Temperature-independent Inhibition of L-Type Calcium Currents by Halothane and Sevoflurane in Human Atrial Cardiomyocytes

Right atrial appendages were obtained as surgical specimens from patients (n = 21) undergoing heart surgery. Patient characteristics are summarized in table 1. All patients were in sinus rhythm and had no evidence of right heart failure. The investigations were performed in accordance with the principles outlined in the Declaration of Helsinki and approved by the local institutional review board (Aachen, Germany). All patients gave written informed consent before surgery.

Human atrial cardiomyocytes were prepared according to Hatem et al. ,20as modified and described in detail by Skasa et al.  21Briefly, the myocardial specimens were gently cut into chunks and washed in Ca²⁺-free buffer twice for approximately 5 min. Afterward, the tissue was incubated in Ca²⁺-free buffer that contained protease XXIV (Sigma, Germany) and collagenase V (Sigma, Germany). Incubation was finished as soon as microscopic examination revealed intact cardiomyocytes. After centrifugation cardiomyocytes were resuspended in a buffer containing the following: NaCl 120 mm, KCl 5,4 mm, MgSO⁴5 mm, sodium pyruvate 5 mm, glucose 20 mm, taurine 20 mm, HEPES 10 mm. The pH was adjusted to 7.4 with NaOH. As visible cell damage to these cells would occur if a physiologic Ca²⁺concentration was immediately restituted, a gradual recalcification was performed. Only well-striated, bleb-free, rod-shaped myocytes were used for the studies, performed within 4 h after the isolation.

Cardiomyocytes were allowed to adhere to a glass coverslip that was transferred into a perfusion chamber. The cells were continuously superfused as described previously.22 

L-type Ca²⁺currents (I^Ca,L) were recorded by means of the whole cell patch clamp technique with use of an amplifier (EPC-9; HEKA, Lambrecht, Germany) and a personal computer equipped with Pulse 8.5 software (HEKA) for data acquisition and analysis. The patch pipettes pulled from borosilicate glass and fire polished had a tip resistance of 3–7 MΩ when they were filled with pipette solution.

A holding potential of −60 mV was chosen to minimize Na⁺currents. L-type Ca²⁺currents were evoked by a series of depolarization pulses (each 100 or 200 ms in duration) to potentials ranging from −50 to +60 mV. Ca²⁺currents are given here as peak current amplitude of the respective depolarizing step. For temporal reasons, pulses only to +10 mV were used to evaluate the effects of anesthetics in most experiments.

Experiments were performed either at room temperature (21–22°C) or 36°C. In the latter case, the temperature of the solution in the double-barreled stainless steel-tubes and the bath chamber (volume, 0.4 ml) was kept constant with a feedback-controlled Peltier device (Strothmann, Aachen, Germany). Temperature was continuously monitored with a thermocouple probe mounted in the chamber wall and did not vary by more than 0.3°C during an experiment.

For measurements of I^Ca,L, the bath solution contained tetraethylammonium chloride, 136 mm; CaCl², 1.8 mm; MgCl², 1.8 mm; glucose, 10 mm; and HEPES, 10 mm; this was pH-adjusted to 7.40 with tetraethylammonium hydroxide. The pipette solution contained CsCl, 140 mm; MgCl², 2 mm; adenosine 5′-triphosphate, 0.3 mm; guanosine 5′-triphosphate, 0.3 mm; EGTA, 10 mm; HEPES, 10 mm. This was pH-adjusted to 7.20 with CsOH.

Bath solutions containing halothane, sevoflurane, or xenon were prepared by passing an appropriate gas mixture through the solution in a glass flask equipped with a frit and a membranous septum. Gas left the flask through a valve. An anesthetic gas analyzer (Capnomac Ultima; Datex Ohmeda, Duisburg, Germany) was used to continuously monitor the concentrations of halothane and sevoflurane in the gas phase. The glass flask was located in a temperature-controlled water bath (36°C) for experiments at physiologic temperature. After at least 30 min of gassing, a fraction (10 ml) of the solution was taken through the septum into gas-tight glass syringe (1010 TLL; Hamilton, Bonaduz, Switzerland) and immediately used as superfusate of the cells. The preparation of solutions containing volatile anesthetics and xenon and analysis of gas concentrations in solutions by head space gas chromatography were described previously.22Standards of anesthetics were prepared by transferring halothane or sevoflurane (in liquid form) into methanol/distilled water at defined concentrations. Samples were taken up into gas-tight flasks. Anesthetic content in the standard solutions were verified immediately afterwards by gas chromatography.23 

