Direct Negative Chronotropic Action of Desflurane on Sinoatrial Node Pacemaker Activity in the Guinea Pig Heart

THE heartbeat is normally initiated by an electrical excitation that originates from the sinoatrial node located in the right atrium and then propagates through the conduction system to the ventricles. The primary pacemaker cells in the sinoatrial node thus play an important role in determining the intrinsic rate of heartbeat. The sinoatrial node pacemaker cells exhibit spontaneous electrical activity which depends on a gradual depolarization of membrane potential toward the threshold level for a subsequent action potential, namely, the slow diastolic depolarization (pacemaker potential).^1–4  Multiple ionic mechanisms have been implicated in this process: an activation of inward currents such as the hyperpolarization-activated cation current (I_f),¹  T-type and L-type Ca²⁺ currents (I_Ca,T and I_Ca,L, respectively)^5–10  as well as a time-dependent decay of the delayed rectifier K⁺ current composed of rapid and slow components (I_Kr and I_Ks, respectively).^11,12  In addition, recent evidence indicates that local subsarcolemmal Ca²⁺ releases from the sarcoplasmic reticulum also contribute to the rhythmic activity of sinoatrial node. Local subsarcolemmal Ca²⁺ releases are thought to stimulate the forward mode of the electrogenic Na⁺/Ca²⁺ exchange current (I_NCX) that generates an inward current to depolarize cell membrane toward the action potential threshold.¹³  At present, the relative contributions of sarcolemmal ion channels and local subsarcolemmal Ca²⁺ releases to sinoatrial node automaticity have yet to be fully elucidated.

The intrinsic activity of sinoatrial node that controls heart rate is modulated by the autonomic nervous system; sympathetic β-adrenergic stimulation accelerates the electrical activity of sinoatrial node and thereby increases heart rate, whereas parasympathetic muscarinic stimulation decelerates the sinoatrial node activity and heart rate.⁴  Some of the ionic currents are targets for regulation by autonomic nervous system. For example, sympathetic activation enhances I_f and I_Ca,L, which seems to be importantly involved in mediating the increases in sinoatrial node automaticity and heart rate.^1,4,7 

Much attention has been given to the effects of volatile anesthetics on heart rate, which could be a major determinant of the myocardial oxygen consumption.^14,15  A number of studies have demonstrated that desflurane administration is accompanied by periods of sympathetic excitation and tachycardia in healthy volunteers,^16–19  patients,²⁰  and experimental animals^21,22  when the inspired concentration is increased rapidly. Accordingly, it has been demonstrated that the induction of anesthesia with desflurane without opioids is associated with a greater risk of myocardial ischemia in patients undergoing coronary artery bypass surgery compared with the risk in patients given only sufentanil.²⁰  This action of desflurane is considerably different from that of another halogenated volatile anesthetic, sevoflurane, which exerts a relatively small influence on heart rate.^14,15 

At present, there is little information available regarding the direct effects of desflurane on the intrinsic sinoatrial node automaticity and its underlying ionic mechanisms. We hypothesized that whereas desflurane produces a transient increase in heart rate through sympathetic activation in vivo, desflurane itself has a direct negative chronotropic action on sinoatrial node pacemaking activity by inhibiting multiple ionic currents, such as I_f, I_Ca,T, I_Ca,L, and I_NCX. In this study, the effects of desflurane on cardiac automaticity were systematically investigated in sinoatrial node cells and ex vivo and in vivo hearts.

All experimental procedures and protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Shiga University of Medical Science (Otsu, Shiga, Japan; approval number; 2010-12-5), and the investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996). Single cells were isolated from 5- to 8-week-old female Hartley guinea pigs (250 to 400 g body weight) with the use of an enzymatic dissociation procedure as described previously.^10,23,24  Cells obtained from the entire sinoatrial node region showed heterogeneity in terms of their morphology, and spindle-shaped cells, which are assumed to be primary pacemaker cells in sinoatrial node,²⁵  were selected for the present experiments.

