40 mV). Ni (0.5 mM) also induced a significant shift in I(Ca,L) inactivation: by 6 mV negative in cAMP-free solution and by 4 mV positive in cells dialyzed with 100 microM cAMP. These data suggest that, in addition to blocking channel conductance by binding to a site in the channel pore, Ni may bind to a second site that influences the voltage-dependent gating of the L-type Ca channel. They also suggest that Ca channel phosphorylation causes a conformational change that alters some effects of Ni. The results may be relevant to excitation-contraction coupling studies, which have employed internal cAMP dialysis, and where Ni has been used to block I(Ca,L) and Ca entry into cardiac cells.
Animals↗
Comparison of Na+-Ca2+ exchange current elicited from isolated rabbit ventricular myocytes by voltage ramp and step protocols.
In this study, the effects of three different voltage protocols on the Na+-Ca2+ exchange current (INa-Ca) of rabbit right ventricular myocytes were studied. Whole-cell patch-clamp recordings were made using a Cs+-based internal dialysis solution and external solutions designed to block major interfering currents. INa-Ca was measured at 35-37 degrees C as (5 mM) Ni-sensitive current elicited by: a 2 s descending ramp (DR: +80 to -120 mV); a 2 s ascending ramp (AR: -120 to +80 mV) and 500 ms voltage steps (VS) between -120 and +80 mV. DR and AR were applied from -40 mV and elicited INa-Ca with reversal potentials (Erev) of -17.6+/-2.5 mV (mean+/-SEM; n=16) and -46.2+/-4. 1 mV (n=10; P=0.0001) respectively. This difference was maintained when the holding potential was -80 mV (-44.0+/-2.1 mV, n=24 and -86. 3+/-4.8 mV, n=10; P=0.0001), when the internal Ca chelator (EGTA) was replaced with BAPTA (-19.5+/-1.8 mV and -46.3+/-1.6 mV, n=6; P=0. 0003) and when DR and AR were applied alternately to the same cell. Experiments using modified ramp waveforms suggested a possible mechanism for these differences. Increases in subsarcolemmal Ca caused by Ca entry (coupled to Na extrusion) during the initial positive potential phase of the DR might have induced INa-Ca reversal at less negative potentials than observed with AR, during the initial phase of which subsarcolemmal Ca would not have accumulated. These data suggest that INa-Ca during voltage-clamp experiments can be significantly influenced by the type of voltage protocol chosen, as the protocol appears to induce subsarcolemmal changes in Ca and Na concentration that are independent of Ca buffering in the bulk cytosol and can occur on a pulse-to-pulse basis.
Animals↗
Characteristics of a transient outward current (sensitive to 4-aminopyridine) in Ca2+-tolerant myocytes isolated from the rabbit atrioventricular node.
A transient outward current (Ito) has been observed in the atrioventricular node (AVN), but its characteristics in Ca-tolerant AVN myocytes have not been investigated previously. In this study, Ito was measured from Ca-tolerant rabbit AVN myocytes at 37 degrees C, using the whole-cell patch-clamp technique. With interfering currents inhibited, 500-ms voltage-clamp pulses applied from -80 mV elicited Ito at potentials positive to -30 mV, which increased in magnitude with test potential amplitude. This current was completely blocked by external application of 5 mM 4-aminopyridine (4-AP). During a command pulse, Ito activated rapidly then inactivated with a bi-exponential time-course. Fast and slow time constants of current inactivation (tauf and taus, respectively) showed voltage dependence. At 0 mV, tauf was 14.5+/-2.7 ms and taus was 112.8+/-21. 2 ms, whilst at +60 mV tauf was 6.7+/-1.1 ms and taus was 63.7+/-9.2 ms (n=25). The steady-state inactivation relationship showed half-maximal inactivation at -33.8 mV (n=8). Re-activation of Ito after an inactivating pre-pulse showed a bi-exponential time-course of recovery: tau1 was 196+/-70 ms, and tau2 was 2707+/-1010 ms (n=6, at -80 mV). Repetitive application of voltage-clamp test pulses showed that Ito inactivation accumulated on repetitive stimulation, but reached a steady state rapidly for a given pulse frequency (0. 2-1.0 Hz). AVN Ito was sensitive to the class 1 anti-arrhythmic flecainide (EC50 for peak current of 24 microM), which showed selectivity for the rapidly inactivating current component. Quinidine also inhibited Ito in a dose-dependent fashion, but did not affect the current time-course. Under voltage-clamp conditions, a simulated diastolic depolarisation from -70 to -45 mV did not significantly reduce Ito amplitude, and under current-clamp conditions 4-AP inhibited spontaneous action potentials. Although this is consistent with a significant role for Ito in shaping AVN activity, under the conditions of this study 4-AP also partially blocked the "rapid" delayed rectifier current, IKr, and so the effects of 4-AP on action potentials could not be attributed exclusively to its effects on Ito.
