Ionic mechanisms responsible for the electrocardiographic phenotype of the Brugada syndrome are temperature dependent.

The Brugada syndrome is a major cause of sudden death, particularly among young men of Southeast Asian and Japanese origin. The syndrome is characterized electrocardiographically by an ST-segment elevation in V1 through V3 and a rapid polymorphic ventricular tachycardia that can degenerate into ventricular fibrillation. Our group recently linked the disease to mutations in SCN5A, the gene encoding for the alpha subunit of the cardiac sodium channel. When heterologously expressed in frog oocytes, electrophysiological data recorded from the Thr1620Met missense mutant failed to adequately explain the electrocardiographic phenotype. Therefore, we sought to further characterize the electrophysiology of this mutant. We hypothesized that at more physiological temperatures, the missense mutation may change the gating of the sodium channel such that the net outward current is dramatically augmented during the early phases of the right ventricular action potential. In the present study, we test this hypothesis by expressing Thr1620Met in a mammalian cell line, using the patch-clamp technique to study the currents at 32 degrees C. Our results indicate that Thr1620Met current decay kinetics are faster when compared with the wild type at 32 degrees C. Recovery from inactivation was slower for Thr1620Met at 32 degrees C, and steady-state activation was significantly shifted. Our findings explain the features of the ECG of Brugada patients, illustrate for the first time a cardiac sodium channel mutation of which the arrhythmogenicity is revealed only at temperatures approaching the physiological range, and suggest that some patients may be more at risk during febrile states.

Electrophysiologic aspects of moricizine HCl.

Moricizine HCl, a phenothiazine derivative, is a new antiarrhythmic drug that has quinidine-like effects on the myocardium. Moricizine HCl, 1 X 10(-7) g/ml, significantly decreases Vmax of the transmembrane action potential of canine Purkinje fibers and reduces the duration of the action potential at 50% and 90% of repolarization, whereas resting potential is unchanged. The drug decreases the fast inward sodium current (lNa), as measured by the double sucrose gap technique in frog atrium trabeculae; the processes of activation, inactivation and reactivation of current did not change even when a high concentration of drug (5 X 10(-6) g/ml) was used. At the same dose, the slow inward calcium current (lCa) increased, but the total outward current did not change. Using the patch-clamp technique, it was shown that moricizine HCl, 1 X 10(-4) g/ml, did not alter single-channel conductance, but essentially decreased the mean open tonic and open-state probability of potassium channels either at positive or negative holding potentials. lNa measured in a single myocyte preparation decreased faster when drug was administered in the external bath compared with intracellular injection. Moricizine HCl action on lNa of the single cell and on Vmax of the action potential is frequency dependent. When lNa was recorded directly, there was a cumulative effect of drug. Similar to other quinidine-like agents, moricizine HCl enhanced arrhythmogenicity within the first few minutes after coronary artery occlusion, but prevented arrhythmias 24 hours after acute myocardial infarction.(ABSTRACT TRUNCATED AT 250 WORDS)

[The combined effect of 2 first-line anti-arrhythmia drugs on the conduction velocity of myocardial stimulation]. / Deistvie kombinatsii dvukh antiaritmicheskikh preparatov I klassa na skorost' provedeniia vozbuzhdeniia po miokardu.

Experiments with recording the time of intraventricular reentry of the canine heart by a special protocol of stimulation showed that the first-line antiarrhythmic agents having various kinetics, ethacizine and lidocaine, compete for their binding to the Na+ channels of cardiac fibers. As a result of this competition, additivity was not found in the effects of ethacizine, 1.5 mg/kg, and lidocaine, 12 mg/kg, on the rate of intraventricular conduction at heart rate under 180/min. At higher cardiac rhythm values, the total effects of the two agents on the conduction were observed. The experimental evidence confirm the results of mathematical modelling of the combined effects of ethacizine and lidocaine on the intraventricular conduction velocity, which were calculated on the basis of kinetic constants for binding and dissociation of the agents to Na+ channels. The findings show that when the two first-line antiarrhythmics having a substantially different kinetics were used in combination, their antiarrhythmic effect may be enhanced without their total effects on the rate of normal intraventricular conduction velocity reentry.