Perforated and conventional (ruptured) whole-cell patch-clamp techniques^26,27  were used to record the spontaneous action potentials and ionic currents, respectively, at 36 ± 1°C.^10,11 

The spontaneous action potential was recorded in normal Tyrode solution containing: 140 mM of NaCl, 5.4 mM of KCl, 1.8 mM of CaCl₂, 0.5 mM of MgCl₂, 0.33 mM of NaH₂PO₄, 5.5 mM of glucose, and 5 mM of HEPES (pH adjusted to 7.4 with NaOH). The pipette solution contained: 70 mM of potassium aspartate, 50 mM of KCl, 10 mM of KH₂PO₄, 1 mM of MgSO₄, and 5 mM of HEPES (pH adjusted to 7.2 with KOH), to which amphotericin B (Wako Pure Chemical Industries, Osaka, Japan) was added to obtain a final concentration of 100 μg/ml.^10,24 

I_f was recorded with a K⁺-rich pipette solution containing: 70 mM of potassium aspartate, 50 mM of KCl, 10 mM of KH₂PO₄, 1 mM of MgSO₄, 5 mM of adenosine 5’-triphosphate (disodium salt; Sigma, St Louis, MO), 0.1 mM of guanosine 5’-triphosphate (dilithium salt; Roche Diagnostics GmbH, Mannheim, Germany), 5 mM of EGTA, 1.2 mM of CaCl₂, and 5 mM of HEPES (pH adjusted to 7.2 with KOH). The bath solution was normal Tyrode solution supplemented with 2 mM NiCl₂ and 0.5 mM BaCl₂, which eliminated the voltage-dependent Ca²⁺ current and the Ba²⁺-sensitive K⁺ current, respectively. I_f was elicited by 2,000-ms hyperpolarizing steps applied from a holding potential of −40 mV to test potentials of −50 to −140 mV. I_f was measured as the difference between the instantaneous and steady-state current levels during each voltage step.²⁸  The I_f conductance (g_f) was calculated at each test potential according to the following equation: g_f = I_f/(V_t − V_rev), where I_f is current density, V_t is test potential, and V_rev is reversal potential for I_f. The voltage dependence of I_f activation was assessed by fitting g_f to a Boltzmann equation: g_f = g_f,max/(1 + exp((V_t − V_h)/k)), where g_f,max is the fitted maximal conductance of I_f, V_h is the voltage at half-maximal activation, and k is the slope factor.

I_Ca,T and I_Ca,L were measured with a Cs⁺-rich pipette solution containing: 90 mM of cesium aspartate, 30 mM of CsCl, 20 mM of tetraethylammonium chloride, 2 mM of MgCl₂, 5 mM of adenosine 5’-triphosphate (Mg salt; Sigma), 5 mM of phosphocreatine (disodium salt; Sigma), 0.1 mM of guanosine 5’-triphosphate (dilithium salt; Roche), 5 mM of EGTA, and 5 mM of HEPES (pH adjusted to 7.2 with CsOH). The bath solution was a Na⁺- and K⁺-free solution containing: 140 mM of Tris-hydrochloride, 1.8 mM of CaCl₂, 0.5 mM of MgCl₂, 5.5 mM of glucose, and 5 mM of HEPES (pH adjusted to 7.4 with Tris-base), to which tetrodotoxin (Wako) was added at a concentration of 10 μM to eliminate the possible contamination of the voltage-gated Na⁺ conductance. Depolarizing voltage steps (200 ms in duration) were initially applied from a holding potential of −90 mV to test potentials of −70 to +40 mV to activate the voltage-dependent Ca²⁺ current (I_Ca, composed of I_Ca,T and I_Ca,L), and then depolarizing voltage steps were applied from a holding potential of −60 mV to test potentials of −50 to +40 mV to activate I_Ca,L in a given cell.^5,8–10,29 I_Ca,T was obtained by digitally subtracting I_Ca,L from I_Ca at each test potential. The voltage dependence of I_Ca,T and I_Ca,L activation was evaluated by the conductance (g_Ca,T or g_Ca,L)–voltage relationships that were fitted to a Boltzmann equation: g_Ca,T (or g_Ca,L) = g_Ca,T,max (or g_Ca,L,max)/(1 + exp((V_h − V_t)/k)), where g_Ca,T,max and g_Ca,L,max are the fitted maximal conductance for I_Ca,T and I_Ca,L, respectively.