4-Aminopyridine↗
An investigation of the role played by the E-4031-sensitive (rapid delayed rectifier) potassium current in isolated rabbit atrioventricular nodal and ventricular myocytes.
The aim of this study was to measure and compare the profile of rapid delayed rectifier potassium current (IKr) elicited by action potential (AP) waveforms applied to isolated rabbit atrioventricular nodal (AVN) and ventricular myocytes. All measurements were made using whole-cell patch-clamp recordings at 37 degrees C. In AVN myocytes, IKr during voltage steps and slow ramp depolarisations showed "inward rectification" (characteristic for this channel) at positive potentials. The E-4031-sensitive current showed half-maximal activation at -10.8 +/- 0.86 mV, with a slope factor for the activation relation of 6.5 +/- 0.77 mV (n = 7). During AVN APs, IKr rapidly reached a peak after the AP upstroke and remained at similar amplitude until late in AP repolarisation. At the maximum diastolic potential following the AVN AP, a component of IKr remained which decayed during the pacemaker depolarisation, consistent with a role for the current in generating AVN pacemaker activity. In ventricular myocytes IKr was small at the beginning of the AP, and increased slowly during the AP plateau. Measurement of Ba-sensitive-inward rectifier K current (IK1) in ventricular myocytes revealed that IK1 rapidly increased during the final AP repolarisation phase, whilst IKr declined. It is concluded that IKr may participate in both AP repolarisation and the pacemaker depolarisation in AVN cells, whilst in ventricular myocytes, IKr and IK1 participate in controlling early and final AP repolarisation respectively.
Animals↗
Inhibition of the current of heterologously expressed HERG potassium channels by imipramine and amitriptyline.
1 Tricyclic antidepressants (TCAs) are associated with cardiovascular side effects including prolongation of the QT interval of the ECG. In this report we studied the effects of two TCAs (imipramine and amitriptyline) on ionic current mediated by cloned HERG potassium channels. 2 Voltage clamp measurements of HERG currents were made from CHO cells transiently transfected with HERG cDNA. HERG-encoded potassium channels were inhibited in a reversible manner by both imipramine and amitriptyline. HERG tail currents (IHERG) following test pulses to +20 mV were inhibited by imipramine with an IC50 of 3.4+/-0.4 microM (mean+/-s.e.mean) and a Hill coefficient of 1.17+/-0.03 (n = 5). 3 microM amitriptyline inhibited IHERG by 34+/-6% (n = 3). The inhibition showed only weak voltage dependence. 3 Using an 'envelope of tails' comprised of pulses to +20 mV of varying durations, the tau of activation was found to be 155+/-30 ms for control and 132+/-26 ms for 3 microM imipramine (n = 5). Once maximal channel activation was achieved after 320 ms (as demonstrated by maximal tail currents), further prolongation of depolarization did not increase imipramine-mediated HERG channel inhibition. 4 Taking current measurements every second during a 10 s depolarizing pulse from -80 mV to 0 mV, block was observed during the first pulse in the presence of imipramine and the level of IHERG block was similar throughout the pulse (n=5). 5 A three pulse protocol (two depolarizing pulses to +20 mV separated by 20 ms at -80 mV) revealed that imipramine did not significantly alter the kinetics of IHERG inactivation. The tau of inactivation was 8+/-2 ms and 5.6+/-0.4 ms (n = 5) in the absence and presence of 3 microM imipramine, respectively, and currents inactivated to a similar extent. 6 Our data are consistent with TCAs causing components of block of the HERG channel in both the closed and open states. Any component of open channel block occurs rapidly upon depolarization. Inhibition of IHERG by the prototype TCAs imipramine and amitriptyline may suggest a mechanism for QT prolongation associated with risks of arrhythmia and sudden death that accompany high concentrations of TCAs following overdose.