[Effect of creatine phosphate on the slow inward calcium current, action potentials and the strength of myocardial contraction]. / Vliianie kreatinfosfata na medlennyi vnutr' napravlennyi kal'tsievyi tok, potentsial deistviia i silu sokrashcheniia miokarda.

The effect of creatine phosphate on the frog heart muscle involved maintenance of its contraction at high level even after inhibition of mitochondrial oxidative phosphorylation by sodium cyanide. The effect of creatine phosphate on the contractile force and action potential is similar for the frog heart ventricle and atrium. The voltage--clamp technique showed that creatine phosphate controlled the slow inward calcium current through the surface membrane of the frog atrium cells.

[Effect of a diethylamino analog of ethmozine on the force of contraction and action potential of the guinea pig myocardium]. / Vliianie diétilaminovogo analoga étmozina na silu sokrashcheniia i potentsial deistviia miokarda morskoi svinki.

Ethacizine applied in the concentration range between 1 X 10(-7) and 1 X 10(-5) g/ml decreased the rate of action potential growth (Vmax) of the mammal myocardium. Inhibition of the Vmax was accompanied by the diminution of the force of contraction which involved two phases. Ethacizine also decreased the overshoot of slow response action potential with the time constant similar to that in the first rapid phase of force reduction. It is assumed that the first rapid phase occurred due to the blockade of the slow calcium channels, while the second slow phase resulted from the inhibition of Na--Ca exchange because of the decrease of intracellular Na+ concentration after the blockade of the fast sodium channels by ethacizine.

[Effect of ethmozine on action potentials and myocardial contraction in guinea pigs]. / Vliianie étmozina na potentsial deistviia i silu sokrashcheniia miokarda morskoi svinki.

Ethmozine decreased the maximum rate of action potential rise (Vmax) in a dose-dependent manner. Using the Scatchard plot the apparent dissociation constant was calculated to be 1.52 X 10(-5) g/ml. Ethmozine also decreased the force of contraction in the concentration range between 1 X 10(-6) and 1 X 10(-4) g/ml with the apparent dissociation constant obtained from the Scatchard plot being equal to 1.48 X 10(-5) g/ml. The linear correlation coefficient between the decrease in Vmax and the decrease in the force of contraction was found to be equal to 0.998. Negative inotropic action of ethmozine was less pronounced when the stimulation frequency had been switched from 0.8 to 0.1 Hz. The decrease in Vmax under the action of ethmozine (3 X 10(-5) g/ml) was diminished from 56 +/- 7% (0.8 Hz) to only 3 +/- 8% (0.1 Hz). This was accompanied by the decrease in the negative inotropic effect: from 58 +/- 9% (0.8 Hz) to 16 +/- 15% (0.1 Hz). It was assumed that the negative inotropic action of ethmozine was mediated by the Na--Ca exchange, which was inhibited by the decrease of the intracellular Na+ concentration due to the blockade of sodium channels by ethmozine.

[Decreased sodium conductance of the cardiomyocyte membrane: the cause of the negative inotropic action of quinidine-like anti-arrhythmia agents]. / Umen'shenie natrievoi provodimosti membrany kardiomiotsitov--prichina otritsatel'nogo inotropnogo deistviia khinidinopodobnykh antiaritmicheskikh sredstv.

The action of tetrodotoxin on the guinea-pig muscle caused a decrease in the maximum rate of action potential, which was accompanied by a negative inotropic effect. The magnitude of such an effect correlated well with the decrease of sodium permeability, measured by the maximum rate of depolarization (r = 0,995). The effect was enhanced under the action of veratrine and diminished under that of isoproterenol. It was suggested that the negative inotropic effect of tetrodotoxin was the result of the inhibition on Na-Ca exchange due to diminished intracellular Na+ concentration.

Transmural heterogeneity of ventricular repolarization under baseline and long QT conditions in the canine heart in vivo: torsades de pointes develops with halothane but not pentobarbital anesthesia.