I_NCX was recorded with a modified Cs⁺-rich pipette solution containing: 90 mM of cesium aspartate, 20 mM of CsCl, 10 mM of NaCl, 20 mM of tetraethylammonium chloride, 2 mM of MgCl₂, 5 mM of adenosine 5’-triphosphate (Mg salt; Sigma), 5 mM of 1,2-bis(O-aminophenoxy) ethane- N,N,N’,N’-tetraacetic acid, 1.75 mM of CaCl₂, and 10 mM of HEPES (pH adjusted to 7.2 with CsOH; pCa was estimated to be 6.82 [approximately 150 nM]).^30,31  The bath solution contained: 140 mM of NaCl, 5.4 mM of CsCl, 1.8 mM of CaCl₂, 0.5 mM of MgCl₂, 5.5 mM of glucose, 0.02 mM of ouabain (to inhibit Na⁺/K⁺ pump), 0.01 mM of ryanodine (to inhibit ryanodine receptor), 0.01 mM of nifedipine (to inhibit I_Ca,L), and 5 mM of HEPES (pH adjusted to 7.4 with NaOH).^32,33  With these bath and pipette solutions, major overlapping currents were blocked, and the reversal potential of I_NCX (E_NCX) is calculated to be −33.6 mV, using the Nernst equation: E_NCX = 3E_Na − 2E_Ca, where E_Na and E_Ca represent the equilibrium potentials for Na⁺ and Ca²⁺, respectively. Membrane currents were recorded during the hyperpolarizing phase (+40 to −120 mV) of voltage-ramp protocol (dV/dt = −0.25 V/s).³⁴  After recording the baseline current, the cell was exposed to desflurane at 6 or 12%, which was subsequently washed out. The cell was then exposed to 5 mM NiCl₂ to totally block I_NCX. The fractional block induced by desflurane was expressed as percent of total I_NCX.

I_Kr was recorded with a K⁺-rich pipette solution during superfusion with normal Tyrode solution supplemented with 1 μM HMR 1556 (Hoechst Marion Roussel, Frankfurt, Germany) and 0.4 μM nisoldipine (Sigma) to minimize I_Ks and I_Ca,L, respectively. HMR 1556 at 1 μM fully inhibits I_Ks without producing significant effects on I_Kr.³⁵ I_Kr was activated by 250-ms depolarizing steps to test potentials of −40 to +40 mV applied from a holding potential of −50 mV. E-4031 was used to confirm that the current recorded under these experimental conditions was due to I_Kr.¹¹ 

I_Ks was measured with a K⁺-rich pipette solution during superfusion with normal Tyrode solution supplemented with 5 μM E-4031 (Wako) and 0.4 μM nisoldipine (Sigma) to eliminate I_Kr and I_Ca,L, respectively.^10,11 I_Ks was activated during 2,000-ms depolarizing steps to test potentials of −40 through +50 mV applied from a holding potential of −50 mV.

The amplitudes of I_Kr and I_Ks tail currents, elicited on return to the holding potential (−50 mV), reflect the degree of current activation at the preceding depolarizing test potential, and voltage dependence of I_Kr and I_Ks activation was evaluated by fitting the amplitude of tail current (I_tail) to a Boltzmann equation: I_tail = I_tail,max/(1 + exp((V_h − V_t)/k)), where I_tail,max is the fitted maximal tail current density of I_Kr or I_Ks.

The current amplitude was normalized with reference to the cell membrane capacitance and was expressed as current density (in pA/pF). The zero-current level is indicated to the left of the current records by a horizontal line.

Desflurane (Baxter, Deerfield, IL) was equilibrated in bathing solutions in a reservoir by passing air (flow rate, 0.5 l/min) through a calibrated vaporizer for at least 15 min before entering a recording chamber for the patch-clamp experiments.³⁶  In the present experiments, the effects of desflurane on spontaneous action potentials and ionic currents were examined at 6 and 12%, which have been reported to correspond to approximately 1 and 2 minimum alveolar concentrations, respectively, in guinea pigs.³⁷  The concentration of desflurane in a recording chamber superfused with normal Tyrode solution at 36 ± 1°C was measured to be 0.35 ± 0.05 mM (n = 4) and 0.82 ± 0.11 mM (n = 4) for 6 and 12% desflurane, respectively, using gas chromatography.

Electrocardiogram was recorded from Langendorff-perfused guinea pig hearts with the use of two silver electrodes attached to the ventricular apex and to the metal aortic cannula, as described previously.^10,24 

Guinea pigs were initially anesthetized via an intraperitoneal injection of sodium pentobarbital (80 mg/kg) and were artificially ventilated through a tracheotomy with a respirator (tidal volume, 2.5 ml; rate, 60/min; flow rate, 0.8 l/min of an air–oxygen mixture [60% inspired oxygen]). The surface electrocardiogram was recorded by placing wire electrodes in the subcutaneous spaces in a lead II configuration. A period of approximately 30 min was allowed for the stabilization of surface electrocardiogram recordings, and desflurane was then successively administered in 0.8 l/min of an air–oxygen mixture, at concentrations of 6 and 12% in a randomized order for a period of 10 to 20 min for each concentration. In some experiments, propranolol (Sigma), dissolved in sterile saline, was administered intraperitoneally, at a concentration of 10 mg/kg to induce β-adrenergic blockade before the inhalation of desflurane.³⁸ 

The Maltsev and Lakatta model³⁹  was coded using simBio⁴⁰  and was used for a computer simulation study. The spontaneous action potentials of sinoatrial node cells in the presence of desflurane were simulated by decreasing the conductances for I_f, I_Ca,T, I_Ca,L, I_NCX, I_Kr, and I_Ks by the same degree as detected in the present voltage-clamp experiments, without altering any other parameters.

