Myocardial Depressant Effects of Desflurane: Mechanical and Electrophysiologic Actions In Vitro

According to a procedure approved by the Yonsei University College of Medicine Animal Research Committee (Seoul, Korea), the heart was removed from 400- to 450-g female GPs or rats (Sprague-Dawley) after halothane anesthesia (2.5–4 vol%). Next, the right ventricular papillary muscles were excised, mounted horizontally in a tissue bath, and superfused (8 ml/min) at 37°C with modified standard Tyrode solution (118 mm NaCl, 5 mm KCl, 2 mm CaCl², 1.2 mm MgSO⁴, 25 mm NaHCO⁻³, 11 mm glucose, 0.10 mm EDTA). EDTA was used to chelate any trace contaminant heavy metals. The solution was circulated through a bath from an unsealed reservoirs through which a mixture of 95% O²and 5% CO²was bubbled (flow rate: 0.5 l/min) to maintain a pH of 7.4 ± 0.05. The tendinous end of the papillary muscle was attached to a Grass FT03 force transducer (Grass Instruments, Quincy, MA) while the other end of the muscle was pinned to the bottom of the tissue bath. The length of the muscle was adjusted to the lowest resting force at which maximum twitch force was obtained. The muscles were field-stimulated using a Grass S44 stimulator (Grass Instruments) at 120% of stimulus threshold at 0.5 Hz for 60 min for stabilization. After a 15-min rest, a rested state (RS) contraction was elicited, followed by stimulation rates of 0.1, 0.5, 1, 2, and 3 Hz, sequentially. At each stimulation rate, the duration of stimulation was preset and kept constant during the experiment. After baseline measurements, the muscles were successively exposed to 6% and 12% desflurane (vaporizer setting) or 1.15% and 2.3% isoflurane (vaporizer setting), each for 15 min, at which the response were recorded. In pilot experiments, a 15-min application of desflurane was sufficient to produce a stable and consistent effect. Recovery responses were measured after a 20-min washout period. Effects on contractile function were estimated from the peak force (PF) and maximum rate of force development (dF/dt-max).

To examine the effects of desflurane on catecholamine release from the ventricular myocardium,10both receptor blockade and catecholamine depletion were used. In an initial control study, a positive inotropic response to tyramine (30 μm) was used to verify that the GP ventricular myocardium has catecholamine-containing sympathetic nerve endings.

In the first experimental series, muscles were exposed to phentolamine (1 μm) and propranolol (1 μm) to prevent catecholamine activation of α and β adrenoceptors on the cardiac membrane. After a 15-min pretreatment and measurement of baseline contractile forces, contractions were recorded after a 15-min application of desflurane. Recovery was observed after a 20-min washout period (phentolamine and propranolol were maintained throughout). In a separate experimental group, a lack of response to a 15-min application of tyramine (30 μm) was used to verify that the blockade of adrenoceptors by phentolamine and propranolol was adequate.

In the second experimental series, the ventricular myocardium was depleted of catecholamines by an intraperitoneal injection of reserpine (4 mg/kg) into the GPs 24 h before the experiment.16The depletion of catecholamine was verified by the absence of a positive inotropic response to an occasional threefold increase of stimulus voltage during the 60-min stabilization period, because high stimulus intensity can activate catecholamine release from sympathetic nerve endings. Contractile measurements were taken during control conditions, after a 15-min application of 6% desflurane, and after a 20-min washout. In a separate experimental group, the depletion of catecholamine by reserpine was also verified by a lack of contractile enhancement after treatment with tyramine.

Rat papillary muscles were used to evaluate the effects of desflurane on contractions caused almost exclusively by Ca²⁺release from the SR. After baseline measurements at RS, the muscles were exposed to 12% desflurane for 15 min. Recovery responses were measured after a 20-min washout.

Experiments using GP papillary muscles in low-Na⁺(25 mm) Tyrode solution (25 mm NaHCO⁻³, 5 mm KCl, 2 mm CaCl², 1.2 mm MgSO⁴, 11 mm glucose, 0.1 mm EDTA, 234 mm sucrose) were performed to confirm the effect of desflurane in rat force studies and to rule out the species-dependent differences in SR Ca²⁺release. After obtaining baseline measurements at RS after a 15-min rest, muscles were exposed to 12% desflurane for 15 min. Recovery responses were measured after a 20-min washout.

