Down-regulation of sodium current in chronic heart failure: effect of long-term therapy with carvedilol.

Evidence has accumulated recently about the importance of alterations in Na+ channel function and slow myocardial conduction for arrhythmias in the infarcted and failing heart. The present study tested a hypothesis that Na+ current (INa/C) density decreases in chronic heart failure (HF) and that Na+ channel (NaCh) functional density can be restored by long-term therapy with carvedilol, a mixed alpha- and beta-adrenergic blocker. Studies were performed using a canine model of chronic HF produced in dogs by sequential intracoronary embolizations with microspheres. HF developed approximately 3 months after the last embolization (left ventricle, LV, ejection fraction = 28 +/- 1%). Ventricular cardiomyocytes (VCs) were isolated enzymatically from LV mid-myocardium, and INa was measured by whole-cell patch-clamp. The maximum INa/C was decreased in failing (n = 19) compared to normal (n = 12) hearts (33.1 +/- 1.6 vs 48.5 +/- 5.1 pA/pF, mean +/- SE, p < 0.001). The steady-state inactivation and activation of INa remained unchanged in failing compared to normal hearts. Long-term treatment with carvedilol (1 mg/kg, twice daily for 3 months) normalized INa/C in dogs with HF. INa/C in HF dogs (n = 6) treated with carvedilol was higher compared to that of non-treated HF dogs (n = 6) (49.4 +/- 0.9 vs 29 +/- 4.8 pA/pF, p < 0.007). In vitro culture of VCs of failing hearts for 24 h did not restore INa/C. However, INa/C was partially restored when VCs were incubated for 24 h with BAPTA-AM, an intracellular Ca2+ buffer. Thus, we conclude that experimental chronic HF in dogs results in down-regulation of the functional density of NaCh that can be restored by long-term therapy with carvedilol. The mechanism of NaCh down-regulation in HF may be linked to poor Ca2+ handling in this stage of disease.

Late sodium current is a novel target for amiodarone: studies in failing human myocardium.

V. A. Maltsev, H. N. Sabbah and A. I. Undrovinas. Late Sodium Current is a Novel Target for Amiodarone: Studies in Failing Human Myocardium. Journal of Molecular and Cellular Cardiology (2001) 33, 923-932. The authors recently reported the existence of a novel late Na(+)current (I(NaL)) in ventricular cardiomyocytes (VC) isolated from both normal and failing human hearts. Both in failing human and canine VC, partial block of I(NaL)normalized action potential (AP) duration and abolished early after depolarizations (EADs). The most recent computer simulation studies indicate a significant contribution of the persistent Na(+)current into the ion current balance on the plateau of VC AP as well as its important role in the dispersion of AP duration across the ventricular wall. The data thus indicate a possibility for I(NaL)to be a new therapeutic target. The present study tested a hypothesis that I(naL)could be a novel target for amiodarone (AMIO). Midmyocardial VC isolated from left ventricle of explanted failing human hearts were measured by a whole-cell clamp. I(NaL)was effectively blocked by AMIO in therapeutic concentrations, with IC(50)being 6.7+/-1.1 microM (mean+/-S.E.M., n=16 cells). At the same time, AMIO (5 microM ) produced almost no effect on the transient Na(+)current (IC(50)=87+/-28 microM, n=8). AMIO significantly shifted the steady-state inactivation (SSI) curve of I(NaL)towards more negative potentials and accelerated decay time course in a dose-dependent manner. At 5 microM, AMIO shifted SSI by 21+/-3 mV (n=7) and decreased the decay time constant from 0.67+/-0.05 s to 0.37+/-0.04 s (n=5, P<0.004). Evaluation of AMIO binding to different Na(+)channel (NaCh) states by means of mathematical models describing dose-dependent SSI shift and decay acceleration was consistent with an action that AMIO blocks NaCh preferentially in inactivated and activated states rather than in resting state. The authors conclude that the late Na(+)current is effectively blocked by AMIO and represents a new target for the drug in patients with chronic heart failure (HF).

Cardiac contractility modulation with the impulse dynamics signal: studies in dogs with chronic heart failure.