Results are presented as the means ± SD, with the number of animals and experiments indicated by N and n, respectively. Two to three sinoatrial node cells (n) were used for the patch-clamp experiments from one cell isolation (animal, N) in a given protocol. The effects of one or two concentrations of desflurane were measured in each experiment using sinoatrial node cells (patch-clamp), isolated hearts (Langendorff perfusion), or animals (in vivo inhalation), and one measurement was obtained for each concentration of desflurane in a given experiment. The error bars in the figures indicate SD with n given in parentheses. Our previous study showed that the volatile anesthetic sevoflurane (3%) significantly decreases the spontaneous firing rate of guinea pig sinoatrial node cells by 20%.¹⁰  A power analysis predicted that a group size of n = 6 was necessary to detect differences of 20% between group means of spontaneous firing rate of sinoatrial node cells and heart rate in ex vivo Langendorff-perfused and in vivo hearts, assuming a statistical power of 0.8 at a significance level (α) of 0.05 (StatMate Version 2.0; GraphPad Software, La Jolla, CA). A group size of n = 4 would allow for the detection of a difference of 30% between group means of ionic currents. Statistical comparisons were performed using a one-way ANOVA, followed by Dunnett test (Prism Version 5.0; GraphPad). We used two-tailed hypothesis testing for all tests. P value less than 0.05 was considered to be statistically significant.

We first examined the effects of desflurane on sinoatrial node automaticity using the whole-cell patch-clamp method. In the experiments shown in figure 1, a spontaneously active sinoatrial node cell was successively exposed to 6 and 12% desflurane for approximately 5 min, with a washout period of approximately 8 min. The firing rate of spontaneous action potentials was reduced by desflurane at both concentrations in a reversible manner (fig. 1, A, lower panel, and B). Under control conditions, the firing rate of spontaneous action potentials averaged 183.2 ± 15.1/min (n = 13, N = 5), which was significantly decreased to 154.1 ± 14.5/min (n = 11, N = 5) and 132.7 ± 20.6/min (n = 10, N = 5) by 6 and 12% desflurane, respectively (fig. 1C).

The sinoatrial node pacemaker cells generate a slow diastolic depolarization (pacemaker potential) that drives the membrane potential toward a threshold level for subsequent action potential (fig. 1B). The slope of the diastolic depolarization, namely the diastolic depolarization rate (DDR), determines the time interval between successive action potentials and acts as the major determinant of the firing rate.^41,42  As illustrated in figure 1D, DDR was significantly reduced from the control value of 76.8 ± 16.1 mV/s (n = 13, N = 5) to 61.1 ± 12.3 mV/s (n = 11, N = 5) and 44.2 ± 11.1 mV/s (n = 10, N = 5), during the exposure to 6 and 12% desflurane, respectively. Thus, a decrease in the firing rate was accompanied by a reduction in DDR in spontaneous action potentials of sinoatrial node cells during the administration of desflurane, which suggests that desflurane depresses the DDR and thereby reduces the firing rates of sinoatrial node action potentials.

We then examined the effects of desflurane on the ionic currents that are involved in the electrical activity of sinoatrial node pacemaker cells, namely I_f, I_Ca,T, I_Ca,L, I_NCX, I_Kr, and I_Ks.^2–4  Molecular genetic and pharmacological studies support the view that I_f provides an inward current that determines the DDR, which in turn controls the spontaneous firing rate of sinoatrial node action potentials.^1–4,43 Figure 2A illustrates superimposed current traces of I_f recorded during voltage-clamp steps to test potentials of −50 through −140 mV before and during exposure to 12% desflurane, and after its washout. Desflurane at 12% modestly decreased the amplitude of I_f in a reversible manner. We evaluated the effects of desflurane on I_f by constructing the conductance–voltage relationships (fig. 2B), assuming the reversal potential to be −24 mV. This analysis confirmed that I_f conductance was modestly but significantly reduced by desflurane at 12% but not at 6% (fig. 2C). Neither the half-activation voltage nor the slope factor was significantly affected by either concentration of desflurane (table 1).