INTRODUCTION: In vitro studies have provided evidence for the existence of M cells. The present study examines the contribution of the M cell to transmural dispersion of repolarization (TDR) and to the development of torsades de pointes (TdP) in the canine heart in vivo in animals anesthetized with either pentobarbital or halothane. METHODS AND RESULTS: Monophasic action potentials (MAPs) were recorded from 4 to 7 transmural sites, before and after d-sotalol. Cells displaying the longest MAP duration (MAPD) generally were localized to the deep subendocardium to mid-myocardium (M region) in the anterior wall of the left ventricle. d-Sotalol preferentially prolonged the MAPD of the M region, increasing TDR significantly more (P < 0.05) in animals anesthetized with halothane (31+/-5 to 88+/-17 msec) than in those receiving pentobarbital (24+/-9 to 53+/-7 msec; basic cycle length 1,500 msec). In halothane-anesthetized dogs, a remarkable transient increase in M cell MAPD followed interpolation of one or more extrasystole(s), leading to a transient increase in TDR and TdP. TdP was never observed with pentobarbital anesthesia. CONCLUSION: Our results demonstrate that transmural heterogeneity of repolarization is amplified under acquired long QT conditions and that the increase in TDR underlies the development of TdP in halothane- but not pentobarbital-anesthetized dogs. The data support an important contribution of M cells to TDR and to the development of TdP in the canine heart in vivo. Our data also highlight the importance of acceleration-induced prolongation of MAPD (a phenomena observed principally in M cells) in the development of TdP.

A proarrhythmic response to sodium channel blockade: modulation of the vulnerable period in guinea pig ventricular myocardium.

The vulnerable period (VP) is an interval of time during the cardiac cycle within which premature stimulation may lead to trains of responses (one: many stimulus-response coupling). Although the VP parallels the recovery of sodium channel availability, modulators of its boundaries remain unclear. Numerical studies of a uniform cable demonstrated that reduction in sodium channel availability increased the range of premature stimuli, resulting in unidirectional block, a precursor of reentrant activation. Consequently, we hypothesized that the kinetics of use-dependent sodium channel blockade could reflect one dimension of a drug's proarrhythmic potential. In strips from guinea pig right ventricle, we probed the boundaries of the VP in the presence of use-dependent sodium channel antagonists utilizing a train of stimuli followed by a premature stimulus. Under drug-free conditions when the sites of drive and premature stimulation were the same, the VP was less than 4 ms in duration. When the drive and premature sites were different, the drug-free VP was greater than 5 ms in 22 of 24 preparations and 0 in the other two, with an average VP duration of 16 +/- 10 ms (mean +/- SD). In the presence of 1 microM moricizine, VP = 17 +/- 4 ms; 12 microM moricizine, VP = 35 +/- 4 ms; 3 microM flecainide, VP = 50 +/- 17 ms; and 4 microM quinidine, VP = 2 +/- 1 ms. These results suggest that residual unsuppressed premature ventricular contractions (PVCs) in the presence of some class 1 drugs have a greater potential for initiating a proarrhythmic response than PVCs in the absence of a class 1 drug.

[Effect of the ionophore A23187 on the contractility and slow action potential of the guinea pig papillary muscle]. / Deistvie ionofora A23187 na silu sokrashcheniia i medlennyi potentsial deistviia papilliarnoi myshtsy morskoi svinki.

Calcium ionophore A23187 (2.10(-6) M) increases contractility of the isolated guinea-pig papillary muscle 3.13 +/- +/- 0.47-fold at 25 degrees C and 1.88 +/- 0.28-fold at 37 degrees C. Prolonged exposure (30 min) to 2 mM of caffeine failed to eliminate positive inotropic effect of ionophore. A23187 (2.10(-6) M) also increased slow response action potential overshoot (18 mM K+ in Tyrode solution, resting potential 46 +/- 1 mV); at 37 degrees C the peak overshoot value was reached by the 2nd min, being equal to 5.2 +/- 0.4 mV; at 25 degrees C the peak value was attained by the 15th min, being equal to 15.5 +/- 0.9 mV with an abrupt increase in the repolarization rate. Overshoot enhancement within the first minutes of A23187 action is concluded to be due to the increased slow inward Ca-current rather than to the decreased outward currents. It is assumed that A23187 facilitates Ca2+ transport to the lipid phase thereby increasing the amount of sarcolemma bound Ca2+ that participates in the development of Ca-current.