When isolated strips of myocardium are rapidly cooled, a contracture develops as a result of sudden release of Ca²⁺from the SR,17used as an index of the SR Ca²⁺content. Using GP papillary muscles, stimulation was gradually increased from 0.1 to 2 Hz until a stable contraction was obtained. The duration of stimulation at each stimulation rate was preset and kept constant during the experiment. Immediately after the abrupt cessation of the stimulus, a rapid cooling contracture (RCC) was triggered by rapidly changing the perfusion of modified standard Tyrode solution (flow rate: approximately 50 ml/min) from 37°C to 0°C, which decreased the temperature to less than 5°C within 2–4 s. Cooling was maintained for 60 s, after which perfusion with 37°C modified standard Tyrode solution was resumed. After determination of the control response, the identical protocol was repeated after a 15-min exposure to 6% desflurane, a 15-min exposure to 12% desflurane, and lastly after a 20-min washout. Isometric force and bath temperature were continuously recorded during the experiment.

β-Adrenergic stimulation with 0.1 μm isoproterenol enhances contractility by increasing Ca²⁺entry via  L-type Ca²⁺(I^Ca,L) channels18,19as well as an increasing Ca²⁺uptake into and release from the SR.20When applied in 26 mm K⁺Tyrode solution (97 mm NaCl, 26 mm KCl, 2 mm CaCl², 1.2 mm MgSO⁴, 25 mm NaHCO⁻³, 11 mm glucose, 0.1 mm EDTA), there is partial depolarization of the myocardium to −40 to −45 mV, which inactivates fast Na⁺channels, while permitting conduction of slow action potentials (APs) (mediated by I^Ca,L). After recording the biphasic contractions12,13,15,21under control conditions at RS up to 3-Hz stimulation rate, contractions at the same stimulation rates were recorded after application of 6% and 12% desflurane (applied sequentially for 15 min each). Recovery responses were measured after a 20-min washout.

After each force experiment, muscle cross-sectional area was estimated from muscle length, weight, and density (1.04 g/ml), assuming a cylindrical form. Mean cross-sectional areas were 0.82 ± 0.37 mm²(n = 65, from 0.24 to 1.99 mm²) and 0.81 ± 0.26 mm²(n = 5, from 0.54 to 1.15 mm²) in GPs and rats, respectively.

Papillary muscle membrane potential was monitored with conventional 3 m KCl-filled glass microelectrodes (10–20 MΩ) attached to a VF-1 preamplifier (World Precision Instruments, Sarasota, FL). Membrane potential and its maximum rate of rise during the AP (dV/dt-max) were monitored on a digital storage oscilloscope. Because the vigorous contractions at higher stimulation rates (2 and 3 Hz) often caused dislodgement of impaled microelectrodes, only results from an impalement maintained throughout the whole sequence at 0.25-Hz stimulation rate were tabulated. Stimulation intensity was maintained at approximately 120% of the lowest intensity necessary to elicit a response and then adjusted to maintain approximately the same latency (stimulus-action potential interval, usually 5–10 ms). AP amplitude, dV/dt-max, and AP duration at 50% and 90% of repolarization (APD⁵⁰and APD⁹⁰, respectively) were measured at 0.25-Hz stimulation rate in modified standard Tyrode solution and in 26 mm K⁺Tyrode solution with 0.1 μm isoproterenol.

Guinea pigs (approximately 300 g) were anesthetized with halothane at 2.5–4 vol%. The heart was quickly excised and retrogradely perfused using a Langendorff perfusion system. The heart was perfused for 5 min at 37°C at a rate of 7 ml/min with modified Tyrode solution (143 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl², 0.5 mm MgCl², 5 mm HEPES, 0.18 mm glucose, pH 7.4). The perfusate was then switched for 5 min to a nominally Ca²⁺-free Tyrode solution followed by perfusion with the same solution to which collagenase (0.4 mg/ml, Worthington type II; Worthington Biochemical Corporation, Lakewood, NJ) and protease XIV (0.04 mg/ml; Sigma-Aldrich Co., St. Louis, MO) had been added. After a 10- to 12-min enzymatic treatment, 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, 10 mm HEPES, pH 7.4) was perfused for 5 min. The ventricles were then cut off and minced with scissors and agitated in a small beaker with Kraftbrühe solution, and the resulting slurry was filtered through a 200-μm nylon mesh. The isolated ventricular cells were stored in Kraftbrühe solution for 1 h at room temperature (21°–22°C), kept at 4°C, and used within 8 h. Only the rod-shaped cells with apparent striations that remained quiescent in the solution containing 2 mm CaCl²were used for the experiments. All patch clamp experiments were conducted at room temperature.