Because I_f is a mixed cationic conductance carried by Na⁺ and K⁺ under normal physiological conditions, its reversal potential reflects the relative permeability of I_f to Na⁺ and K⁺.^4,29  The reversal potential of I_f was measured by tail currents of fully activated I_f in the absence and presence of desflurane. The membrane was first hyperpolarized to −130 mV for 2,000 ms to fully activate I_f, and was then clamped back to various test potentials between +5 and −45 mV (fig. 3). The tail current reversed at approximately −25 mV under both conditions (fig. 3A), and this may be more clearly seen in figure 3B, in which amplitude of the tail currents is plotted against the test potentials. There were no appreciable differences in the reversal potentials for I_f in the absence and presence of desflurane (−24.2 ± 2.2 mV vs. −24.4 ± 2.1 mV, n = 6, N = 3). This observation indicates that desflurane did not appreciably alter the ion selectivity of the channel for Na⁺ and K⁺.

Experimental evidence has been presented to show that both I_Ca,T and I_Ca,L contribute to the slow diastolic depolarization and spontaneous activity of sinoatrial node pacemaker cells.^2–4 I_Ca,T and I_Ca,L exhibit different voltage dependency for inactivation and activation; I_Ca,T is available with a holding potential of −90 mV but is fully inactivated at −60 mV, whereas I_Ca,L is activated during depolarizing steps from a holding potential of −60 mV. Therefore, both I_Ca,T and I_Ca,L are measured during depolarizing steps applied from a holding potential of −90 mV, whereas I_Ca,L is measured during depolarization from a holding potential of −60 mV.^{5,6,8–10,29} 

Figure 4A shows the superimposed current traces for I_Ca, I_Ca,L, and I_Ca,T, recorded before and after a 5-min exposure to 12% desflurane at various test potentials. The peak potentials of I_Ca,T and I_Ca,L were −30 mV and −10 mV, respectively, similar to those reported in mouse sinoatrial node cells,^8,9,29  and they were not affected by desflurane (fig. 4, B and C). Desflurane at 6% significantly reduced the maximal conductance for I_Ca,T and I_Ca,L to 81.1 ± 6.1% and 80.2 ± 9.6% (n = 4, N = 2) of the control values, respectively. Desflurane at 12% also significantly decreased the maximal conductance for I_Ca,T and I_Ca,L to 70.4 ± 11.1% and 68.6 ± 10.7% (n = 4, N = 2) of the control levels, respectively (fig. 4D). However, both the half-activation voltage and the slope factor were not significantly affected by desflurane (table 1).

The effects of desflurane on I_NCX, which has also been proposed to contribute to the pacemaker automaticity in sinoatrial node cells,⁴  were then examined. I_NCX operates in a bidirectional way under the present ionic conditions, where Na⁺ and Ca²⁺ were present in both the external and pipette solutions. Figure 5 shows results of a representative experiment examining the effect of 12% desflurane on I_NCX. After recording the baseline current, the cell was exposed to 12% desflurane, which was subsequently washed out. The cell was then exposed to 5 mM NiCl₂ to fully block I_NCX (fig. 5A). Figure 5C illustrates current–voltage relationships for I_NCX inhibited by 12% desflurane (a–b) and 5 mM NiCl₂ (a–d), obtained by digitally subtracting the current traces shown in figure 5B. Both current–voltage relationships crossed the voltage axis at approximately −30 mV, which is close to the predicted reversal potential of I_NCX (−33.6 mV), supporting the view that the measured currents were primarily due to I_NCX.³²  The effects of 6 and 12% desflurane on I_NCX in the forward and reverse modes were assessed by measuring the fractional block at 60 mV on either side of the reversal potential.³³  As summarized in figure 5D, desflurane at 6 and 12% significantly reduced both the forward and reverse mode I_NCX. It should also be noted that the forward and reverse mode I_NCX were inhibited by similar degrees by each concentration of desflurane (fig. 5D).