Amitriptyline↗
Effect of phosphorylation on L-type calcium current in ventricular myocytes dialysed with proteolytic enzymes.
1. L-Type Ca2+ channels play important roles in cardiac excitation and conduction. The present study used the whole-cell patch-clamp technique to investigate properties of Ca2+ channels in guinea-pig isolated ventricular myocytes. The effects of internal application of the proteolytic enzymes trypsin and carboxypeptidase (CBP) on the whole-cell L-type Ca2+ current (ICa) were determined. When the effects of the enzymes on ICa had reached steady state, the effects of isoprenaline (ISP) or 2,3-butane-dione monoxime (BDM), which increase and decrease channel phosphorylation, respectively, were examined. The effects of these agents were compared with those observed in the absence of enzyme pretreatment. 2. The amplitude and inactivation characteristics of ICa during depolarizing voltage-clamp commands to +10 mV (0.1 Hz) were determined at 37 degrees C. 3. Trypsin and CBP (both at concentrations of 1 mg/mL in the pipette solution) increased the amplitude of ICa 4.2- and 2.8-fold, respectively, and each enzyme increased the time constant of the slowly inactivating current by 50%. 4. Trypsin decreased the potential at which ICa was half maximally activated from (mean +/- SD) -1.4 +/- 2.2 mV (n = 9) to -11.3 +/- 2.5 mV (n = 7). Although CBP increased ICa amplitude, it did not shift the half-maximal activation voltage. Maximum conductance was increased 5.3-fold by trypsin and 2.2-fold by CBP. 5. Isoprenaline (1 mumol/L) had no effects in myocytes dialysed with trypsin, but significantly increased the current in myocytes dialysed with CBP by 8%. 6. At 12 mmol/L, BDM had no effect on current amplitude in the presence of trypsin, but decreased the time constant of slow inactivation to control values. After dialysis with CBP, BDM significantly decreased the maximum current by 11% and also decreased the rate of slow inactivation towards control values. 7. These data suggest that trypsin and CBP may have digested a part of the calcium channel that normally restricts current flow, but to different extents. The enzymes interacted with BDM and ISP in a fashion suggesting that two sites may influence the amplitude of the current and at least two other sites may influence the time course of the slowly inactivating current.
Animals↗
Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37 degrees C, dialysed internally with cAMP-free and cAMP-containing solutions.