Isolated myocytes were allowed to settle to the bottom of a recording chamber mounted on an inverted microscope where the bathing solutions could be exchanged. The chamber was continuously perfused at a rate of 2 ml/min. Standard whole cell voltage clamp methods were used.22After the initiation of the whole cell recording configuration, an interval of 4–6 min was allowed to establish a stable baseline. Voltage clamp measurements were performed using an Axopatch 200B patch clamp amplifier (Axon Instruments, Foster City, CA). Patch electrodes were prepared from a borosilicate glass model KIMAX-51 (American Scientific, Charlotte, NC), which have a typically 2–3 MΩ resistance when filled with an internal solution. After fabrication with a two-stage micropipette puller, the pipette tips were heat-polished with a microforge. Data acquisition was performed using a pCLAMP system version 6.0 (Axon Instruments) coupled with a Pentium III personal computer.

Simultaneous recordings of delayed outward K⁺current (I^K) and inward rectifier K⁺current (I^KI) were obtained using a linear voltage ramp protocol in which the cells were held at −40 mV before sweeping the membrane potentials from −130 to +50 mV at a rate of 30 mV/s. For the detailed evaluation of desflurane effect on I^K, the I^Kwas measured by step depolarizations from −30 to +80mV with a holding potential of −40 mV in 20-mV increments, 5-s intervals, and 4-s pulse duration. The voltage-dependent I^Ca,Lwas evoked by step depolarization lasting 200 ms from a holding potential of −40 to +10 mV in one step at a frequency of 0.1 Hz.

An external bathing 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 1N NaOH) was used before establishing the whole cell recording. 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 KOH) was used before establishing the whole cell recording configuration. After establishing the whole cell voltage clamp, 0.2 mm CdCl²was added to the external bathing solution to eliminate any confounding Ca²⁺current. Inward Ca²⁺currents were measured using a patch pipette solution (30 mm CsCl, 100 mm aspartic acid, 100 mm CsOH, 10 mm BAPTA, 10 mm HEPES, 10 mm phosphocreatinine, 1 mm Na²GTP, 5 mm Na²ATP, 10 mm glucose, 2 mm MgCl², adjusted to pH 7.25 with 1 m CsOH). Upon obtaining the whole cell recording, the external bathing solution was exchanged to a solution (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) to measure I^Ca,L.

After the baseline measurement, the myocytes were exposed to 6% or 12% desflurane for 2 min. Recovery responses were measured after a 2-min washout. In pilot experiments, a 2-min application of desflurane was sufficient to produce a stable and consistent effect.

In contraction and AP experiments at 37°C, desflurane or isoflurane was equilibrated with solution in one reservoir by passing the 95% O²–5% CO²gas mixture (flow rate: 0.5 l/min) through a desflurane (Devapor Type M32600; Dräger AG, Lübeck, Germany) or isoflurane (Isotec 3; Ohmeda, West Yorkshire, United Kingdom) vaporizer for 15 min. The end-tidal concentrations of desflurane or isoflurane were monitored with a gas analyzer (Capnomac; Datex, Helsinki, Finland) that had been calibrated before use. Isoflurane was used to compare contractile parameters with approximately equianesthetic concentrations of desflurane.

As determined by gas chromatographic measurement, desflurane concentrations in the 37°C Tyrode superfusate that had been equilibrated for 15 min were 0.42 ± 0.06 mm (n = 3) and 0.91 ± 0.05 mm (n = 4) for 6% and 12% desflurane, respectively. Considering that the Tyrode solution/gas partition coefficient is 0.2 at 37°C,23desflurane at 0.42 mm and 0.91 mm correspond to gas phase concentrations of 5.3% (0.89 minimum alveolar concentration [MAC]) and 11.6% (1.93 MAC), respectively. As previously reported, the equilibrated concentrations of isoflurane at 37°C were 0.22 mm for 1.3% and 0.42 mm for 2.5%,14corresponding to 1.0% (0.77 MAC) and 1.9% (1.45 MAC) gas phase isoflurane at 37°C, respectively, based on a Tyrode solution/gas partition coefficient of 0.57 at 37°C.23 