The intravenous use of positive inotropic agents, such as sympathomimetics and phosphodiesterase inhibitors, in heart failure is limited by pro-arrhythmic and positive chronotropic effects. Chronic use of these agents, while eliciting an improvement in the quality of life of patients with advanced heart failure, has been abandoned because of marked increase in mortality when compared to placebo. Nevertheless, patients with advanced heart failure can benefit from long-term positive inotropic support if the therapy can be delivered 'on demand' and in a manner that is both safe and effective. In this review, we will examine the use of a novel, non-stimulatory electrical signal that can acutely modulate left ventricular (LV) contractility in dogs with chronic heart failure in such a way as to elicit a positive inotropic support. Cardiac contractility modulation (CCM) with the Impulse Dynamic(trade mark) signal was examined in dogs with chronic heart failure produced by intracoronary microembolizations. Delivery of the CCM signal from a lead placed in the great coronary vein for periods up to 10 minutes resulted in significant improvements in cardiac output, LV peak+dP/dt, LV fractional area of shortening and LV ejection fraction measured angiographically. Discontinuation of the signal resulted in a return of all functional parameters to baseline values. In cardiomyocytes isolated from dogs with chronic heart failure, application of the CCM signal resulted in improved shortening, rate of change of shortening and rate of change of relengthening suggesting that CCM application is associated with intrinsic improvement of cardiomyocyte function. The improvement in isolated cardiomyocyte function after application of the CCM signal was accompanied by an increase in the peak and integral of the Ca(2+) transient suggesting modulation of calcium cycling by CCM application. In a limited number of normal dogs, intermittent chronic delivery of the CCM signal for up to 7 days showed chronic maintenance of LV functional improvement. In conclusion, pre-clinical results to date with the Impulse Dynamics CCM signal indicate that this non-pharmacologic therapeutic modality can provide short-term positive inotropic support to the failing heart and as such, may be a useful adjunct in the treatment of advanced heart failure. Additional, long-term studies in dogs with heart failure are needed to establish the safety and efficacy of this therapeutic modality for the chronic treatment of this disease syndrome.

Repolarization abnormalities in cardiomyocytes of dogs with chronic heart failure: role of sustained inward current.

We previously showed that a canine model of chronic heart failure (HF) produced by multiple coronary microembolizations manifests ventricular arrhythmias similar to those observed in patients with chronic HF. In the present study, we used single canine cardiomyocytes isolated from the left ventricle (LV) of normal dogs (n = 13) and dogs with HF (n = 15) to examine the cellular substrate of these arrhythmias. Action potentials (APs) and ion currents were measured by perforated and whole cell patch clamp, respectively. We found prolonged APs and alterations of AP duration resulting in early afterdepolarizations (EADs) at the low pacing rates of 0.5 Hz and 0.2 Hz. Na+ channel blockers saxitoxin (STX, 100 nM) and lidocaine (90 microM) reduced AP duration dispersion and abolished EADs in HF cardiomyocytes. The steady-state current (Iss)-voltage relation, in the voltage range from -25 mV to 25 mV analogous to the AP plateau level, was significantly shifted inward in HF cardiomyocytes. STX and lidocaine shifted the Iss-voltage relationship in an outward direction. The shifts produced by both drugs was significantly greater in cardiomyocytes of dogs with HF, indicating an increase in inward current. In the experimental configuration in which K+ currents were blocked, the density of the steady-state Ca2+ current (ICa) was found to decrease in HF cardiomyocytes by approximately 33%. In contrast, the density of the steady-state Na+ current (INa) significantly (P < 0.01) increased in HF cardiomyocytes (0.17 +/- 0.06 pA/pF) compared with normal cells (0.08 +/- 0.02 pA/pF). The relative contribution of INa to the net inward current was greater in HF cardiomyocytes, as evident from the increased ratio of INa/ICa (from 0.22 to 0.68). These observation support a hypothesis that anomalous repolarization of HF cardiomyocytes is due, at least in part, to an increased steady-state inward Na+ current.

Novel, ultraslow inactivating sodium current in human ventricular cardiomyocytes.