In many mammalian tissue types an integral membrane protein--the sodium/calcium (Na/Ca) exchanger--plays a key role in intracellular Ca homeostasis, and evidence suggests that Na/Ca exchange function can be modulated by cAMP-dependent phosphorylation. External Nickel (Ni) ions are used widely to inhibit the exchange but little is known about the mode of Ni action. In guinea-pig ventricular myocytes, we investigated inhibition of Na/Ca exchange by external Ni under phosphorylated (cells dialysed with cAMP) and non-phosphorylated conditions. Ventricular myocytes were isolated from adult guinea-pig hearts, recordings were made at 37 degrees C using the whole-cell patch clamp technique. Internal and external solutions were used which allowed Na/Ca exchange current (INaCa) to be measured during a descending voltage ramp protocol (+80 to -120 mV) applied from a holding potential of -40 mV. The application of 10 mM Ni caused a maximal block of INaCa since inhibition was identical to that when a Na- and Ca-free (0Na/0Ca) solution was superfused externally. Kinetics of Ni-block of INaCa were assessed using applications of different external [Ni] to cells dialysed internally with cAMP-free and 100 microM cAMP-containing solutions. At +60 mV, Ni inhibited INaCa in cells dialysed with a cAMP-free solution with a dissociation constant (KD) of 0.29 +/- 0.03 mM and the data were fitted with a Hill coefficient of 0.89 +/- 0.07 (n = 9 cells). In cells dialysed with 100 microM cAMP the exchange was inhibited by Ni with a KD of 0.16 +/- 0.05 mM, the Hill coefficient was 0.82 +/- 0.16 (n = 6-7 cells). The KD and Hill coefficient values obtained in cells dialysed with cAMP-free and cAMP-containing solutions were not significantly different. Inhibition of INaCa by Ni did not appear to be voltage-dependent, was maximal within 3-4 s of application and was rapidly reversible. With cAMP-free internal dialysate, inhibition was 'mixed' showing competition with external Ca and a degree of non-competitive block. With 100 microM cAMP the inhibition appeared to be more non-competitive. We conclude that, under these experimental conditions, a concentration of external Ni of 10 mM is sufficient to produce maximal inhibition of INaCa in guinea-pig cardiac cells.
Animals↗
Alteration of HERG current profile during the cardiac ventricular action potential, following a pore mutation.
HERG is believed to encode the major sub-unit of the cardiac 'rapid' delayed rectifier K channel (I(Kr)). Both I(Kr) and HERG exhibit marked inward rectification at positive membrane potentials due to rapid inactivation and this is thought to influence significantly the contribution of the current to cardiac action potential (AP) repolarisation. We investigated directly the role played by rapid inactivation, by measuring current activated by a ventricular AP waveform, from Chinese Hamster Ovary cells transfected with HERG cDNA with a point-mutation (S631A) in the pore region. Square command pulses elicited HERG-S631A current which increased progressively in magnitude with test potential up to +30/+40 mV (n=6). During test pulses to +40mV, HERG-S631A exhibited little inactivation compared to wildtype HERG. During an action potential command, WT-HERG current developed progressively during the AP plateau and slow repolarisation phase, showing maximal current between -30mV and -40 mV (n=10). In contrast, HERG-S631A current increased earlier during the AP plateau, with a maximal amplitude near +30mV (n=7). Current then declined as the AP proceeded, giving rise to a 'bow'- or 'inverted-U-' shaped current profile. A mathematical model with inactivation removed from the HERG current reproduced the I-V profile of HERG-S631A. These data provide a direct demonstration that rapid inactivation normally plays a critical role in determining both time-course and voltage dependence of HERG/I(Kr) -current during the cardiac ventricular AP.
Action Potentials↗
Whole-cell membrane currents from human osteoblast-like cells.
Bone cells share common responses to external stimuli with most other cells. Among these are changes in membrane ion channel activity, although at present, relatively little is known about their nature or significance in human bone cells. Using the whole-cell configuration of the patch-clamp technique, we have revealed two types of membrane current in MG63 human osteoblast-like cells. With a potassium-based dialysis solution and a holding potential of -40 mV, voltage commands to more negative potentials elicited an inward current. This current showed little inactivation with time during the command pulse and exhibited some characteristics of an inwardly rectifying K current, including sensitivity to external K and Ba. The second type of current was outward, activated by depolarizing pulses from -40 mV. This current was transient in nature, activating in the first 50 ms of the pulse and then showing rapid inactivation to reach a steady-state level after 4 to 5 seconds. The transient outward current was sensitive to block by TEA, CTX, and to a lesser extent, Ba. These data suggest that a large proportion of this outward current is carried by K ions through channels that may be sensitive to the internal Ca ion concentration. The transient outward current was enhanced by setting the holding potential at -100 mV, and greatly inactivated by setting it at 0 mV. Increased understanding of the significance of these membrane currents may allow development and use of agents to modulate their action and therefore influence bone cell behavior in disease states such as osteoporosis.
4-Aminopyridine↗
Actions of myristyl-FRCRCFa, a cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchanger, tested in rabbit ventricular myocytes.