We next investigated the effects of desflurane on I_Kr and I_Ks, which are the major repolarizing outward currents in mammalian sinoatrial node cells.^4,10–12  The effect of desflurane on I_Kr was examined at test potentials ranging from −40 to +40 mV in the presence of the I_Ks blocker HMR 1556 (1 μM)³⁵  and I_Ca,L blocker nisoldipine (0.4 μM). Figure 6A shows superimposed current traces during 250-ms depolarizing steps to −10 mV applied from a holding potential of −50 mV under control conditions and during administration of 12% desflurane, initially without and then with 5 μM E-4031. I_Kr was determined as E-4031–sensitive difference current in these experiments. Figure 6B illustrates I_Kr at a test potential of −10 mV under control conditions and in the presence of desflurane (12%), as obtained by digital subtraction of the two appropriate current traces as indicated. Single exponential fit of the tail current showed that the deactivation kinetics of I_Kr at −50 mV was not appreciably affected by desflurane (control, τ = 193 ± 23 ms, n = 8, N = 3; 12% desflurane, τ = 197 ± 19 ms, n = 6, N = 3; fig. 6B, inset). The amplitudes of I_Kr tail currents at various test potentials in the absence and presence of 6 and 12% desflurane were plotted and fitted with the Boltzmann equation (fig. 6C). As demonstrated in figure 6D, there were no appreciable differences in the peak amplitude of I_Kr tail currents, as estimated by Boltzmann fit, in the absence (control) and presence of desflurane at 6 and 12%. Furthermore, half-activation voltage and slope factor for I_Kr were not affected by desflurane (table 1).

In a separate set of experiments, we measured I_Kr at test potentials of −40 through +40 mV as an E-4031 (5 μM)–sensitive current in the absence of desflurane using the same voltage-clamp protocol as shown in figure 6 (see fig. 1, Supplemental Digital Content 1, https://links.lww.com/ALN/B33). The deactivation time constant of I_Kr, determined in the absence of desflurane, averaged 216 ± 21 ms (n = 6, N = 3; see fig. 1, A and B, Supplemental Digital Content 1, https://links.lww.com/ALN/B33), which is not significantly different from that measured for I_Kr in the presence of desflurane. The amplitudes of I_Kr tail currents at each test potential were similar in the absence and presence of desflurane (see fig. 1C, Supplemental Digital Content 1, https://links.lww.com/ALN/B33). In addition, half-activation voltage and slope factor for I_Kr in the absence of desflurane averaged −26.3 ± 2.3 mV and 6.0 ± 0.6 mV (n = 6, N = 3), respectively, and these values were not significantly different from those of I_Kr, determined in the presence of desflurane (table 1). Thus, the properties and amplitudes of I_Kr identified as an E-4031 (5 μM)–sensitive current were similar in the absence and presence of desflurane, thereby supporting the view that desflurane has no appreciable effect on the inhibitory actions of E-4031 on I_Kr.

We next investigated the effects of desflurane on I_Ks in sinoatrial node cells in the presence of 5 μM E-4031 and 0.4 μM nisoldipine. As demonstrated in figure 7A, 12% desflurane markedly reduced the amplitudes of I_Ks, activated during 2,000-ms depolarizing steps to −40 to +50 mV, without appreciably affecting the deactivation kinetics of I_Ks measured at −50 mV (control, τ = 143 ± 25 ms, n = 8, N = 3; 12% desflurane, τ = 147 ± 29 ms, n = 6, N = 3; fig. 7A, inset). Figure 7B shows the current–voltage relationships for I_Ks tail currents recorded in the absence and presence of 6 and 12% desflurane. Desflurane at 6 and 12% significantly decreased the maximal amplitude of I_Ks tail current from the control value of 7.69 ± 1.97 pA/pF (n = 8, N = 3) to 4.23 ± 0.72 pA/pF (n = 6, N = 3) and 2.08 ± 0.65 pA/pF (n = 6, N = 3), respectively (fig. 7C), without producing appreciable effects on half-activation voltage and slope factor (table 1).

We conducted experiments to examine the effects of desflurane on the heart rates ex vivo in Langendorff-perfused hearts and in vivo in desflurane-anesthetized guinea pigs (fig. 8). Desflurane significantly reduced the heart rates in the Langendorff-perfused hearts, in a similar way as in sinoatrial node cells (figs. 8, A and D, and 1C). In contrast, in whole animals, the heart rate was transiently and significantly increased on the inhalation of 12% desflurane in vivo (fig. 8, B and D). As demonstrated in figure 8C, in guinea pigs pretreated with the β-adrenergic blocker propranolol (10 mg/kg, via intraperitoneal injection), the heart rate was monotonically and significantly reduced during inhalation of 12% desflurane, without a transient increase (fig. 8D). It should be noted that the basal heart rate in guinea pigs pretreated with propranolol was significantly lower than that without propranolol pretreatment (196.4 ± 6.9/min, n = 12 vs. 220.8 ± 15.2/min, n = 12).