Proarrhythmic response to sodium channel blockade. Theoretical model and numerical experiments.

BACKGROUND: The use of flecainide and encainide was terminated in the Cardiac Arrhythmia Suppression Trial because of an excess of sudden cardiac deaths in the active treatment group. Such events might arise from reentrant rhythms initiated by premature stimulation in the presence of anisotropic sodium channel availability. Drugs that bind to sodium channels increase the functional dispersion of refractoriness by slowing (a result of the drug-unbinding process) the transition from an inexcitable state to an excitable state. It is interesting that encainide and flecainide unbind slowly (15-20 seconds), whereas lidocaine and moricizine unbind rapidly (0.2-1.3 seconds). METHODS AND RESULTS: With a computer representation of a cable with Beeler-Reuter membrane properties, we found a small (6 msec) vulnerable window that occurred 338 msec after the last drive stimulus. Premature stimuli falling within the vulnerable window resulted in unidirectional block and reentrant activation. In the presence of a slowly unbinding drug, the window was delayed an additional 341 msec, and its duration was extended to 38 msec. The delay (antiarrhythmic effect) before the onset of the vulnerable window and its duration (proarrhythmic effect) were both dependent on the sodium channel availability and the recovery process. Both effects were also prolonged when sodium channel availability was reduced by membrane depolarization. Defining the proarrhythmic potential as the duration of the vulnerable window, we found that hypothetical use-dependent class I drugs have a greater proarrhythmic potential than non-use-dependent drugs. CONCLUSIONS: The antiarrhythmic and proarrhythmic properties of pure sodium channel antagonists are both dependent on sodium channel availability. Consequently, the price for increased antiarrhythmic efficacy (suppressed premature ventricular contractions) is an increased proarrhythmic vulnerability to unsuppressed premature ventricular contractions.

Use of ionic currents to identify and estimate parameters in models of channel blockade.

Models of ion channel blockade are frequently validated with observations of ionic currents resulting from electrical or chemical stimulation. Model parameters for some models (modulated receptor hypothesis) cannot be uniquely determined from ionic currents. The time course of ionic currents reflects the activation (fraction of available channels that conduct in the presence of excitation) and availability of channels (the ability of the protein to make a transition to a conducting conformation and where this conformation is not complexed with a drug). In the presence of a channel blocking agent, the voltage dependence of availability appears modified and has been interpreted as evidence that drug-complexed channels exhibit modified transition rates between channel protein conformations. Because blockade and availability both modify ionic currents, their individual contributions to macroscopic conductance cannot be resolved from ionic currents except when constant affinity binding to a bindable site is assumed. Experimental studies of nimodipine block of calcium channels and lidocaine block of sodium channels illustrate these concepts.

Larger late sodium conductance in M cells contributes to electrical heterogeneity in canine ventricle.

Action potentials and whole cell sodium current were recorded in canine epicardial, midmyocardial, and endocardial myocytes in normal sodium at 37 degrees C. Tetrodotoxin (TTX) reduced the action potential duration of midmyocardial cells to a greater degree than either epicardial or endocardial cells. Whole cell recordings in potassium-free and very-low-chloride solutions revealed a slowly decaying current that was completely inhibited by 5 microM TTX or replacement of external and internal sodium with the impermeant cation N-methyl-D-glucamine. Late sodium current density at 0 mV was 47% greater in midmyocardial cells and averaged -0.532 +/- 0.058 pA/pF in endocardial, -0.463 +/- 0.068 pA/pF in epicardial, and -0.785 +/- 0.070 pA/pF in midmyocardial cells. Neither the frequency dependence of late sodium current nor its recovery from inactivation exhibited transmural differences. After a 4.5-s pulse to -30 mV, late sodium current recovered with a single time constant of 140 ms. We conclude that a larger late sodium conductance in midmyocardial cells will favor longer action potentials in these cells. More importantly, drugs that slow inactivation of sodium channels will produce a nonuniform response across the ventricular wall that is proarrhythmic.

Ethacizin blockade of calcium channels: a test of the guarded receptor hypothesis.