In cardiac muscle, the electrogenic Na-Ca exchanger plays important roles in determining action potential shape and in the beat-to-beat homeostasis of intracellular calcium. In this study we tested the actions of a putative cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchange, "Myristyl- (Myr-) FRCRCFa". Experiments were performed using isolated rabbit right ventricular myocytes and whole-cell patch-clamp at 35-37 degreesC. The Na-Ca exchange current (INa-Ca), L-type calcium current (ICa,L), inward rectifier potassium current (IK1) and delayed rectifier potassium current (IK) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. With other major currents blocked, INa-Ca was measured as the Ni-sensitive component of current during a voltage ramp applied from the holding potential of -40 mV, between +80 and -120 mV (ramp velocity 0.1 V s-1). In untreated cells, INa-Ca at +60 mV was 7.1+/-0.6 pA/pF and at -100 mV was -2.7+/-0.3 pA/pF (n=9). After a 15-min pre-incubation with 20 microM Myr-FRCRCFa, INa-Ca was reduced to 4.2+/-0.3 pA/pF at +60 mV and -1. 5+/-0.2 pA/pF at -100 mV (P<0.02; n=7). After incubation with 20 microM Myr-FRCRCFa for 1 h, INa-Ca at both potentials was further reduced (2.3+/-0.8 pA/pF at +60 mV; -0.9+/-0.3 pA/pF at -100 mV; P<0. 008 compared with control; n=4). Under selective recording conditions for ICa,L, there was little difference in ICa,L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the ICa,L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. IK1 was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. IK, measured as deactivating tail current following 1-s test depolarisations to a range of test potentials, was also not significantly altered by Myr-FRCRCFa. The suppression of INa-Ca in cells incubated in Myr-FRCRCFa suggests that addition of the myristyl group to FRCRCFa peptide conveys cell permeancy to the peptide and that Myr-FRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an INa-Ca blocker. ICa,L, IK1 and IK were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained hexapeptides may, therefore, provide a means of producing cell-permeant inhibitors of the cardiac Na-Ca exchanger.
Animals↗
Time course and voltage dependence of expressed HERG current compared with native "rapid" delayed rectifier K current during the cardiac ventricular action potential.
It is widely believed that HERG (human ether-a-go-go-related gene) encodes the major subunit of the cardiac "rapid" delayed rectifier K channel. The aims of the present study were threefold: (1) to record directly the time course and voltage dependence of expressed HERG current in a mammalian cell line, during an imposed ventricular action potential (AP); (2) to compare this with native rapid delayed rectifier current (IKr) elicited by applying an AP command to isolated guinea-pig ventricular myocytes; (3) to provide mechanistic information regarding the profile of HERG/IKr during the AP. We used the AP clamp technique and conventional whole-cell patch-clamp recordings at 32-34 degreesC. HERG was transiently expressed in Chinese hamster ovary (CHO) cells. There was an outward current in transfected CHO cells, which developed progressively during the AP plateau and slow repolarisation phase. The instantaneous current-voltage (I-V) relation for both leak-subtracted HERG current (n=10) and E-4031-sensitive current (n=6) during AP repolarisation was maximal between -30 mV and -40 mV. The conductance-voltage (G-V) relation was maximal at potentials between -60 and -75 mV. A similar voltage dependence for HERG current was observed during a descending ramp from +60 to -80 mV (n=5), but not during either an ascending ramp (n=5), or a reversed AP waveform (n=8). These data suggest that instantaneous HERG current during the AP does not depend on the instantaneous command voltage alone, but upon the previous voltages during the applied waveform. The time course of activation of HERG current at potentials near the AP plateau was rapid. Tail currents recorded on premature repolarisation at different time points in the AP showed directly that HERG also activates rapidly during the AP. The I-V profiles of fully activated HERG and of current during the AP were very similar. IKr from guinea-pig ventricular myocytes was measured as E-4031-sensitive current during the AP clamp command. The current had a similar I-V and G-V profile to HERG current in CHO cells. These data indicate that HERG current and native IKr are similar during an applied AP waveform. Activation of HERG is rapid during the AP. However, due to rapid inactivation relatively little current flows until the potential becomes less positive than 0 mV. The removal of inactivation then allows more current to flow, giving rise to the distinct instantaneous I-V profile during the AP. The correlation between the voltage dependence of HERG during the AP and the fully activated I-V relation indicates that the contribution of HERG/IKr to AP repolarisation is more significantly determined by the open-channel I-V relation, than the precise activation time course of the current.