Our final investigations explored the implications of desflurane-induced changes in ionic conductances for its negative chronotropic action, using the sinoatrial node cell model of Maltsev and Lakatta³⁹  (fig. 9). The firing rate of spontaneous action potentials was decreased (fig. 9A) by simulating the inhibitory effects of 6% desflurane on I_f, I_Ca,T, I_Ca,L, I_NCX (forward mode), I_Kr, and I_Ks in the sinoatrial node cell model (fig. 9, C–H). It is interesting to note that net inward current during diastolic depolarization phase, which is responsible for DDR, is modestly but appreciably decreased in the computer simulation of desflurane effect on sinoatrial node action potentials (fig. 9B). Figure 9I compares the percent decreases in the firing rate and DDR induced by 6% desflurane between patch-clamp experiments and computer simulations. The sinoatrial node cell model was able to reasonably well simulate the experimental data concerning the inhibitory effects of desflurane on the spontaneous action potentials of sinoatrial node cells (fig. 1).

The present patch-clamp experiments revealed that desflurane produces a direct inhibitory action on sinoatrial node pacemaker activity by depressing the diastolic depolarization (fig. 1). We previously demonstrated that sevoflurane, another halogenated volatile anesthetic, also suppresses DDR and thereby decelerates the spontaneous activity of sinoatrial node pacemaker cells.¹⁰  It is generally accepted that the slow diastolic depolarization is driven by a net inward current produced by a complex but coordinated interaction of multiple inward and outward ionic currents, such as I_f, I_Ca,T, I_Ca,L, I_NCX, I_Kr, and I_Ks, although the relative contribution of each ionic current remains to be fully elucidated.⁴  However, clinical evidence has shown that loss-of-function mutations and/or pharmacological blockade of I_f,^44,45 I_Ca,T,⁴⁶  or I_Ca,L^7,47  result in a sinus dysfunction and/or bradycardia. Experiments using molecular genetic and/or pharmacological approaches have supported the functional significance of I_f, I_Ca,T, I_Ca,L, and I_NCX in the regulation of DDR and spontaneous activity of sinoatrial node cells.^1–10,43  It is therefore reasonable to assume that the inhibitory action of desflurane on multiple ionic currents, including I_f, I_Ca,T, I_Ca,L, and I_NCX (figs. 2–7), is responsible for the reduction of DDR and deceleration of spontaneous activity in sinoatrial node cells. The computer simulation study also supports the implication of the desflurane-induced changes in ionic currents for its negative chronotropic action on sinoatrial node cells (fig. 9).

It is generally believed that the inhibitory action of volatile anesthetics on ion channel/transporter arises from direct binding to channel/transporter proteins or is mediated by altering the behavior and dynamics of plasma membrane lipids. In addition, there is good evidence that volatile anesthetics indirectly affect the function of ion channels by modifying regulatory signaling molecules/pathways.⁴⁸  The present experiments found that the degree of inhibition by desflurane varied among the ionic currents in sinoatrial node cells; desflurane potently inhibited I_Ks (fig. 7), moderately inhibited I_Ca,T, I_Ca,L (fig. 4), and I_NCX (fig. 5), modestly inhibited I_f (figs. 2 and 3), but had little effect on I_Kr (fig. 6). Our previous study shows that sevoflurane also inhibits I_f, I_Ca,T, I_Ca,L, and I_Ks to different degrees in sinoatrial node cells¹⁰ ; sevoflurane modestly inhibits I_f, whereas potently suppressing I_Ca,T, I_Ca,L, and I_Ks (see table 1, Supplemental Digital Content 1, https://links.lww.com/ALN/B33). Among these ionic currents, I_f seems to be less sensitive to inhibition by desflurane and sevoflurane. In contrast, I_Ks seems to be more sensitive to inhibition by these two volatile anesthetics. These differences in the sensitivity of ionic currents to inhibition by desflurane and sevoflurane suggest that the inhibitory mechanisms of these volatile anesthetics differ among the individual ionic currents. Future studies are needed to elucidate the precise mechanisms underlying the actions of volatile anesthetics, which may include direct interaction with ion channels and/or the indirect modification of regulatory signaling molecules/pathways.