Gassing a solution with halothane, 0.75% (vol/vol in air; corresponding to 1 MAC in humans) at 36°C resulted in a concentration of 0.21 mm halothane. When gassing a solution with sevoflurane, 2.1% (vol/vol in air; 1 MAC in humans), the concentration yielded 0.21 mm at 36°C. Xenon solutions were prepared by gassing with xenon (95%)/O²(5%). Some loss of xenon (∼30%) occurred during superfusion of the cells; the final concentration of xenon in the immediate neighborhood of the cell was 2.2–2.3 mm at 36°C.

Current amplitude denotes the peak current during one depolarizing pulse. Current densities were calculated by dividing current amplitudes by the whole cell capacitance. Values are expressed as mean ± SD. To analyze the kinetics of the time-dependent inactivation during the pulse, the currents were fitted with the double-exponential decay function:

where I denotes the current; t, the time; and τ, a time constant. The subscripts fast and slow refer to the fast and slow components of the total current, respectively. Details of the analysis have been described previously.24In modification of the previous study, the right boundary of the fit was 93 ms after the peak. Fitting was performed with IgorPro 3.15 software (WaveMetrics Inc., Lake Oswego, OR). Statistical comparisons were carried out with Prism 3.02 software (GraphPad Software Inc., San Diego, CA) using the paired Student t  test. A probability of error of P < 0.05 was considered significant.

First, we measured the concentrations of halothane and sevoflurane in the standard bath solution after equilibration with various concentrations (corresponding to 1–3 MAC at 37°C) of the anesthetics in the gas phase. The aqueous concentrations differed markedly dependent on whether equilibration was performed at 36°C (fig. 1A) or at room temperature (21°C; fig. 1B). The slope of the relation shown in figure 1indicates how many mmoles per liter of halothane and sevoflurane are found in the aqueous phase per volume percent of the anesthetics in the gas phase. Respective values are 0.29 and 0.09 mm/vol % for halothane and sevoflurane at 36°C. At room temperature, the corresponding values were 0.52 and 0.12 mm/vol % (n = 3 separate experiments for each concentration).

The L-type Ca²⁺currents in cardiomyocytes from human right atria exhibited different characteristics at 36°C compared to 21°C (fig. 2A). Most strikingly, the amplitude of the peak current during depolarizing pulses was considerably increased by a rise in the temperature. In atrial cardiomyocytes from four patients measured at 36°C (n = 5) or at 21°C (n = 5), maximum peak current density was −4.2 ± 1.2 pA/pF at 36°C and −1.8 ± 0.3 pA/pF at 21°C (P < 0.01). Moreover, current-voltage relation was slightly shifted to the left at 36°C (fig. 2B) which might, however, indicate a lack of voltage control in the cardiomyocytes. Still, maximal current amplitudes were observed between +10 and +20 mV such that a depolarizing pulse to +10 mV was used as standard stimulus to evoke peak I^Ca,L.

Finally, the inactivation kinetics of I^Ca,Lrecorded during depolarizing pulses to +10 mV were temperature-sensitive. The decline of I^Ca,Lafter the maximum peak was assessed by a fit of the current amplitudes with a double exponential function (see Methods). The fit yielded two time constants, τ^fand τ^s, that represent the fast and slow inactivating component of I^Ca,L. The inactivation kinetics were markedly slower at 21°C than at 36°C as both τ^fand τ^swere significantly larger at 21°C. For example, τ^fand τ^swere 5.6 ms and 28.6 ms, respectively, at 36°C for the current tracing labeled with b  in figure 2A. The corresponding time constants at 21°C were 41.9 ms and 61.6 ms (fig. 2A, tracing a ). Summarized values (n = 5) for τ^fand τ^sat both temperatures are shown in figure 2C.