During myocyte experiments at room temperature, desflurane was equilibrated with an external bathing solution in one reservoir by passing 100% O²(flow rate: 0.2 l/min) through the desflurane vaporizer for 15 min. Concentrations of 0.78 ± 0.05 mm (n = 3) and 1.23 ± 0.03 mm (n = 3) were measured for 6% and 12% desflurane, respectively. Given that the Tyrode solution/gas partition coefficient is 0.27 at 22°C,23the aforementioned desflurane concentrations were equivalent to gas phase concentrations of 7.0% and 11.0%, respectively. An aqueous concentration of 0.78 mm desflurane at 22°C is equivalent to approximately 9.9% desflurane gas phase at 37°C.

Tyramine, phentolamine, propranolol, and reserpine were purchased from the Sigma-Aldrich Chemical Company (St. Louis, MO). To obtain a 1-mg/ml solution of reserpine, reserpine (20 mg) was dissolved in 0.5 ml glacial acetic acid and 1 ml propylene glycol, and then brought to a volume of 20 ml with distilled water. The pH was adjusted to 4.98 with 10N NaOH. Unless otherwise stated, all chemicals were purchased from the Sigma-Aldrich Chemical Company.

Because of the variations in baseline values from one muscle to another, alterations in contractile responses were expressed as a percentage of baseline. In papillary muscle and isolated myocyte experiments, repeated-measures analysis of variance followed by Student-Newman-Keuls test was applied to test for statistically significant differences among the baseline, drug application, and washout period as well as among the stimulation rates. An unpaired t  test was used to compare the contractile forces between the equianesthetic concentrations of desflurane and isoflurane, and to compare the differences between 6% and 12% concentrations of desflurane. To compare the effects of desflurane among control conditions, groups with adrenoceptor blockade, and reserpine-treated groups, one-way analysis of variance followed by Student-Newman-Keuls test was applied. All results were expressed as mean ± SD. A P  value less than 0.05 was considered significant.

Desflurane caused concentration-dependent depression of PF to approximately 70% and 40% of baseline from RS to 3-Hz stimulation rates, respectively (n = 7, P < 0.05; fig. 1A). Equianesthetic concentrations of isoflurane depressed contractions to approximately 75% and 50% of baseline, respectively (n = 5, P < 0.05; fig. 1B). At each stimulation rate, the contractile depression caused by equianesthetic concentrations of desflurane and isoflurane were not different (not significant [NS]). The effects of desflurane and isoflurane on contractile forces did not seem to depend on the stimulation frequencies. Whereas the contractile force recovered to 50–60% of baseline after a 20-min washout of desflurane, equianesthetic concentration of isoflurane exhibited complete recovery. There was no further recovery after an additional 20-min washout of desflurane (data not shown). The baseline values of PF are shown in table 1.

Tyramine increased the contractile forces to approximately 200% of the baseline from RS to 3-Hz stimulation rates (RS: 179 ± 43%, 0.1 Hz: 168 ± 39%, 0.5 Hz: 186 ± 36%, 1 Hz: 217 ± 70%, 2 Hz: 210 ± 61%, 3 Hz: 162 ± 23%, n = 6). The contractile forces returned to baseline values after washout for 20 min. Treatment with tyramine (30 μm) after α- and β-adrenoceptor blockade (n = 6) or in reserpinized myocardium (n = 6) did not alter the contractile force at each stimulation rate (fig. 2A). Whereas application of α- and β-adrenoceptor blockers did not alter the contractile force at RS and 0.1-Hz stimulation rates, a modest but statistically significant depression to approximately 85% of baseline was observed at 1- to 3-Hz stimulation rates (fig. 2B). Application of desflurane (5.3%) after α- and β-adrenoceptor blockade depressed the contractile force to approximately 50% and 40% of baseline at RS to 1 Hz and higher stimulation rates, respectively (n = 6, P < 0.05; fig. 2B). After α- and β-adrenoceptor blockade (n = 8) or in reserpinized myocardium (n = 8), the contractile depression by 5.3% desflurane did not differ from the effects observed under control conditions (fig. 3). To eliminate the contribution of contractile depression by α- and β-adrenoceptor blockade, fractional depression by desflurane was compared with α- and β-blockade baseline values. The baseline values of PF among these three groups are shown in table 2. Similar to the muscles in the absence of pharmacologic sympathetic blockade, partial recovery was observed in the three aforementioned groups at all stimulation rates (data not shown).