BACKGROUND: Alterations in K+ channel expression and gating are thought to be the major cause of action potential remodeling in heart failure (HF). We previously reported the existence of a late Na+ current (INaL) in cardiomyocytes of dogs with chronic HF, which suggested the importance of the Na+ channel in this remodeling process. The present study examined whether this INaL exists in cardiomyocytes isolated from normal and failing human hearts. METHODS AND RESULTS: A whole-cell patch-clamp technique was used to measure ion currents in cardiomyocytes isolated from the left ventricle of explanted hearts from 10 patients with end-stage HF and from 3 normal hearts. We found INaL was activated at a membrane potential of -60 mV with maximum density (0.34+/-0.05 pA/pF) at -30 mV in cardiomyocytes of both normal and failing hearts. The steady-state availability was sigmoidal, with an averaged midpoint potential of -94+/-2 mV and a slope factor of 6.9+/-0.1 mV. The current was reversibly blocked by the Na+ channel blockers tetrodotoxin (IC50=1.5 micromol/L) and saxitoxin (IC50=98 nmol/L) in a dose-dependent manner. Both inactivation and reactivation of INaL had an ultraslow time course (tau approximately 0.6 seconds) and were independent of voltage. The amplitude of INaL was independent of the peak transient Na+ current. CONCLUSIONS: Cardiomyocytes isolated from normal and explanted failing human hearts express INaL characterized by an ultraslow voltage-independent inactivation and reactivation.

Cytochalasin D alters kinetics of Ca2+ transient in rat ventricular cardiomyocytes: an effect of altered actin cytoskeleton?

The effects of cytochalasin D, a specific F-actin depolymerizing agent, on Ca2+ transients in rat ventricular cardiomyocytes were investigated. Cytochalasin D (20 microM) significantly slowed decay of Ca2+ transients (tau decay control cells=28.1+/-1.3, n=28tau decay=47.3+/-2.8 ms, n=20, P<0.001). The rising phase of Ca2+ transients was also significantly slower in cytochalasin D treated cells (tau rise=5.1+/-0.6 ms, n=17nu tau rise in control cells=3.6+/-0.2, n=21,P<0.01). Phalloidin (100 microM), an F-actin stabilizer, prevented cytochalasin D-induced alterations of Ca2+ transient kinetics. The cytochalasin D effect was not related to the l-type Ca2+ current since the current density and kinetics were not altered by the drug. We conclude that integrity of F-actin-based cytoskeleton is an important factor for sarcoplasmic reticulum function.

Relationship between action potential, contraction-relaxation pattern, and intracellular Ca2+ transient in cardiomyocytes of dogs with chronic heart failure.

Abnormalities of contractile function have been identified in cardiomyocytes isolated from failed human hearts and from hearts of animals with experimentally induced heart failure (HF). The mechanism(s) responsible for these functional abnormalities are not fully understood. In the present study, we examined the relationship between action potential duration, pattern of contraction and relaxation, and associated intracellular Ca2+ transients in single cardiomyocytes isolated from the left ventricle (LV) of dogs (n = 7) with HF produced by multiple sequential intracoronary microembolizations. Comparisons were made with LV cardiomyocytes isolated from normal dogs. Action potentials were measured in isolated LV cardiomyocytes by perforated patch clamp, Ca2+ transients by fluo 3 probe fluorescence, and cardiomyocyte contraction and relaxation by edge movement detector. HF cardiomyocytes exhibited an abnormal pattern of contraction and relaxation characterized by an attenuated initial twitch (spike) followed by a sustained contracture ('dome') of 1 to 8 s in duration and subsequent delayed relaxation. This pattern was more prominent at low stimulation rates (58% at 0.2 Hz, n = 211, 21% at 0.5 Hz, n = 185). Measurements of Ca2+ transients in HF cardiomyocytes at 0.2 Hz manifested a similar spike and dome configuration. The dome phase of both the contraction/relaxation pattern and Ca2+ transients seen in HF cardiomyocytes coincided with a sustained plateau of the action potential. Shortening of the action potential duration by administration of saxitoxin (100 nM) or lidocaine (30 microM) reduced the duration of the dome phase of both the contraction/relaxation profile as well as that of the Ca2+ transient profile. An increase of stimulation rate up to 1 Hz caused shortening of the action potential and disappearance of the spike-dome profile in the majority of HF cardiomyocytes. In HF cardiomyocytes, the action potential and Ca2+ transient duration were not significantly different from those measured in normal cells. However, the contraction-relaxation cycle was significantly longer in HF cells (314 +/- 67 ms, n = 21, vs. 221 +/- 38 ms, n = 46, mean +/- SD), indicating impaired excitation-contraction uncoupling in HF cardiomyocytes. The results show that, in cardiomyocytes isolated from dogs with HF, contractile abnormalities and abnormalities of intracellular Ca2+ transients at low stimulation rates are characterized by a spike-dome configuration. This abnormal pattern appears to result from prolongation of the action potential.

