The rat hearts (Sprague-Dawley, weighing 250-300 g) were quickly excised following enflurane anesthesia and retrogradely perfused using a Langendorff perfusion system for 5 min at 37℃. Perfusion was done at a rate of 7 mL/min with a modified Tyrode solution (143 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 5 mM HEPES, and 5.5 mM glucose, pH 7.4). The perfusate was then switched to a nominally Ca²⁺-free Tyrode solution for 5 min, followed by perfusion with the same solution containing collagenase (0.4 mg/mL, Worthington type II, Worthington Biochemical Corporation, Lakewood, NJ, USA) and hyaluronidase (0.4 mg/mL, Sigma type II, Sigma-Aldrich Co., St. Louis, MO, USA). After a 10-12 min enzymatic treatment, a final perfusion was performed for 5 min with Kraftbrühe solution (10 mM taurine, 10 mM oxalic acid, 70 mM glutamic acid, 35 mM KCl, 10 mM H₂PO₄, 11 mM glucose, 0.5 mM EGTA, and 10 mM HEPES, pH 7.4). The ventricles were then cut off, minced with scissors, and agitated in a small beaker of Kraftbrühe solution. The resulting slurry was filtered through a 200-µm nylon mesh. The isolated ventricular cells were stored in Kraftbrühe solution for 1 hour at room temperature (21-22℃), then kept at 4℃, and used within a period of 8 hours. Only rod-shaped cells with apparent striations that remained quiescent in the solution containing 2 mM CaCl₂ were used for the experiments. The whole-cell voltage-clamp studies were performed at room temperature to enhance the viability of the isolated myocytes during recording.

Fifty-seven ventricular cells from 30 rats were used in this study. Single cells were used in most experiments, but replicate experiments in single cells were carried out in part in I_KI measurements. In nine cells used for measurement of I_KI, we replicated measurement of I_KI in six cells following measurements of I_to, because recovery of I_to following wash-out was usually complete. We confirmed that the results of I_KI between three single cells and six replicate cells were consistent with no alteration of I_KI by desflurane.

Isolated myocytes were allowed to settle to the bottom of a recording chamber and mounted on an inverted microscope, where bathing solutions could be exchanged. The chamber was continuously perfused at a constant rate (2 mL/min). Standard whole-cell voltage-clamp methods were used.12 In order to establish a stable baseline, an interval of 4-6 min was allowed after initiating the whole-cell recording configuration. Voltage-clamp measurements were performed using an Axopatch 200B patch clamp amplifier (Axon Instruments Inc., Foster City, CA, USA). Patch electrodes were prepared from a borosilicate glass model KIMAX-51 (American Scientific, Charlotte, NC, USA) with a two-stage micropipette puller. The pipette tips were heat-polished, using a microforge, giving a typical resistance of 2-3 MΩ when filled with an internal solution. Data acquisition was performed using a pCLAMP system version 6.0 (Axon Instruments Inc., Foster city, CA, USA).

The APs were elicited in current-clamp mode by 5-ms, 800-pA current injections at a frequency of 1 Hz.

To examine the I_to, the cells were depolarized to -40 mV for 50 ms from a holding potential of -80 mV to inactivate the Na⁺ current, then depolarized to test potentials up to +60 mV in 10-mV increments for 300 ms. To obtain more information about the possible mechanism of the desflurane-induced voltage blockade of K⁺ currents, the voltage dependence of steady-state inactivation was determined using a standard double-pulse protocol. The membrane potential was initially clamped at -80 mV and stepped to different potentials ranging from -100 to 0 mV in 10-mV increments for 500 ms followed by a 200-ms test pulse to +80 mV. The voltage clamp protocol was repeated every 2 s. Steady state inactivation data were fitted to a Boltzmann distribution of the following equation: I/I_max=I/{1+exp [(V-V_1/2)]/κ}, where I_max is the maximal current, V_1/2 is the membrane potential producing 50% inactivation, and κ is the slope factor.