Unlike GP muscle, rat muscle SR does not become depleted of Ca²⁺during rest, but rather accumulates it due to its higher resting intracellular [Na⁺],24,25resulting in a large RS contraction. Rat papillary muscles also exhibit a negative force-frequency relation, which may be related to hypoxia.26Because of the potential for hypoxic alteration, particularly at high stimulation rates, the effect of desflurane (11.6%) was only examined at RS. Alteration of the RS contractile force was not observed in either the presence of desflurane (7.65 ± 2.09 vs.  6.99 ± 1.91 mN/mm², n = 5, NS) or after a 20-min washout (7.65 ± 2.09 vs.  7.19 ± 1.91 mN/mm², n = 5, NS).

In GP myocardium, Ca²⁺extrusion via  the Na⁺-Ca²⁺exchange is markedly reduced by low extracellular Na⁺concentration so that Ca²⁺is accumulated in SR during rest,19,25leading to an enhanced RS contraction similar to the behavior of rat myocardium. Contractions in low-Na⁺Tyrode solution require a modest Ca²⁺current as evidenced by the fact that these contractions are hardly inhibited by nifedipine.19Desflurane (11.6%) did not alter the RS contraction (7.34 ± 2.33 vs.  6.96 ± 2.03 mN/mm², n = 6, NS).

After a 2-Hz stimulation rate, desflurane (5.3%) markedly depressed the RCC (13 ± 7% of baseline, 3.60 ± 0.52 vs.  0.46 ± 0.10 mN/mm², n = 7, P < 0.05), and similar depression was shown with 11.6% desflurane (11 ± 7% of baseline, 3.60 ± 0.52 vs.  0.36 ± 0.07 mN/mm², n = 7, P < 0.05). The time to peak contractures could not be determined because the shape of the RCC was almost abolished so that no peak was definable (fig. 4). There was incomplete recovery of the RCC amplitude after a 20-min washout of 5.3% desflurane (28 ± 12% of baseline, 3.60 ± 0.52 vs.  0.97 ± 0.18 mN/mm², P < 0.05; fig. 4).

With β-adrenergic stimulation in 26 mm K⁺Tyrode solution, the pattern of force after rest and up to 0.5- to 1-Hz stimulation rate consistently demonstrated a distinct early and late phase of force development, defined as dF^E/dt-max and dF^L/dt-max, respectively.12,13,15,21Whereas the early peak of the biphasic contraction elicited is activated by Ca²⁺accumulated during previous depolarizations and released immediately upon rapid depolarization of the membrane, the late peaking force is attributed to the Ca²⁺that enters the cell during the initial phase of the AP, is taken up into the SR, and is subsequently released late in depolarization.15,19,21This late peaking force is depressed by L-type Ca²⁺channel blockers such as nifedipine.15,19,21In contrast, ryanodine decreases the initial force development15,21by putting the cardiac Ca²⁺release channel (RyR2) in a low conductance state and decreasing the SR Ca²⁺available for immediate release, although the late release is not inhibited.15The baseline values of PF, dF^E/dt-max, and dF^L/dt-max are shown in table 3. Desflurane at 5.3% and 11.6% resulted in a marked depression of late force development (5.3%: approximately 40% of baseline, 11.6%: approximately 20% of baseline, n = 7) with a modest to moderate depression of early force development (5.3%: approximately 80% of baseline, 11.6%: approximately 60% of baseline) (figs. 5A–C). After a 20-min washout, near-complete (P < 0.05) and complete (NS) recovery of dF^E/dt-max and dF^L/dt-max, respectively, were present.

In normal APs, desflurane (11.6%) altered neither the amplitude nor the dV/dt-max at 0.25-Hz stimulation rate, although the APD⁵⁰and APD⁹⁰were prolonged significantly (APD⁵⁰: 113 ± 7% of baseline, APD⁹⁰: 111 ± 6% of baseline, n = 6; table 4). No changes in resting membrane potential, which ranged from −87 to −92 mV, were observed. In slow APs, whereas desflurane (11.6%) altered neither the AP amplitude nor the dV/dt-max at 0.25-Hz stimulation rate, the APD⁵⁰and APD⁹⁰were also prolonged (APD⁵⁰: 109 ± 29% of baseline, APD⁹⁰: 115 ± 15% of baseline, n = 6; table 4). No changes in resting membrane potential, which ranged from −39 to −44 mV, were observed.

