Volatile anesthetics may directly inhibit cardiac fast Na+ inward current (INa) and, consequently, may be responsible for slowing impulse conduction and dysrhythmias due to abnormal conduction and reentry . Halothane produces a modest depression of conduction in both ventricular and Purkinje fibers [2,3] consistent with the reported actions of halothane in reducing action potential amplitude and overshoot . Previous reports that volatile anesthetics affect Vmax in heart cells are conflicting, and do not clearly establish that INa is involved since anesthetics can also affect electrical uncoupling between cells [2-5]. On the other hand, anesthetic effects on the peak INa current during the upstroke of the cardiac action potential cannot be excluded . Although the Vmax of the action potential represents a reasonable index for peak INa, it is not a reliable measure for the quantitative assessment of Na+ channel behavior .
The ion conductance of the fast INa current is controlled by two experimentally separable processes: 1) activation, which regulates ion channel opening, is a voltage-dependent step in response to changes in membrane potential, and 2) inactivation, which is responsible for rate and extent of ion channel closure during maintained depolarization. These characteristics ensure a rapid activation of the ion channel in response to membrane potential changes. Changes in inactivation and/or activation properties of the voltage-dependent Na+ channel can, therefore, alter the cardiac action potential and set the stage for dysrhythmias, especially under conditions of myocardial ischemia .
The present study was designed to characterize and compare the effects of three volatile anesthetics, halothane, isoflurane, and sevoflurane, at clinically relevant concentrations on the fast cardiac INa. The whole-cell patch clamp technique was used to measure directly the effects of anesthetics on INa in single cardiac cells. In patch clamp experiments of cardiac Na+ channels, spontaneous shifts of both steady-state inactivation and activation toward more negative potential occur . Therefore, it was necessary to evaluate the rates at which these shifts occur under control conditions. We report that all three anesthetics depressed I (Na), and that halothane was the most potent of the group at equianesthetic concentrations. In all three cases, block of INa was concentration- and voltage-dependent. Furthermore, each anesthetic shifted the steady-state inactivation and activation curves to more hyperpolarized potentials.
After approval by our institutional animal care and use committee, single ventricular cells were isolated from enzymatically treated hearts from adult guinea pigs, weighing 200-300 g. The procedure of the cell isolation is a modification of that of Mitra and Morad . The guinea pigs were anesthetized with sodium pentobarbital (250 mg/kg), their hearts rapidly removed, mounted on a Langendorff apparatus, and perfused through the aorta with oxygenated buffer solution (37 degrees C) containing Joklik solution (Gibco Minimum Essential Medium). After the blood was cleared from the hearts, they were then perfused in an enzyme solution containing Joklik, 0.6 mg/mL collagenase (Life Technologies, Grand Island, NY) and 0.12 mg/mL protease, Type XIV (Sigma Chemical Co., St. Louis, MO) for 14 min. The digested hearts were then removed, and the ventricles chopped coarsely and shaken for approximately 8 min in the enzyme solution. The digested heart tissue was then filtered, and the cells were separated and collected by centrifugation, after which they were placed in a recovery solution containing Joklik 1 mM CaCl2 and 1 g/100 mL bovine albumin fraction V (Pentex[R], Bayer Corp., Kankakee, IL). Further centrifugation and washing in Tyrode solution was performed before the cells were ready for experiments. Cells were then transferred to a plexiglass chamber mounted on the stage of an inverted microscope (Olympus IMT-2, Tokyo, Japan). Only rod-shaped cells with clear borders and striations were selected for experiments and they were used within 12 h after isolation.