Relationship between steady-state activation and availability of cardiac sodium channel: evidence of uncoupling.

The coupling between steady-state activation and availability from inactivation was characterized for the cardiac Na+ channel. To evaluate this coupling, we plotted the relationship between the conductance and availability curve midpoint potentials measured in 92 rat ventricular cardiomyocytes and applied a correlation analysis. We found a high correlation between the midpoints (correlation coefficient = 0.86, slope = 0.95) within the availability midpoint potential range positive to -100 mV. In contrast, the midpoints were not correlated in the myocytes (37 of 92 cells) having midpoint potential negative to -100 mV, indicating an uncoupling between activation and availability.

Cytoskeleton modulates coupling between availability and activation of cardiac sodium channel.

The aim of this study was to investigate modulation of voltage-dependent steady-state activation and availability from inactivation of the cardiac Na+ channel by the cytoskeleton. As an experimental approach, we used long-lasting monitoring [63 +/- 5 (SE) min] of the half-point potentials of the steady-state availability curve (V(1/2A)) and normalized conductance curve (V(1/2G)) in 116 rat ventricular cardiomyocytes by whole cell patch clamp at 22-24 degrees C. Both half-point potentials shifted in the negative direction with time as an exponentially saturating change, with the shift of V(1/2G) being smaller and faster. An F-actin disrupter, cytochalasin D (Cyto-D, 20 microM), accelerated the rate of the V(1/2A) shift but decreased the range of the V(1/2G) shift. An F-actin stabilizer, phalloidin (100 microM), temporarily (for 28.2 +/- 2.2 min, n = 15) prevented the V(1/2A) shift but did not influence the V(1/2G) shift. The best fit for the V(1/2G)-V(1/2A) relationship in untreated cells (1,021 data points measured in 51 cells) was a second-degree (2.06) power function. Cytoskeleton-directed agents modified the relationship. In Cyto-D-treated cells, the V(1/2G)-V(1/2A) relationship was shifted (by 2.5 mV) toward positive V(1/2G). On the contrary, a microtubule stabilizer, taxol (100 microM), shifted the relationship toward negative V(1/2G) (by 12.2 mV). We conclude that coupling between availability and activation is modulated by F-actin-based and microtubular cytoskeleton.

Blockade of lysophosphatidylcholine-modified cardiac Na channels by a lidocaine derivative QX-222.

Single Na channels from rat and rabbit ventricular cells were studied with use of the excised inside-out patch-clamp technique. To investigate local anesthetic interactions with Na channels modified by the ischemic metabolite lysophosphatidylcholine (LPC), the quaternary ammonium lidocaine derivative QX-222 [2-(trimethylamino)-N-(2,6-dimethylphenyl)acetamide] was applied to the cytoplasmic side of patches from untreated cells and from those treated with LPC for approximately 1 h. Single-channel amplitudes and kinetics for unmodified channels were similar to those reported previously for cardiac cells with a single-component, mean-channel open time. LPC-modified channels showed prolonged open channel bursting with a two-component, mean open time, suggesting two open states. Conductance sublevels to the 60-70% level of the main conductance were found in both unmodified and LPC-modified channels and also with and without QX-222 present. QX-222 reversibly shortened the open time of the unmodified channel and for both open times of the LPC-modified channel without decreasing single-channel amplitude. Calculated association rates for QX-222 with the channel were found to be greater for the open states of the modified channel than those for the unmodified channel. Thus the lidocaine analogue QX-222 interacts with and blocks the open state of both unmodified and LPC-modified, cardiac Na channels. The blocking effect on LPC-modified channels would be predicted to be greater both because of the longer dwell time in the high-affinity open states for modified channels and also because of an intrinsically greater association rate in the modified channels.

Rapid onset of lysophosphatidylcholine-induced modification of whole cell cardiac sodium current kinetics.