Using a linear voltage ramp protocol, peak inward I^KIat −130 mV was not significantly reduced by 0.78 mm (n = 13) or 1.23 mm desflurane (n = 13) from the control values of −4.74 ± 1.08 and −4.35 ± 1.32 nA, respectively. The peak outward component of I^KIelicited at potentials ranging from −60 or −50 mV was also unchanged by 0.78 mm (n = 13) or 1.23 mm desflurane (n = 13) from the control values of 0.29 ± 0.13 and 0.34 ± 0.19 nA, respectively. A typically observed response is shown in figures 6A and B.

Whereas I^KIdid not seem to be significantly reduced by desflurane, the current-voltage relation presented in figure 6indicates that the outward K⁺current recorded at membrane potentials positive to −20 mV, which primarily represents I^K, was depressed by desflurane. The peak outward I^Kassessed at +60 mV was reduced significantly to 63 ± 19% (n = 13, P < 0.05) and 58 ± 12% (n = 13, P < 0.05) of the baseline values by 0.78 mm and 1.23 mm desflurane from its baseline values of 0.37 ± 0.27 nA and 0.37 ± 0.27 nA, respectively (fig. 6C). Complete recovery was observed after washout (fig. 6A). A more detailed study of time-dependent I^Kwas performed by measuring the amplitude of I^Kat the end of a step depolarization to +80 mV. Desflurane (0.78 mm) reduced the I^Kto 57 ± 22% (n = 5, P < 0.05) from its baseline value of 1.08 ± 0.31 nA, whereas 1.23 mm desflurane reduced I^Kto 61 ± 13% (n = 7, P < 0.05) from its baseline value of 0.71 ± 0.12 nA (figs. 7A–D). Washout of desflurane resulted in a complete recovery.

At a membrane potential of +10 mV, peak I^Ca,Lwas reduced to 58 ± 6% of baseline (n = 5, P < 0.05) by 1.23 mm desflurane from its baseline value of −0.45 ± 0.09 nA (figs. 8A and B). Desflurane (0.78 mm) reduced peak I^Ca,Lto 77 ± 6% (n = 5, P < 0.05) of baseline from its baseline value of −0.4 ± 0.07 nA (fig. 8B). Complete recovery was shown after washout (fig. 8A).

The results of the current study demonstrate that desflurane depresses the contractile force in a concentration-dependent manner, similar to the effect of equianesthetic concentration of isoflurane. Myocardial depression by desflurane has been well characterized in isolated atrial9and ventricular tissues,8,10,11in intact animal studies,3–6and in clinical settings.2,7Whereas most in vivo  animal3,5,6and human studies2have reported similar degree of contractile depression with equianesthetic concentration of desflurane and isoflurane, other in vivo  animal studies have demonstrated conflicting results showing greater27or less4depression than isoflurane. Using a GP heart Langendorff preparation, Bovan et al.  8demonstrated that desflurane caused a slightly greater depression of isovolumic left ventricular pressure than did an equianesthetic concentration of isoflurane, although greater contractile depression by isoflurane has been reported in rat10and hamster myocardium11as well as human atrial tissue.9The contractile depression observed in this study is compatible with the report by Bovan et al.  8(approximately 40% of control with 11.7% desflurane), and slightly greater than the depression documented by Pagel et al.  6(approximately 75% of baseline by 1 MAC desflurane) in chronically instrumented dogs with pharmacologic blockade of the autonomic nervous system.

A curious observation was the incomplete recovery of normal contractions that we observed after the 20-min washout of desflurane. In previous studies examining isoflurane12or sevoflurane,13a complete recovery of contractions was observed after a 20-min anesthetic washout. In the current study, identical experiments using isoflurane showed a complete reversal of contractile depression, indicating that there is no time-dependent deterioration in our experimental preparation. To verify the incomplete recovery, we examined four more muscles (control, 11.6% desflurane, and washout) and reconfirmed similar depression (approximately 40%) and recovery (approximately 60%) from RS to 3-Hz stimulation rates. In previous work performed in the same superfused preparation, the depressant effects of propofol were only partially reversible with washout.28Like propofol, desflurane is highly hydrophobic and, once absorbed by the tissue, will diffuse from the tissue and dissolve in the aqueous medium very slowly. In isoproterenol-stimulated muscles in 26 mm K⁺Tyrode solution in the current study, the effects of desflurane seemed to be more reversible on washout. The much more vigorous contractions would increase solution contact and diffusion from the tissue, whereas with weak contractions and minimal motion, diffusion into solution would be restricted by unstirred layers of solution inside and outside the tissue.