High-resistance seals and voltage clamp were attained in Tyrode solution containing (in mM): 132 NaCl, 4.8 KCl, 1.2 MgCl2, 10 HEPES, 5 dextrose, 1 CaCl (2); pH = 7.4 with NaOH. After establishment of whole-cell voltage clamp, the external bath solution was changed to one that isolated for Na+ current. This solution contained (in mM): 115 CsCl, 10 NaCl, 10 HEPES, 1 MgCl2, 1.8 CaCl2, 5.5 glucose, and 3 CoCl; pH = 7.2 with CsOH. Cobalt and cesium were used as blockers for L-type calcium and potassium channel currents, respectively. The standard pipette solution contained (in mM): 11 EGTA, 1 CaCl2, 10 HEPES, 2 Mg-ATP, 90 CsF, 60 CsCl, 10 NaF; pH = 7.3 with CsOH. To compare halothane, isoflurane, and sevoflurane at equianesthetic concentrations, drugs were prepared in the following final bath concentrations (mM): halothane 0.6 and 1.2, isoflurane 0.5 and 1.0, and sevoflurane 0.6 and 1.2. The concentrations of these anesthetics at 22 degrees C in the Tyrode solution are equivalent to the following percentages in the gas phase (vol%) : halothane 0.98, 1.96, isoflurane 1.30, 2.60, and sevoflurane 1.98, 3.96, respectively. Anesthetic potencies are not expressed as minimum alveolar concentrations (strongly temperature-dependent) but as aqueous concentrations, which are relatively temperature-insensitive . Thus, the concentration can be extrapolated to body temperature . Solution samples of the chamber were taken after every experiment and analyzed by gas chromatography to verify the anesthetic concentration surrounding the cell.
Current measurements were obtained in the whole-cell configuration of the patch clamp procedure as described by Hamill et al. . To optimize voltage control of the cardiac cells: (a) small cells (60-80 pF) were selected to decrease membrane capacitance; (b) external Na+ concentration was reduced to 10 mM to decrease magnitude of Na+ current to avoid saturation of the amplifier and loss of clamp control (the average current amplitude of cells included in this study was 1.83 +/- 0.04 nA); (c) pipette resistances ranged from 1.0 to 1.5 M Omega to decrease access resistance; (d) experiments were performed at room temperature (22 degrees C); (e) series resistance compensation was adjusted (about 80%) to give the fastest possible capacity transients without producing ringing. Under these conditions, the voltage error was <3 mV. Linear leak currents were estimated by extrapolation of a linear curve fit to currents negative to -60 mV and subtracted from total current measurements similar to the method of Arena and Kass . Experiments were acceptable only in cells in which leak currents were unchanged. The average access resistance in control and during exposure to anesthetics calculated from a 10-mV test pulse was 3.38 +/- 0.82 M Omega and 3.33 +/- 0.76 M Omega, respectively. In physiological concentrations of K+ (5 mM extracellular, 140 mM intracellular), the average membrane potential was -72.7 +/- 0.42 mV (n = 6). Pipettes from borosilicate glass were pulled with a multistage puller (Sachs-Flaming, PC-84) and heat polished (Narishige microforge, MF-83). Current was measured by a List EPC-7 patch clamp amplifier (Adams & List Assoc., Great Neck, NY), and the out-put was low-pass filtered at 3 kHz to reduce high-frequency noise. All data were digitized and stored for later analysis with the pCLAMP software package (Axon Instruments, Inc., Foster City, CA).
After rupturing the membrane and achieving voltage clamp conditions, stability of Na+ current amplitude was monitored for 4 min before recordings for experiments were initiated. Whole-cell currents were elicited by 30-ms test pulses from a holding potential of -110 mV to +20 mV in 10-mV increments. To examine time-dependent changes of peak INa, 50 ms test pulses were applied every 15 s from -110 mV to a test potential of -30 or -40 mV, where peak INa occurred. Steady-state activation was obtained by calculating conductance from: Equation 1 where INa is the current amplitude, V is the test potential, and Erev is the reversal potential for sodium. The conductance was normalized to maximum conductance gmax. To monitor steady-state inactivation of INa, cells were subjected to preconditioning pulse potentials between -130 (the holding potential) and +20 mV for 500 ms and then stepped to the test pulse potential (24 ms) at -30 or -40 mV, the potential at which peak Na+ current occurred. Peak currents from the test potentials were normalized to the maximum current (Imax) obtained during the test potentials. Both steady-state activation and inactivation were fitted to a Boltzmann distribution  described by: Equation 2 where V is the test (for activation) or preconditioning (for inactivation) potential, V1/2 is the potential at which half maximum activation or inactivation occurs and k is the slope factor.