Lysophosphatidylcholine (LPC), an ischemic metabolite implicated in arrhythmogenesis, has been shown to modulate aspects of Na+ channel gating, but its effects on steady-state availability (h infinity), recovery from inactivation, and the timing of onset and possible reversibility, have not been characterized. We studied Na current (INa) by the whole-cell patch clamp technique on isolated rat ventricular myocytes at 22 degrees C with reduced Na+ (45 mM out, 5 mM in) from a holding potential of -150 mV. Changes in the electrophysiological parameters were measured after LPC 10 microM was added to the bath and compared to time controls (TC) taken from the time of seal formation. LPC decreased peak current for a test potential to -30 mV by about 20%. The peak current voltage relationship shifted in a positive direction by about 5 mV after LPC as compared to a small 2 mV negative shift in TC cells. LPC shifted the steady-state availability curve in the hyperpolarizing direction by about 6 mV. LPC perfusion caused a slowing of the decay of INa, and also a slowing of recovery from inactivation. Onset of the effects occurred within 6 min after adding LPC to the bath and were statistically significant with respect to TC cells between 12 and 16 min. In three cells, some of the effects on INa were either arrested or partially reversed by washout and cell survival was less than 20 min if LPC was not removed from the bath. These LPC induced changes in INa would tend to slow conduction and increase refractoriness, effects also seen in acutely ischemic myocardium. We therefore conclude that LPC action on INa may potentiate the arrhythmogenic substrate and that the onset of these changes are sufficiently rapid to play a role in the electrical instability of acute ischemia.

Cytoskeleton modulates gating of voltage-dependent sodium channel in heart.

To investigate the role of the cytoskeleton in cardiac Na+ channel gating, the action of cytochalasin D (Cyto-D), an agent that interferes with actin polymerization, was studied by whole cell voltage clamp and cell-attached and inside-out patches from rat and rabbit ventricular cardiac myocytes. Cyto-D (20-40 microM) reduced whole cell peak Na+ current by 20% within 12 min and slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation. Brief treatments (< 10-15 min) of cell-attached patches by Cyto-D (20 microM) in the bath induced short bursts of Na+ channel openings and prolonged decays of ensemble-averaged currents. Bursting of the Na+ channel was more pronounced when the cell suspension was pretreated with Cyto-D (20 microM) for 1 h before seal formation. Application of Cyto-D on the cytoplasmic side of inside-out patches resulted in more dramatic gating changes. Peak open probability was reduced by > 50% within 20 min, and long bursts of openings occurred. Washout of Cyto-D did not restore ensemble-averaged current amplitude, but burst duration decreased toward control values. Cyto-D also induced an additional slower component to open and closed times. These results suggest that Cyto-D, through effects on cytoskeleton, induced cardiac Na+ channels to enter a mode characterized by a lower peak open probability but a greater persistent activity as if the inactivation rate was slowed. The cytoskeleton, in addition to localizing integral membrane proteins, apparently also plays a role in regulating specific detailed functions of integral membrane proteins such as the gating of Na+ channels.

Inward sodium current at resting potentials in single cardiac myocytes induced by the ischemic metabolite lysophosphatidylcholine.

To investigate possible ionic current mechanisms underlying ischemic arrhythmias, we studied single Na+ channel currents in rat and rabbit cardiac myocytes treated with the ischemic metabolite lysophosphatidylcholine (LPC) using the cell-attached and excised inside-out patch-clamp technique at 22 degrees C. LPC has been reported previously to reduce open probability and to induce sustained open channel activity at depolarized potentials. We now report two new observations for Na+ currents in LPC-treated patches: 1) The activation-voltage relation of the peak of the ensemble currents is shifted in the negative (hyperpolarizing) direction by approximately 20 mV compared with control currents. This effect was observed in all patches for depolarizations from a holding potential of -150 mV to different test potentials. 2) In some LPC-treated patches, Na+ channels exhibited sustained bursting activity at potentials as negative as -150 mV, giving a nondecaying inward current. This bursting activity was accompanied by double and triple simultaneous openings and closings, suggesting tight cooperativity in channel gating. These LPC-modified channels were identified as Na+ channels, because their unitary conductance was the same as Na+ channels in control solutions, because the single channel current-voltage relation was extrapolated to reverse at the Na+ Nernst potential, and because the current was blocked by the local anesthetic QX-222. This novel depolarizing current may play a role in the electrophysiological abnormalities in ischemia, including abnormal automaticity and reentrant arrhythmias, and could be a target for antiarrhythmic drugs.

Modulation of cardiac sodium channel gating by lysophosphatidylcholine.