Interestingly, I_Kr in guinea pig sinoatrial node cells is insensitive to inhibition by desflurane (fig. 6). Previous investigations have also shown that both isoflurane and sevoflurane have a minimal effect on I_Kr in guinea pig ventricular myocytes^49–51  and its molecular correlate human ether-a-go-go-related gene channels exogenously expressed in Chinese hamster ovary cells.⁵¹  Although I_Kr and human ether-a-go-go-related gene channels are susceptible to blockade by a wide variety of compounds, including clinical drugs, associated with drug-induced long QT syndrome,⁵²  it seems likely that I_Kr and human ether-a-go-go-related gene channels are less sensitive to inhibition by the halogenated volatile anesthetics.

The negative chronotropic effect of desflurane was also observed in the isolated Langendorff perfusion model (fig. 8), similar to the previous observations.³⁷  Experimental results obtained from sinoatrial node cells and isolated Langendorff-perfused hearts, where the neural and humoral influences are mostly abolished, strongly supports the view that desflurane has a direct inhibitory effect on the intrinsic cardiac automaticity produced by sinoatrial node pacemaker cells.

Both sympathetic and parasympathetic branches of the autonomic nervous system densely innervate sinoatrial node, and thereby control the electrical activity of the pacemaker cells in the sinoatrial node.⁴  The in vivo heart rate is therefore determined by the interaction of the autonomic nervous tone with the intrinsic electrical activity of sinoatrial node pacemaker cells. A number of investigations have demonstrated that a rapid increase in desflurane concentration elicits a transient increase in heart rate and mean arterial pressure in both humans and experimental animals, which is ascribed to the activation of sympathetic nervous system.^14,16–22  When the autonomic nervous system is pharmacologically blocked in dogs, desflurane inhalation even at high concentrations (1.75 minimum alveolar concentration) produces negative chronotropic and inotropic actions.²²  In the present experiments, the heart rate in guinea pig was transiently and significantly increased during inhalation of 12% desflurane, whereas heart rate was monotonically and significantly decreased during desflurane inhalation after β-adrenergic blockade (fig. 8), indicating that β-adrenergic-mediated increase in the heart rate occurs in guinea pig during higher inspired concentration of desflurane. These findings suggest that guinea pig also represents a suitable animal model for investigating the mechanisms for desflurane-induced changes in heart rate, like other experimental animal models such as dog and swine.^21,22  It therefore seems reasonable to extrapolate the present results to explain the action of desflurane on heart automaticity in the clinical settings.

Several mechanisms have been proposed to explain the desflurane-induced augmentation in sympathetic outflow that occurs upon the rapid increase in the inspired concentrations of desflurane, including (1) a reflex response initiated by irritant receptors in the airway, (2) a baroreflex response to the lower arterial pressure caused by higher concentrations of desflurane, and (3) a direct stimulation of the sympathetic medullary centers.^16–19  In the clinical settings, the desflurane-induced increases in heart rate and arterial pressure can be effectively attenuated by the administration of the drugs that interfere with sympathetic responses, namely, the β-adrenergic blocker esmolol, α₂-adrenergic agonist clonidine, and μ-opioid fentanyl.^18,19  β-adrenergic blockers have been shown to have a direct peripheral action, whereas α₂-adrenergic agonists suppress the sympathetic impulses arising from the brain.^18,19  However, fentanyl has been suggested to attenuate the desflurane-induced increases in heart rate and blood pressure by producing a vagomimetic action (vagal-sympathetic-accentuated antagonism) and/or by reducing the efferent sympathetic activity to the heart.¹⁸  Because clinically used concentrations of desflurane have a direct negative chronotropic effect on the heart (figs. 1 and 8), it seems reasonable to blunt the tachycardia induced by higher concentrations of desflurane by reducing the sympathetic influence to the heart. The present study may also draw attention to the possibility that the negative chronotropic action of desflurane could be significant in patients being treated with pharmacological blockers for ion channels involved in sinoatrial node pacemaking or associated with compromised channel functions caused by gene mutations.

In conclusion, the present investigation demonstrates that clinically used concentrations of desflurane have a direct inhibitory effect on sinoatrial node automaticity, which is mediated through the inhibition of ionic currents, and may provide an electrophysiological basis for the effectiveness of drugs that prevent the sympathetic influences to the heart in blunting desflurane-induced tachycardia in clinical practice.

This study was supported by a Grant-in-Aid for Young Scientists (B) (No. 24791590 to Dr. Kojima) and Grant-in-Aid for Scientific Research (C) (No. 24592335 to Dr. Kitagawa, Nos. 24591086 and 25460287 to Dr. Matsuura) from The Japan Society for the Promotion of Science (Tokyo, Japan).

The authors declare no competing interests.