Action Potentials↗
Cultured adult cardiac myocytes: future applications, culture methods, morphological and electrophysiological properties.
Isolated adult cardiac myocytes maintained in primary culture have been used as a model of the adult myocardium for 20 years. With the recent advances and current interest in using molecular biological techniques to investigate cardiac physiology, culturing myocytes is becoming an increasingly important technique. Acutely isolated myocytes do not remain viable for the time needed for the changes in gene expression to occur, and therefore it is necessary to maintain myocytes in culture. The aims of this review are: (1) To describe a method for isolating and culturing myocytes in serum-free medium. This section is targeted at new researchers in the field, with particular emphasis on aspects of the isolation procedure which are important for optimising myocyte culture. (2) To review current knowledge of how contractile, electrophysiological and morphological properties of adult myocytes are preserved in culture. Over the past 5 to 10 years significant advances have been made in developing novel techniques which help maintain the in-vivo properties of myocytes in culture. Efficient methods for transporting exogenous genes and anti-sense oligonucleotides into adult myocytes are now available. We anticipate that in future these advances will make cultured myocytes more attractive for use in biophysical and molecular investigations of cardiac physiology.
Animals↗
Voltage dependence of the Fura-2 transient in rabbit left atrial myocytes at 37 degrees C.
We used the whole-cell patch-clamp technique and monitoring of Fura-2 fluorescence to investigate the voltage dependence of the L-type Ca current (ICa,L) and intracellular Ca (Cai) transient in rabbit atrial myocytes at 37 degrees C. Imaging the atrial cell membrane with Di-4-ANNEPS showed (in contrast to ventricular cells) that atrial cells had very few transverse tubules. We measured ICa,L using a Cs-based internal dialysis solution to eliminate interfering K currents. The voltage dependence of peak ICa,L amplitude was bell-shaped: ICa,L was maximal at +10 mV and declined at more negative and positive potentials. For measuring the Fura-2 (Cai) transient, we used a K-based internal dialysis solution to preserve normal excitation-contraction coupling. Ryanodine (20 microM) plus thapsigargin (2 microM) (blockers of the sarcoplasmic reticulum, SR) abolished the phasic component of the Fura-2 transient (n = 5), demonstrating that the phasic Fura-2 transient provided an index of the magnitude of SR release. The Fura-2 transient also showed bell-shaped voltage dependence, but this was different from that for ICa,L. The Fura-2 transient peaked at +30 mV and partially declined at more positive potentials; but at potentials where inward ICa,L was small (if not absent), the phasic Fura-2 transient still attained a significant amplitude. We used a rapid application of nifedipine (32 microM), and of nifedipine plus 5 mM Ni, to assess the ability of ICa,L and reverse-mode Na-Ca exchange to trigger SR Ca release. With test pulses to +10 mV and +60 mV, a rapid switch to nifedipine (which blocked ICa,L) produced no significant reduction in phasic Fura-2 transient amplitude. This suggests that in the absence of ICa,L, another mechanism was able to trigger SR release. With pulses to +10 and +60 mV, a single beat switch to nifedipine plus 5 mM Ni almost completely abolished the phasic transient. Since 5 mM Ni inhibits Na-Ca exchange, this suggests that, in the absence of ICa,L, trigger Ca entry via reverse Na-Ca exchange was able to activate SR Ca release in atrial cells at 37 degrees C. The mechanisms underlying the Fura-2 transient in atrial cells, and differences with pre-existing data from rabbit ventricular cells, are discussed.