Statistical analysis was computed using paired t-tests when comparing two sample means in the case where a cell served as its own control, and unpaired t-tests when comparing samples of one anesthetic at two different concentrations. A one-way analysis of variance was used when different groups of anesthetics were compared. Differences between group means were evaluated with Bonferroni's test. For experiments comparing shifts in steady-state inactivation and activation, the predicted background shift was subtracted from the obtained shifts, and a paired t-test was performed. The test for normality and equality of variance within groups was not satisfied in experiments comparing voltage-dependent effects of anesthetics. In this case, the Mann-Whitney rank sum test and two-way repeated measure of analysis on the ranks were used. A test was considered to be significant when P < 0.05. Data are presented as mean +/- SEM.
(Figure 1) shows representative whole-cell Na+ current traces and the corresponding I-V relationship recorded in the absence (control) and presence of the anesthetics halothane, isoflurane, or sevoflurane. Na+ current was elicited in response to a series of stepwise (10 mV) depolarizing test pulses from a holding potential of -110 mV to +20 mV. In the example shown, all three anesthetics reduced peak INa (Figure 1). The I-V relationships show effects of the anesthetics at the various membrane potentials. The peak current was obtained in the range of -30 to -40 mV.
(Figure 2A) illustrates the time course of the peak INa current during control, halothane exposure (1.2 mM), and washout from a representative cell. Maximum anesthetic effects were observed within 2.5-3 min after drug application. The effects of halothane on peak INa were reversible. Under control conditions without application of drugs, INa was stable (constant amplitude) for at least 30 min (data not shown). The summary of the effects of all three anesthetics on peak INa is shown in Figure 2B. In all cases the depressant effects of isoflurane and sevoflurane on INa also were reversible upon washout (data not shown). Halothane and isoflurane showed significant concentration-dependent effects with increased potency at the higher concentration tested. The mean effect of sevoflurane also showed concentration-dependence, but the results were not statistically significant. Halothane at 0.6 mM and 1.2 mM produced significant depressions of peak INa of 12.3% +/- 1.8% and 24.4% +/- 4.1% (mean +/- SEM, n = 12 cells) versus control, respectively. Isoflurane and sevoflurane were less potent than halothane, decreasing peak INa by 4.8% +/- 1.1% (0.5 mM, n = 12 cells), 11.4% +/- 1.4% (1.0 mM, n = 15 cells) and 3.0% +/- 0.7% (0.6 mM, n = 14), 10.7% +/- 3.9% (1.2 mM, n = 12 cells), respectively. At both concentrations, the depressant effect of halothane on INa was significantly different from isoflurane and sevoflurane. There were no significant differences between the latter two groups.
The current-voltage relationships in Figure 1 indicate that the effects of the volatile anesthetics may be dependent on the membrane potential. Figure 3A demonstrates Na+ current traces during control and after halothane exposure (0.6 mM) from the same cell elicited from a holding potential of -110 mV to three different test potentials. At a test potential of -40 mV (Figure 3, left panel) no blocking effect of halothane was observed and there was even an increase of I (Na). The inhibition of INa by halothane at -30 mV where peak Na+ current occurred was 11% (Figure 3, middle panel). At a test potential of -10 mV (Figure 3, right panel), the blocking effect appears more potent (approximately 50%). To evaluate this voltage-dependent effect, the effects of low and high concentrations of anesthetics on INa were plotted against various membrane potentials (Figure 3B). To account for variability of the potential at which peak INa occurred (-30 or -40 mV), the results are plotted as Delta V = [vertical bar] Vm,peak [vertical bar] + Vm, where IVm,peak I is the absolute value of the potential at which peak INa was recorded and Vm is the test potential. In all cases, the blocking action of anesthetics on INa became increasingly greater at depolarized potentials. At a potential 10 mV negative to Vm,peak, all three anesthetics at the low concentrations caused significant enhancements of the current relative to control. At high concentrations of isoflurane and sevoflurane, the increase of INa was not significant versus control. At the higher concentration (1.2 mM) halothane, on the other hand, produced a significant block. Inhibition (or increase) in Na+ current among different levels of voltage is significantly different for each anesthetic at both concentrations, respectively.