In heart muscles, the depolarization-activated release of SR Ca²⁺is mediated by Ca²⁺entering the myocyte and triggering the opening of the SR Ca²⁺release channels (type 2 ryanodine receptors, RyR2), a process termed Ca²⁺-induced Ca²⁺release  (CICR). Over repeated cardiac cycles, depression of I^Ca,Lwill also result in a decrease of Ca²⁺accumulation in the SR available for subsequent release, consistent with the contractile depression of normal contractions observed with L-type Ca²⁺channel blockers such as nifedipine.15However, when the SR has a very high Ca²⁺accumulation, such as the RS contraction of rat or GP heart in low Na⁺Tyrode solution, any decrease in I^Ca,Lseems to be less important because those contractions are minimally depressed by nifedipine.19Consequently, the depression of I^Ca,Lby desflurane in rat or GP hearts in low Na⁺Tyrode solution did not reduce CICR, which has been previously observed for sevoflurane.13The lack of depression is likely due to the high concentration of SR luminal Ca²⁺that seems to prime the RyRs for more complete opening and release.29Therefore, even a modest entrance of Ca²⁺may cause complete RyR activation and Ca²⁺release from the SR. In addition, the decreased Na⁺gradient may foster “reverse” flow and Ca²⁺entry through the Na⁺-Ca²⁺exchanger, and this entering Ca²⁺may contribute to CICR, compensating for depression of I^Ca,L.30 

The moderate depression of early tension development observed in our results may be mediated by reduced SR Ca²⁺content secondary to a reduction of inward Ca²⁺current by desflurane during previous contractions. However, desflurane, like isoflurane,12enflurane,15and sevoflurane,13markedly reduced late force development in 26 mm K⁺Tyrode solution with 0.1 μm isoproterenol. This depression may be mediated in part by reduction of the inward Ca²⁺current and/or by the reduction of the late SR Ca²⁺release. Considering the modest effect on SR Ca²⁺release in rat and GP myocardium and the reduction of I^Ca,Lby desflurane in the current results, marked depression of late force development is likely to be mediated by reduction of inward Ca²⁺current.

Cooling has been shown to increase the open probability of the RyRs,29,31and this action likely mediates the Ca²⁺release that activates the cooling contracture. Desflurane virtually abolished the force induced by rapid cooling, a characteristic that also shared with sevoflurane,13isoflurane,32,33and enflurane (unpublished results, C. Lynch III, M.D., Ph.D., Charlottesville, Virginia, 1990). However, in the current study, there was only a moderate depression of the 2-Hz contractions by desflurane immediately before cooling, indicating the presence of significant releasable pool of Ca²⁺in the SR sufficient to elicit depolarization-activated contractile force. The fact that RCC is profoundly inhibited by fluorinated ether anesthetics suggests that the functional properties of the SR Ca²⁺release channel (RyR2) were altered by such anesthetics while leaving the more physiologic CICR largely intact.33The rapid cooling-induced Ca²⁺release associated with cold cardioplegia is associated with decreased recovery of contractile function versus  warm cardioplegia.34Although this is an interesting experimental observation, the inhibition of rapid cooling-induced Ca²⁺release by desflurane may have clinical significance. Anesthetic-induced depression of cooling-induced Ca²⁺release could result in greater recovery of contractile function after cold-induced Ca²⁺release.

The minimal effect of desflurane on rat cardiac RS contractions as well as on GP myocardium in low Na⁺are consistent with a modest effect on SR Ca²⁺uptake and its depolarization-activated CICR. These findings suggest that desflurane has a relatively small effect on the amount of Ca²⁺stored and released, an observation that is consistent with the results of Gueugniaud et al.  10in isolated rat ventricular myocardium. Whereas isoflurane12and sevoflurane13exhibit effects similar to those of desflurane on SR function, halothane and possibly enflurane stand in contrast with these anesthetic agents by causing a more prominent decrease in initial force development,12,15probably by activating the cardiac SR Ca²⁺release channel and depleting the SR Ca²⁺store.35,36 