The effects of lysophosphatidylcholine (LPC) on Na channels have been investigated in inside-out patches of adult rat ventricular cells using the patch-clamp technique. Applying LPC (9-25 microM) to the inner side of the membrane reduced peak Na current (INa) and prolonged the time course of INa inactivation. Both effects increased with duration of LPC exposure. Analysis of single-channel behavior revealed that after 15-20 min of exposure to LPC, Na channels displayed 2 types of gating behavior. One type consisted of normal kinetics and the other consisted of long-lasting burst (LB) of Na channel openings (up to the 300 ms bursts of the test pulse). This modification in gating resulted in the initial appearance of a slowly decaying and later a noninactivating component of INa in the ensemble average current. The slope conductance and reversal potential of these modified channels remained unchanged from control. Overall open time distribution for long bursting kinetics channels was biexponential, indicating existence of two long-lasting bursting modes, one with fast kinetics (LB-f, mean open time 1.2 ms) and one with slow kinetics (LB-s, mean open time 13.7 ms). These data indicate that exposure to LPC results in the slow interconversion of Na channels between several modes of activity, including those in which inactivation occurs slowly or not at all.

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.

Quinidine blocks adenosine 5'-triphosphate-sensitive potassium channels in heart.

The ATP-sensitive potassium channel current (IK-ATP) was studied in excised inside-out patches from rat ventricular cells at 20-23 degrees C. The bath solution contained 140 mM KF, and the pipette solution contained 140 mM KCl and 1.2 mM MgCl2. ATP (0.5 mM) in the bath inhibited IK-ATP. In the absence of ATP, 10 microM quinidine decreased open probability 67 +/- 1% (n = 6) at -50 mV and 28 +/- 12% at -130 mV (n = 5) without affecting single channel conductance (48-52 pS). The block increased with 25 and 50 microM quinidine and could be reversed on washing quinidine for several minutes. Interburst (closed) intervals were increased by quinidine, whereas open and closed time distributions within bursts were not changed. We conclude that quinidine blocks IK-ATP in a "slow" and voltage-dependent manner in clinically relevant concentrations. Because of the postulated role for IK-ATP in cardiac ischemia, quinidine block of this channel may play a role in ischemic arrhythmias.

[Changes in balance of ionic currents of cardiac cells as one of the mechanisms of arrhythmia]. / Narushenie balansa ionnykh tokov serdechnykh kletok kak odin iz mekhanizmov aritmii.

Changes in balance of transmembrane ionic currents were achieved in two ways: 1) modifying Na current with lysophosphatidylcholine (LPC), a toxic metabolite of ischemia; 2) modifying Ca current with BAY K 8644, an agonist of Ca channels. Elementary current through Na channel were recorded in an inside-out mode using patch clamp techniques. The effects of BAY K 8644 were studied on the transmembrane potential of guinea pig papillary muscle (PM) and sheep Purkinje fibers (PF). Application of LPC (9-25 microns) from the inner side of the membrane caused a peak reduction of averaged Na current and prolonged the time of inactivation. Analysis of single channel behavior revealed that LPC induced long-lasting bursts of openings, resulting in a noninactivated fraction of Na current. The duration of the action potential (AP) of PM increased slightly (20%), while PF AP duration increased progressively with time even at a low BAY K 8644 dose (0.5 microM). Early after-depolarization (EAD) occurred at min 5. After 10-minute drug use, PF became inexcitable at high plateau levels. TTX and ethmozine (1 microM) restored AP. Thus, changes in balance of ion c currents by modifying Na and/or Ca channels may culminate in the occurrence of an additional steady-state potential at plateau levels. The latter may result EAD and re-entry around functionally inexcitable PF. The similarity of mechanisms of arrhythmias induced by ischemia using the presented model is discussed.

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.

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.

Ischemic poison lysophosphatidylcholine modifies heart sodium channels gating inducing long-lasting bursts of openings.

The effects of lysophosphatidylcholine (LPC) on Na channels in inside-out patches of adult rat ventricular cells using the patch-clamp technique have been investigated. Application of LPC (9-25 microM) from the inner side of membrane for 4-15 min caused a reduction of averaged Na current (INa) peak and prolonged the time course of inactivation in the potential range of -50 to -10 mV. Analysis of single channel behaviour revealed that after 30-50 min of exposure, in addition to normally functioning Na channels with short openings, LPC induced long-lasting bursts of Na channel openings (up to the 300 ms duration of the test pulses). This resulted in an appearance of noninactivated component of INa. The slope conductance of these modified channels remained the same as in control (11.3 pS - control; 11.6 pS - LPC-treated). The dwell time for modified channels increased significantly.