Animals↗
Modulation by mexiletine of action potentials, L-type Ca current and delayed rectifier K current recorded from isolated rabbit atrioventricular nodal myocytes.
Using whole-cell patch clamp recordings at 37 degrees C, we have examined the effects of externally applied mexiletine (a class 1b antiarrhythmic agent) on action potentials, L-type Ca current (ICa, L) and delayed rectifier K current (IK) in single isolated rabbit atrioventricular nodal (AVN) myocytes. In spontaneously active AVN cells, 30-100 micro;M mexiletine depolarised the maximum diastolic potential and slowed both action potential upstroke and repolarisation. Under selective recording conditions for ICa,L, mexiletine reduced peak ICa,L (at +10 mV) amplitude in a dose-dependent fashion (41.8 +/- 3.0% inhibition by 100 micro;M and 16.4 +/- 1.8% at 30 micro;M). The voltage dependence of ICa,L activation was unaffected by both concentrations of the drug. Under selective recording conditions, IK amplitude was measured as the peak of the deactivating tail current following a depolarising voltage pulse to +20 mV. 30 micro;M mexiletine inhibited IK by 34.3 +/- 5.8%, whilst 100 micro;M mexiletine reduced the current by 52.7 +/- 6.1%. The effects of mexiletine on ICa,L and IK are likely to contribute significantly to the changes in action potentials observed in spontaneously active cells. These findings are also suggestive of key roles for ICa,L and IK in determining the shape and rate of action potentials in this region of the heart.
Action Potentials↗
"Voltage-activated Ca release" in rabbit, rat and guinea-pig cardiac myocytes, and modulation by internal cAMP.
It is widely believed that Ca release from the sarcoplasmic reticulum (SR) in heart muscle is due to "Ca-induced Ca-release" (CICR), triggered by transmembrane Ca entry. However, in intact guinea-pig cells or cells dialysed with cAMP there may be an additional mechanism - SR release may be activated directly by membrane depolarisation without Ca entry. The first objective of the present study was to investigate whether this "voltage-activated Ca release" (VACR) mechanism is present across species such as rabbit, rat and guinea-pig. The second objective was to characterise the dependence of a VACR mechanism on internal [cAMP]. Membrane current was measured with the whole-cell patch-clamp technique, intracellular [Ca] was monitored with Fura-2 (or a combination of Fluo-3/SNARF-1). Rapid changes of superfusate (within 100 ms) were made using a system which maintained cell temperature at 37 degrees C. We used a train of conditioning pulses to ensure a standard SR load before each test pulse. In rabbit myocytes dialysed with 100 microM cAMP, 89.6 +/- 7.0% of the control intracellular Ca (Cai) transient was still elicited by depolarisation during a switch to 5 mM Ni, which blocked pathways for Ca entry. This suggested that rabbit myocytes possess a VACR mechanism. The percentage of control Cai transient elicited by depolarisation in the presence of 5 mM Ni (i.e. magnitude of VACR) increased in a graded fashion with the pipette [cAMP] between zero and 100 microM. In rat myocytes dialysed with 50 microM cAMP, 64.4 +/- 6.2% of SR release was activated by depolarisation in the presence of 5 mM Ni, suggesting the presence of a VACR mechanism. The extent to which VACR triggered SR release increased with the pipette [cAMP] between zero and 50 microM. In guinea-pig myocytes dialysed with 100 microM cAMP, 74.6 +/- 3.6% of the control Cai transient was elicited by depolarisation in the presence of 5 mM Ni. The degree to which VACR triggered SR release was also graded with the pipette [cAMP] between zero and 100 microM. It therefore appears that each of the three species might possess a VACR mechanism which can be modulated by the internal [cAMP]. This may reflect an effect of cAMP to phosphorylate key proteins involved in excitation-contraction coupling. Under normal physiological conditions with a basal [cAMP] between 2 and 20 microM, VACR may play a role in triggering SR release. The role of VACR may increase under conditions which increase internal [cAMP].
Animals↗
Amiodarone blocks L-type calcium current in single myocytes isolated from the rabbit atrioventricular node.