To determine whether the acceleration of I_to inactivation in the presence of desflurane could be associated with time-dependent closure of the open channels, the magnitude of current inhibition at various times after the initiation of the depolarizing pulse was determined at a membrane potential of +60 mV. The outward current in the presence of each desflurane concentration, normalized to the control outward current, was plotted as a function of time after the start of depolarization. Because inactivation increases exponentially during depolarization, the equation [(I_control-I_drug)/I_control=a₁×(1-e^-t/τ)+a₂] was used to describe the rate of inhibition of I_to at each desflurane concentration where τ is the time constant of inhibition development. (I_control-I_drug)/I_control equals the amount of inhibition at time t, a₁ equals the maximum inhibition at a drug concentration, and a₂ equals the zero intercept. The extrapolation to "time zero" shows the proportion of channels inhibited before the start of the pulse, which is an indirect measure of the proportion of channels inhibited in the resting state.13

The outward currents activated by depolarizing voltage steps in rat ventricular myocytes consist of a rapidly inactivating component, I_to, and a non-inactivating, sustained component, I_sus.14,15 The sustained outward current (I_sus) was recorded with the same voltage protocol after addition of 5 mM 4-aminopyridine in a modified Tyrode solution, which preferentially blocks I_to. I_sus in adult rat ventricular myocytes is responsible for the slower phase of AP repolarization back to the resting membrane potential.16 The I_sus, comprised of I_K and a small time-independent outward current,14 remain in the presence of 4-aminopyridine.

I_KI was measured following step depolarizations from -140 to -40 mV from a holding potential of -40 mV in 20 mV increments, using a 200-ms pulse applied at 5-s intervals.

Voltage-dependent I_Ca,L was evoked by step depolarization for 500 ms from a holding potential of -40 mV to +0 mV in one step at a frequency of 0.1 Hz.

After baseline measurements, the myocytes were exposed to 6% or 12% desflurane for 2-3 min, and recovery responses were measured after washing for 2-3 min to remove the drug. Applying desflurane for 2 min produced a stable and consistent effect in the pilot experiments.

Before establishing the whole-cell recording configuration, modified Tyrode solution (140 mM NaCl, 5.4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, 10 mM glucose, adjusted to pH 7.4 with 1 N NaOH) was used as an external bathing solution. For K⁺ current measurements, a patch pipette solution (20 mM KCl, 110 mM K-aspartate, 10 mM EGTA, 10 mM HEPES, 1 mM MgCl₂, 5 mM K₂ATP, 1 mM CaCl₂, 10 mM NaCl, adjusted to pH 7.2 with 3 N KOH) was used. To measure I_sus, 5 mM 4-aminopyridine was added to the modified Tyrode solution and the pH corrected to 7.4 with HCl. In order to eliminate any confounding Ca²⁺ current from the K⁺ current studies, 0.2 mM CdCl₂ was added to the external solution after establishing the whole-cell voltage-clamp.

The inward Ca²⁺ current was measured using a patch pipette solution (30 mM CsCl, 100 mM aspartic acid, 100 mM CsOH, 10 mM BAPTA, 10 mM HEPES, 10 mM phosphocreatine, 1 mM Na₂GTP, 5 mM Na₂ATP, 10 mM glucose, 2 mM MgCl₂, adjusted to pH 7.2 with 1 M CsOH). Once whole-cell recording was achieved, the bathing solution was exchanged for a solution containing 140 mM NaCl, 5.4 mM CsCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, adjusted to pH 7.4 with 1 M CsOH. Desflurane was purchased from the Baxter Healthcare Corporation (Deerfield, IL, USA). All other chemicals were purchased from Sigma-Aldrich Co.

Before perfusion, the external bathing solution was equilibrated in a reservoir with desflurane, which was supplied by a gas dispersion tube connected to an outlet of the desflurane vaporizer for 15 min. One hundred percent O₂ (flow rate: 0.2 L/min) was passed through a desflurane vaporizer (Devapor Type M32600, Dräger AG, Lübeck, Germany). The end-tidal concentrations of desflurane were monitored using a calibrated gas analyzer (Capnomac, Datex, Helsinki, Finland). Six and 12% desflurane corresponded to 0.78±0.05 mM (n=3) and 1.23±0.03 mM (n=3) respectively. Given a Tyrode solution/gas partition coefficient of 0.27 at 22℃,17 the aforementioned desflurane concentrations are equivalent to gas phase concentrations of 7.0% and 11.0%, respectively.