The results above show that halothane, isoflurane, and sevoflurane depress INa in a voltage-dependent manner, suggesting a possible intrinsic interaction with the channel protein. Thus, we further investigated whether these anesthetics also influenced the steady-state inactivation and activation characteristics of the Na+ channel.
Spontaneous shifts in steady-state inactivation and activation during intracellular dialysis of the cell interior under whole-cell configuration have been reported . Therefore, such shifts were first evaluated under control conditions of this study. After peak Na+ current remained stable for 4 min, inactivation and activation protocols were elicited at 3-min intervals for 24 min under control (anesthetic-free) conditions. Figure 4A represents the spontaneous background shift in V1/2 obtained during steady-state inactivation (anesthetic-free); the rate of shift in V1/2 was -0.265 +/- 0.004 mV/min as determined from a linear fit to data obtained from six cells. Unlike the linear rate of shift in V1/2 under steady-state inactivation conditions, the rate of shift in V1/2 for steady-state activation was better fit with a single exponential function: y = -15.65 + 15.83 x ex/39.37 (n = 6 cells; Figure 4B). No statistically significant changes in the slope factor k were observed for both steady-state inactivation and activation.
(Figure 5) demonstrates the effects of halothane (1.2 mM), isoflurane (1.0 mM), and sevoflurane (1.2 mM) on inactivation and activation of INa under steady-state conditions obtained from three different ventricular myocytes. In the examples shown, each anesthetic produced shifts in both the inactivation and activation curves toward more hyperpolarized potentials. Table 1 summarizes the shifts in the inactivation and activation variables, V1/2 and k. The values for the shifts in V1/2 shown in Table 1 have been corrected for the spontaneous background shifts described above. To correct for the background shifts, the relationships described in Figure 4 were used. Background shifts for both activation and inactivation were determined for the time when data in the presence of anesthetics were recorded. For all cases, the effects of anesthetics reached steady-state within 4 min from control. Hence, at 4 min the predicted background shift in V1/2 for activation and inactivation would be 1.6 and 1.2 mV, respectively. After subtracting the predicted background shift, the shift in inactivation in the presence of anesthetic was still significantly different from control conditions. Hence, the shifts in V1/2 in the steady-state inactivation curve in the presence of anesthetics cannot be accounted for by the spontaneous background shift. Anesthetic-induced shifts in V1/2 for steady-state activation were also significantly greater than from control except in the presence of low concentration of isoflurane. Therefore, only for the low concentration of isoflurane can the induced shift in V1/2 be accounted for by the spontaneous background shift. Within the groups of low and high concentrations of the three anesthetics, no significant difference was found for shifts in both steady-state activation and inactivation, respectively. The slope factor k was not significantly affected by the inhalational anesthetics.
The present study demonstrates that the three inhalational anesthetics halothane, isoflurane, and sevoflurane, at equianesthetic concentrations, decrease the fast cardiac Na+ current of ventricular guinea pig myocytes in a dose- and voltage-dependent manner. The potency of block increased at depolarized membrane potentials. The biggest decrease in INa was induced by halothane. All three anesthetics induced hyperpolarizing shifts in both steady-state activation and inactivation curves. In all but the low concentration of isoflurane, shifts in V1/2 were anesthetic-induced and not due to the spontaneous background shift, which has been reported for the cardiac Na+ channel .