1. Single myocytes were isolated from the rabbit atrioventricular node (AVN) and whole cell patch clamped, using a Cs(+)-based internal dialysis solution. Depolarizing voltage clamps from a holding potential of -40 mV were applied to investigate the effects of the antiarrhythmic agent amiodarone on L-type calcium current (ICa,L). 2. The current-voltage (I-V) relation for ICa,L was bell shaped in normal Tyrode's solution, with a peak at +10 mV. After a 1-min exposure to 10 microM amiodarone, ICa,L at all potentials between -30 mV and +60 mV was largely and reversibly blocked (n = 4). In 13 cells, peak ICa,L was blocked by 85% after amiodarone application. 3. The time course of the blocking effect was monitored during continuous pulsing (at 0.33 Hz) from -40 mV to +10 mV to observe the time course of ICa,L blockade. This could be described by a single exponential function with a rate constant of 0.21 per second. In other cells, amiodarone was applied for 30 sec without any stimulation; and, when stimulation was resumed, the ICa,L amplitude with the first pulse was 22% of the control amplitude (n = 4), indicating that a substantial portion of the blockade of ICa,L by amiodarone was tonic in nature. 4. In addition to the effect of amiodarone on the ICa,L channel, when the beta adrenergic agonist isoprenaline (1 microM) was applied to cells that had been pretreated with amiodarone, ICa,L amplitude was increased by 44.5 +/- 10.8% (n = 4). In cells that had received no such pretreatment with amiodarone, isoprenaline increased the ICa,L amplitude by 101.5 +/- 5.9% (n = 4). Thus, isoprenaline produced a larger increase in ICa,L in the absence of amiodarone than in its presence (P < 0.001). 5. It is concluded that, in single AVN cells, amiodarone exerts a direct ICa,L blocking action. In addition, the attenuated increase in ICa,L with isoprenaline in amiodarone pretreated cells appears consistent with an antagonism of beta adrenergic stimulation of ICa,L.
Adrenergic beta-Agonists↗
Inhibition of L-type calcium current by propafenone in single myocytes isolated from the rabbit atrioventricular node.
1. The atrioventricular node (AVN) is an important part of the conduction system in the heart and is a significant site of antiarrhythmic drug action. The class 1 antiarrhythmic propafenone is effective in treating a variety of arrhythmias, including those involving the AVN. In this study, we have investigated the effects of propafenone on ionic currents in single rabbit AVN cells, focusing in particular on those on L-type calcium current (ICa,L). 2. With a standard K-based internal dialysis solution, exposure to 5 microM propafenone reduced significantly the amplitude of ICa,L. In spontaneously active AVN myocytes, action potential upstroke velocity was decreased by propafenone exposure, consistent with the observed change in ICa,L. 3. By use of a Cs-based internal dialysis solution to record ICa,L selectively, voltage clamp test pulses were applied from a holding potential of -40 mV to +10 mV (stimulation frequency 0.33 Hz). Propafenone 5 microM reduced mean ICa,L density at +10 mV from -9.58 +/- 1.05 pA/pF to -4.19 +/- 0.60 pA/pF (P < 0.002). A range of propafenone concentrations were applied which reduced ICa,L in a dose-dependent manner (IC50 1.7 microM). When test pulses were applied to a range of potentials, propafenone reduced ICa,L at each potential without significantly affecting the activation curve for this current. Thus, propafenone reduced ICa,L conductance, without affecting the voltage-dependent activation properties of the current. 4. ICa,L block by propafenone exhibited tonic-, use- and frequency-dependent characteristics. 5. In the presence of propafenone, the voltage-dependence of inactivation of ICa,L was shifted 8 mV in the hyperpolarizing direction. Also, the recovery of ICa,L from inactivation was slowed by propafenone. 6. The ICa,L blocking properties of propafenone may mediate some of the antiarrhythmic properties of this agent, particularly in regions of the heart such as the AVN in which ICa,L contributes significantly to the action potential upstroke.
Action Potentials↗