The effect on calcium channels of the sodium channel antagonist, ethacizin, was studied in isolated frog ventricular cells using the whole cell voltage-clamp methodology. Ethacizin was found to block inward calcium current in a frequency-, voltage-, and concentration-dependent manner. The frequency-dependent blocking properties were modeled by considering the drug interaction with a voltage-dependent mixture of calcium channels harboring either an accessible or an inaccessible binding site. With repetitive stimulation, the pulse-to-pulse reduction in peak current is shown to be exponential, with a rate linearly related to the interstimulus interval and the drug concentration. Observed frequency- and concentration-dependent blocks were consistent with the predictions of the model, and mixture-specific rate constants were estimated from these data. The negligible shift in channel inactivation and the reduction of apparent binding and unbinding rates with more polarized membrane potentials imply the active moiety of ethacizin blocks open channels and is trapped within the channel at resting membrane potentials. The binding rate at 0 mV is similar to that observed in studies of interactions of other open channel blocking agents with voltage- and ligand-gated channels.

The role of inactivation in open-channel block of the sodium channel: studies with inactivation-deficient mutant channels.

Inactivation has been implicated as an important determinant of the block of Na+ channel by local anesthetic-class drugs. This proposition has been difficult to examine because agents used to modify inactivation change other channel properties and both inactivated and blocked channels do not conduct. We used site-directed mutagenesis of Phe1304 to glutamine in the linker between the third and fourth domains of the mu-1 Na+ channel to slow inactivation. Wild-type and mutant channels were expressed in frog oocytes. Macropatch and single-channel currents were recorded in cell-attached membrane patches. The F1304Q mutation increased mean open time (1.7 fold at -20 mV) and reduced the probability that the channel would fail to open. Closed times were best fit by a double-exponential function, suggesting that the inactivated state transitions were no longer absorbing. In wild-type channels, 100 microM disopyramide decreased mean open time from 1.64 +/- 0.08 to 0.34 +/- 0.04 msec. Total open time per trial was decreased 2-fold. There also was a marked increase in the fraction of null sweeps. In the inactivation-deficient mutant channel, mean and total open times were also reduced. These data indicate that even when inactivation is slowed by a localized specific mutation, open-channel block by disopyramide persists. Inactivation may not be a necessary requirement for open-channel block.

Lidocaine blockade of continuously and transiently accessible sites in cardiac sodium channels.

Lidocaine binds to sodium channels in a voltage dependent manner where depolarization enhances block and hyperpolarization relieves block. Voltage--clamp studies demonstrate that there are two components of block: one involving interaction with a binding site that is accessible for the duration of a depolarizing clamp (continuous access or availability) and one involving interaction with a site that is transiently available or accessible during transitions between polarized and depolarized potentials. Here we report results demonstrating two distinct voltage dependencies of blockade. The voltage dependence of block of the transiently accessible site is similar to that of channel activation and exhibits a maximal binding rate of 1.37 x 10(6)/M/S and an unbinding rate of 39.5/s at -30 mV. Blockade of the sustained site exhibits a voltage dependence similar to inactivation with a maximal binding rate of 3.59 x 10(4)/M/S and an unbinding rate of 0.678/s at -30 mV. Recovery from blockade acquired by either process is voltage dependent and proportional to exp(-0.037 Vm). Drug induced shifts in channel availability and transient site block are accurately predicted from kinetic rates estimated from frequency dependent protocols.

State dependence of ethacizin and ethmozin block of sodium current in voltage clamped and internally perfused cardiac Purkinje cells.

Ethacizin, a positively charged analog of ethmozin, reduces the cardiac action potential upstroke and blocks peak sodium current (INa). We investigated ethacizin block of INa in 11 cells and ethmozin block in 4 cells at 20 degrees C. Rest block measured as the relative INa decrease for the first pulse in drug after 3 to 6 min at the holding potential was negligible for ethacizin but substantial (16% at 5 microM) for ethmozin. Use-dependent block developed exponentially; the time course of block and relative INa remaining were concentration-dependent. Frequency dependence of block between 0.5 and 4 cps was weak for ethacizin. Varying the depolarization duration from 5 to 100 msec, while keeping the recovery interval constant, did not alter the block by ethacizin. In contrast, prolonging the clamp step in ethmozin from 5 to 100 msec increased the rate and depth of block. Apparent binding rates for each drug were calculated using the assumptions of the guarded receptor model. We conclude that ethacizin blocks INa in a use-dependent manner by binding to a transiently available state such as the open state. In contrast, ethmozin block of INa exhibits both rest block and use-dependent block. Use-dependent block can be attributed to binding to a state (or states) maintained during depolarization such as the inactivated state. With these similar drugs, charge appears to be an important determinant of state-dependent binding.