Our results show that volatile anesthetics induce hyperpolarizing shifts in both the steady-state inactivation and activation curves. These shifts in V1/2, especially the shift in steady-state inactivation, are more than those expected from the spontaneous background shifts reported for INa. The slope factor k was not changed, suggesting that the channel's intrinsic voltage sensor was not affected. However, these shifts cannot be explained by alterations of the membrane electric field (surface charge screening effects) because these anesthetics are not charged at pH 7.3. An intriguing aspect of our result is that both the steady-state activation and inactivation shifts in V1/2 are toward hyperpolarizing potentials. Hyperpolarizing shifts in the inactivation curve indicate lesser availability of channels, but such shifts in the activation curve suggest increased conductance at a particular membrane potential. Moreover, because the inhibitory effects of anesthetics on current amplitude were more potent at depolarizing potentials, these results appear to affect INa in an opposing manner. In this respect, mechanisms of inhibition of cardiac Na+ current by volatile anesthetics seem to be different from that of neuronal Na+ current. As in cardiac myocytes, inhalational anesthetics shifted steady-state inactivation curves in neuronal cells in the hyperpolarizing direction, but in contrast to our results, the steady-state activation curves were shifted in the depolarized (positive) direction . Halothane and isoflurane are intermediate to hydrocarbons and alcohols in physicochemical properties. In neurons, the effect of inhalational anesthetics resemble the alcohols much more than the hydrocarbons, shifting the activation curve toward more positive potentials . Interestingly, in ventricular myocytes, inhalational anesthetics seem to act more like hydrocarbons, which have been found to shift the activation curve in the hyperpolarized (negative) direction . The shifts in the steady-state activation curves in ventricular myocytes were consistently less negative than the inactivation curves. Furthermore, the shift in the steady-state activation curve toward hyperpolarized potentials would actually indicate an enhancement rather than inhibition of current amplitude. This may suggest that the depression of INa by the inhalational anesthetics may be in part due to the hyperpolarizing shifts in the inactivation but not due to shifts in the activation curves. Effects of halothane on cardiac INa were previously reported and were suggested to be mainly due to a shift toward negative potentials in the steady-state inactivation curve . However, that study did not evaluate the spontaneous shifts in steady-state inactivation. Effects of steady-state activation were not determined. Consequently, the "true" contribution of the shift in the inactivation curve was not available. Furthermore, the differential effects of anesthetics observed in the current study suggest that shifts in the steady-state inactivation curve cannot, by themselves, account for the depressant effects.
At equianesthetic low or high concentrations, the three anesthetics studied showed no significant differences within groups in shifts in V1/2 for both steady-state activation and inactivation. These findings suggest that the increased arrhythmogenic ability of halothane depends more on its larger effect on peak Na+ current than in shifts of activation and inactivation variables. This would also indicate that the shifts in steady-state inactivation cannot account for the differential effects of halothane compared to isoflurane and sevoflurane.
Voltage-sensitive Na+ channels are responsible for the initiation and propagation of the action potential and, therefore, play a crucial role in cardiac excitability. In the myocardium, volatile anesthetics differ substantially in their ability to potentiate catecholamine-induced dyshythmias in patients and laboratory animals, halothane being more "sensitizing" than isoflurane . Our finding that halothane was more effective in decreasing peak INa compared with isoflurane or sevoflurane may be attributed to the proarrhythmogenic effects of halothane. Despite the significant anesthetic depression of Na+ channel current found in the present study, it has been reported that volatile anesthetics decrease conduction velocity with little change in Vmax[2,19]. This may be due to electrical uncouplings between cells, which can increase Vmax and, thereby, mask the decrease in V (max), indicative of a decrease in Na+ current amplitude . Thus, the depression of conduction may involve a change in cell to cell coupling in addition to the reduction of peak INa during Phase 0 of the action potential.
Cyclic adenosine monophosphate has been shown to modulate cardiac Na (+) channels . Furthermore, a previous report has suggested the involvement of cyclic adenosine monophosphate as one possible site of anesthetic action . An increase in basal adenylate cyclase activity in cardiac membranes was demonstrated after halothane exposure, but not after addition of isoflurane . These differences support, and may in part explain, our results where at approximately equianesthetic concentrations the inhibitory effect on peak Na+ current was about twice as great during exposure to halothane than in the presence of isoflurane and sevoflurane, which had quantitatively similar depressant effects.