One-way repeated measures of ANOVA followed by the Student-Newman-Keuls test were applied to test for significant differences among the control, drug application, and washout. Unpaired t-tests were used to compare differences in I_to currents between 6% and 12% desflurane. All values are expressed as mean±SD. A p value of less than 0.05 was considered significant. In the case of results that were not normally distributed, ANOVA of ranks was used, followed by the Student-Newman-Keuls test. In this case, results are expressed as median (IQR). Statistical comparisons were conducted with Sigmastat (SPSS Inc., Chicago, IL, USA) and all figures were prepared using Origin (Microcal Software Inc., Northampton, MA, USA).

Fig. 1 shows the concentration-dependent prolongation of AP in a rat ventricular myocyte. APD₅₀ and APD₉₀ was prolonged by 42±26% (p<0.05) and 21±13% (p<0.05), respectively, in the presence of 1.23 mM desflurane in 10 cells. AP amplitude and resting membrane potential remained unaltered (Table 1). AP duration returned to the baseline following washout.

At +60 mV, 0.78 mM desflurane reduced the control peak currents of I_to (3.17±0.86 nA) by 20±8% in 10 cells (p<0.05), and the plateau currents measured at the end of depolarization [1.12 (0.92-1.35) nA] by 9% (8.8-9.6%) (p<0.05). Desflurane 1.23 mM reduced the control peak currents of I_to (3.27±1.03 nA) by 32±7% in 10 cells (p<0.05) and the plateau currents (1.37±0.32 nA)] by 13±14% (p<0.05) (Fig. 2). Both peak and plateau currents were completely recovered after wash.

Under control conditions, the voltage required for half-inactivation (V_1/2) and slope factor (κ) for I_to were -29.3±0.6 mV and 7.8±0.5 mV, respectively, in 10 cells. Desflurane 1.23 mM significantly shifted the steady-state inactivation curve in a hyperpolarizing direction (V_1/2=-34.3±0.9 mV, κ=9.95±0.8) (Fig. 3).

Fig. 2A shows that desflurane accelerates decay of the current during the pulse, so we evaluated the effect of desflurane on I_to inactivation kinetics. Individual current records, evoked by a test pulse to +60 mV, were fitted to an exponential function. Unlike the control currents in which the decay could be fitted by a single exponential, inactivation of I_to in the presence of desflurane was better described by a double exponential. Fig. 4A and B show the quality of fit of the single and double exponential functions to the inactivation phase of I_to under the control and 1.23 mM desflurane conditions. The control _τ1 (26±10 ms) was reduced to 9±4 ms by 1.23 mM desflurane in 7 cells (p<0.05). Inactivation kinetics returned to baseline values following washout (24±8 ms) (Fig. 4C).

Fig. 5 shows inhibition of I_to between the control and in the presence of 0.78 mM and 1.23 mM desflurane during the first 60 ms of depolarization. Inhibition induced by desflurane was an exponential function of time. Both the magnitude of the maximum inhibition and its rate of development appeared to be concentration-dependent. The maximum amount of inhibition by 0.78 mM and 1.23 mM desflurane were 0.58±0.11 and 0.82±0.09 in 8 cells, respectively. The time constants for the increased decline in current were 32±13 ms and 12±7 ms, respectively, for 0.78 mM and 1.23 mM desflurane.

Desflurane (1.23 mM) had no effect on I_sus (Fig. 6). Before desflurane exposure, the baseline I_sus values at the end of the plateau at +60 mV were 1.12±0.24 nA in six cells.

At membrane potential ranging from -140 mV to -40 mV, 1.23 mM desflurane did not alter I_KI in nine cells (Fig. 7). I_KI was measured at the end of the pulse duration. Prior to desflurane exposure, the baseline values during test potential of -140 mV were -2.52±0.73 nA.

At +10 mV, 1.23 mM desflurane reduced the I_Ca,L by 40±6% in 8 cells (p<0.05) (Fig. 8). The effects of desflurane on I_Ca,L were completely reversed following washout. The baseline values before exposure to 1.23 mM desflurane were -2.24±0.83 nA.