[Effect of ethmozin on the electrophysiologic parameters of Purkinje cells in the sheep heart. The frequency-dependent blockade of sodium channels]. / Vliianie étmozina na élektrofiziologicheskie parametry kletok Purkin'e serdtsa ovtsy. Chastotno-zavisimaia blokada natrievykh kanalov.

The effect of ethmozin (E) on action potential (AP) duration (ADP90, APD50), AP upstroke velocity (Vmax) and cable properties (Ri, Rm, Rin, Cm, lambda m, tau m) was studied. Vmax was significantly reduced by E (0.2 to 5 mkM). Duration of AP plateau (APD50) was more sensitive to E than APD90. Thus, application of 0.5 mkM E resulted in 30% decrease of APD90 and 60% of APD50. This suggested that E not only inhibits INa, what is reflected by Vmax reduction, but also can affect other currents involved in plateau phase. Cable properties remained unchanged in the wide concentration range of E. Blockade of Vmax by E was use-dependent. Uptake rates of ethmozin by sodium channel for different stimulation frequencies were estimated and kinetic binding and unbinding constants (k = 40322 M-1 ms-1; 1 = 7.17 X 10(-5) ms-1) with unguarded receptor were calculated using novel drug-channel interaction model (Starmer and Grant, 1985).

Use-dependent block of sodium current by ethmozin in voltage-clamped internally perfused canine cardiac Purkinje cells.

Block of sodium current (INa) by ethmozin (moricizine), an antiarrhythmic drug, was investigated in isolated, voltage-clamped, canine cardiac Purkinje cells. Initial block of INa by ethmozin (2 microM) in noninactivated cells (held at -150 mV) was 9.3 +/- 1.2% (S.D.). Additional "use-dependent" block developed in response to repetitive depolarization. This block was both frequency-dependent and dose-dependent with the fall in peak INa greater at increasing depolarization frequencies (0.625 to 4 Hz) and with increasing dose (2 microM to 20 microM). Use-dependent block was modeled according to the guarded receptor hypothesis assuming ingress to the channel binding site during the open state of the channel, and egress from the channel independent of the kinetic state of the channel. The rate constants (on-rate = 2100 +/- 100 (S.D.)/M/ms and off-rate = 1.7 +/- 0.3 (S.D.) 10(-5)/ms) were used to predict the time course of INa block in response to repeated depolarizations and the dose-response relationship of steady-state used-dependent block measured in independent experiments. We conclude that ethmozin blocks INa in Purkinje cells in both a non-use-dependent and a use-dependent manner and that the guarded receptor model is useful in describing the use-dependent block.

Single channel sodium current in rat cardiomyocytes: use-dependent block by ethacizin.

Ethacizin is a phenothiazine derivative antiarrhythmic agent that blocks sodium current. The cell-attached patch clamp of single adult rat ventricular cells was used to investigate mechanisms of use-dependent block of sodium current. Under control conditions peak open probability, first latency, fraction of null sweeps, mean open time and single channel current amplitude were not different at both 1 and 4 Hz. Ethacizin (5 microM) caused a significant decrease in the peak open probability, a significant increase in the first latency and an increase in the fraction of null sweeps at 4 Hz compared with 1 Hz; mean open time and single channel current amplitude were unchanged. These observations support a model of antiarrhythmic action which proposes complete block of single channel conductance resulting from drug binding. A "runs analysis" revealed nonrandom clustering of null traces in the presence of ethacizin and no clustering in control patches. Increasing stimulation frequency makes this nonrandom behavior more apparent. We conclude that the relatively slow cycling of a few channels between blocked and unblocked states induces null sweeps clustering. The implications of these findings for mechanisms of drug block of the Na channel are discussed.