The present results show that anesthetics at clinical concentrations inhibit cardiac INa, although the reductions obtained in our study appear to be relatively small-a depressant effect on peak INa of 3%-24%. However, several related studies have shown that administration of halothane at clinical levels results in alteration of action potential characteristics and conduction of cardiac impulses [3,4]. Ikemoto et al.  found that even 0.5 vol% halothane causes a significant decrease of overshoot and maximum rate of increase, which is largely dependent on the fast inward current carried by sodium. Hirota et al.  correlated their findings of decrease of action potential duration and depression of overshoot by halothane (1% and 2%) with an observed small decrease of the fast inward Na+ current in atrial myocytes from bullfrog hearts. Thus, even a relatively small depressant effect on Na+ current may alter the cardiac action potential profile.
The inhibition of cardiac INa by volatile anesthetics is voltage dependent, increasing at depolarized potentials. However, a significant enhancement of INa at a potential 10 mV negative to Vm,peak was observed at low concentrations for all three anesthetics. The relevance of this current enhancement on the cardiac action potential may be masked during the Phase 0 depolarization by the strongly increasing block of INa at the more depolarized potentials. The voltage-dependent decrease or enhancement of INa by anesthetics may increase the dispersion of conduction velocities throughout the heart. Thus, a proarrhythmogenic effect cannot be excluded. Furthermore, our results suggest that the voltage-dependent block by halothane, isoflurane, and sevoflurane is not due to ionization of the anesthetic molecule. All three anesthetics are relatively neutral at pH 7.3, which is the condition of our experiments. Because Na+ current block was voltage-dependent and more pronounced at more depolarizing potentials, this may suggest that the interaction site of the anesthetics on INa may be from the intracellular side. Further experiments will be needed to test this hypothesis.
When interpreting the present experiments, several factors need to be considered. Our studies were performed under conditions where the Na+ channel is maximally activated. Under physiological conditions, where the resting membrane of cardiac ventricular cells are in the vicinity of -80 to -90 mV, approximately 30% of the Na+ channels are in an inactivated state. Because the inhibition of cardiac INa by anesthetics was more pronounced at membrane potentials positive to the peak potential (between +10 and -20 mV), its actions may be more potent under physiological conditions. The experiments were performed at room temperature, allowing a direct comparison with previous anesthetic studies of Na+ channels in neuronal and cardiac cells that have been conducted at room or lower temperatures [6,17]. The reported effects of these aqueous anesthetic concentrations are relatively temperature-insensitive . However, Na+ channel kinetics change with higher temperatures , and, thus it is difficult to predict how such changes might affect the anesthetic interactions reported here.
In pathophysiological conditions, the effects of volatile anesthetics to reduce cardiac INa and, consequently, conduction velocity and excitability may be enhanced. During the acute phase of myocardial ischemia, external K+ concentrations of approximately 15 mM have been reported in the ischemic zone, resulting in depolarization of heart cells to between -50 and -60 mV . Depolarization of the cell in ischemia would shift the Na+ channels to the inactivated state and, thereby, reduce the number of channels which are available for activation. This would result in the reduction of Na+ current and a slow rate of Phase 0 depolarization . The finding that halothane, at a concentration of 0.39 mM, reduced overshoot and Vmax in infarcted, but not in control canine Purkinje fibers, supports this hypothesis . Given the voltage-dependent effect of the anesthetics on INa-the effects are more potent at the depolarized potentials-anesthetics can profoundly affect Na+ currents under ischemic depolarized conditions. Thus, anesthetics might inhibit the depressed "fast" response of partially depolarized ischemic fibers more so than nonischemic fibers, thereby slowing conduction in the ischemic heart. This could either facilitate or block reentrant dysrhythmias, depending on the conduction and refractory properties of the tissue .
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