In GP myocardium, we observed that tyramine concentration-dependently increased the contractile force (data not shown). Further, pretreatment with α and β blockers or reserpine prevented the increase of contractile force by tyramine, indicating that GP myocardium has releasable catecholamine pools. In rat myocardium, Gueugniaud et al.  10demonstrated modest enhancement of contractile force at various concentrations of desflurane. However, the contractile force of rat myocardium after α- and β-adrenoceptor blockade or reserpine administration was significantly depressed by desflurane, suggesting that intramyocardial catecholamine release induced by desflurane counteracted the anesthetic's contractile depression. In contrast, we did not observe any differences for the effects of desflurane on contractile force among control, adrenoceptor-blocked, and reserpine-treated muscles in GP, indicating a clear species-dependent difference in desflurane effects on releasable intramyocardial catecholamines.

To further assess the sympathomimetic effects of desflurane in vivo  during a rapid increase in desflurane concentration,37–39we observed a change in contractility in two muscles stimulated continuously at 0.5 Hz after a rapid increase in desflurane concentrations from 3% (vaporizer setting) to 12% (vaporizer setting). In contrast to the systemic effects observed in vivo , this rapid increase of desflurane concentration from 3% to 12% decreased the contractile force similarly (data not shown) to that of a higher desflurane concentration in modified standard Tyrode solution (fig. 1A). None of our results suggest that desflurane induces catecholamine release in the GP ventricular myocardium.

I^Kis composed of two different current types: a rapidly activating, inward rectifier current, I^Kr, and a slowly activating current, I^Ks.40In the current study, whole cell voltage clamp experiments indicated that desflurane suppresses the I^K, a property that has been reported for halothane in GP atrial myocytes,41and isoflurane42,43and sevoflurane in GP ventricular myocytes.13Considering the higher voltage used in this study, at which I^Kris usually deactivated, the effects of desflurane primarily represent depression of I^Ks, which has been found to be highly sensitive to other inhalational anesthetics such as isoflurane42,43and sevoflurane.44In contrast, I^Krseems to be less or not sensitive to isoflurane43,44and sevoflurane,44although it is inhibited by halothane.45Further study will be necessary to elucidate the possible effects of desflurane on I^Kr.

Although both sevoflurane and isoflurane have been reported to cause significant depression of inward component and enhancement of outward component of I^KI,46,47the actual differences from the baseline values seemed to be modest, suggesting a minimal effect by these anesthetics on I^KI. Modest effects on the inward and outward components of I^KIby sevoflurane have also been reported in GP ventricular myocytes.13We also found that desflurane caused no change in the inward and outward components of I^KIin GP ventricular myocytes. The inward rectifier K⁺current is the primary current responsible for maintaining a stable cardiac resting membrane potential near the K⁺equilibrium potential. Inhibition of I^KIcan result in diastolic depolarization, which in turn can cause increased cardiac excitability48and lead to dysrhythmias and abnormal automaticity.49The lack of change in the resting membrane potential after application of 0.91 mm desflurane may be attributable to the lack of alteration of I^KI.

Our whole cell voltage clamp experiments revealed a concentration-dependent reduction of peak I^Ca,Ldespite the prolongation of AP duration in normal and slow APs. Although inhibition of I^Ca,Lcan lead to a reduction of AP duration, the reduction of I^Ksseems to have a greater effect, resulting in AP lengthening. Clinically, significant prolongation of QTc after administration of 1 MAC desflurane has also been reported in adults,50and considering that prolongation of AP duration induced by clinically relevant concentrations of desflurane in GP myocytes is most likely due to the suppression of I^K, a similar effect in human myocardium may partially account for the clinical observation of QT prolongation by desflurane.

Overall, the actions of desflurane on the various cellular aspects of Ca²⁺handling and myocardial contractility seem to be similar to those previously observed in studies of isoflurane and sevoflurane. Contractile depression can be attributed to the depression of Ca²⁺channel mediated Ca²⁺influx through the cardiac membrane. The rapid initial release of Ca²⁺from the SR by depolarization seems to be modestly depressed, although alternative Ca²⁺release pathways of the RyR Ca²⁺release channels (cold induced) seem to be markedly depressed. In addition to reduction of I^Ca,L, a major electrophysiologic effect of desflurane seems to be a depression of the delayed outward K⁺current, which in turn results in an increased AP duration.