In the representative experiment given in figure 3A, halothane (1 MAC) reversibly reduced peak I^Ca,Lby 18% at 36°C. However, in another cell from the same patient, halothane (1 MAC) evoked a reduction I^Ca,Lby 30% at 21°C (not shown). In the mean, halothane (1 and 2 MAC) reduced I^Ca,Lby 21.6 ± 2.9% from −4.5 ± 1.1 pA/pF to −3.5 ± 1.0 pA/pF (n = 5, P < 0.05) and by 37.1 ± 12.0% from −4.2 ± 2.3 pA/pF to −2.8 ± 2.0 pA/pF (n = 5, P < 0.05), respectively, at 36°C (fig. 3Band C).

For comparison, halothane (1 MAC) inhibited peak I^Ca,Lby 31.3 ± 4.1% from -1.9 ± 0.5 pA/pF to −1.4 ± 0.4 pA/pF (n = 5, P < 0.05) at 21°C. These results are similar to those of our previous study performed at room temperature.22 

Similarly as halothane, sevoflurane reduced the peak amplitude of I^Ca,L. When we compared the effects of sevoflurane at a concentration of 2 MAC at 21°C versus  36°C, the inhibition of I^Ca,Lwas markedly less extensive at the higher temperature (fig. 4Aand B). Sevoflurane (2 MAC) inhibited peak I^Ca,Lby 33.4 ± 11.4% from −2.6 ± 0.8 pA/pF to −1.7 ± 0.5 pA/pF (n = 5, P < 0.05) at 21°C and by 21.1 ± 7.8% from −5.1 ± 2.5 pA/pF to −3.9 ± 1.8 pA/pF (n = 6, P < 0.05) at 36°C (fig. 4Aand B).

We studied two other concentrations of sevoflurane (1 MAC and 3 MAC, fig. 4Cand D) at 36°C that reduced peak I^Ca,Lin a concentration-dependent manner. Sevoflurane inhibited peak I^Ca,Lby 11.7 ± 6.1% at 1 MAC (n = 5; P < 0.05) and by 33.4 ± 9.9% at 3 MAC (n = 5; P < 0.05). Again, the wash-out of sevoflurane restored I^Ca,Lto the amplitudes observed before the application of the anesthetic gas (fig. 3 and 4).

The reduction of I^Ca,L(i.e. , of the peak amplitude) by halothane and sevoflurane were not accompanied by major effects on the inactivation kinetics. In particular, the major component of the inactivation, τ^fast, was not significantly altered. Significant changes were found for the higher concentrations of either anesthetic on τ^slow(table 2) but these changes were in opposite directions.

In contrast to halothane and sevoflurane, the noble gas xenon (65%) had not affected I^Ca,Lin our previous experiments at 21°C.22As there was a (insignificant) tendency of xenon to accelerate the inactivation kinetics of I^Ca,Lafter its peak, we reasoned that an effect could be validated at 36°C. Equilibration at 36°C of solutions with gases containing xenon at a concentration of 95% and delivery to the bath chamber (some loss during transport) resulted in a final concentration of 2.2 mm xenon in the aqueous phase. Peak I^Ca,Lwas not affected by xenon at 36°C (not shown). Moreover, xenon did not affect the inactivation kinetics and both time constants remained unchanged (table 2).

Because the resulting concentrations of the anesthetics halothane and sevoflurane in the aqueous phase after gassing with a given partial concentration in the gas phase and the inhibition of I^Ca,Lby either anesthetic decreased considerably when the temperature was raised from 21°C to 36°C, we plotted the fractional (percent) inhibition of I^Ca,Lagainst the concentrations of the anesthetics in the aqueous phase (fig. 5 A and B). In this figure, some data are included that derive from our previous studies22,24in which I^Ca,Lin human atrial cardiomyocytes were measured at room temperature (21°C) under identical conditions and with the same set-up as in the present study. Despite the fact that the current amplitudes differed markedly, dependent on the temperature, the plot revealed an almost linear dependence of the percent inhibition of I^Ca,Lon the concentration of halothane or sevoflurane in the bath solution.

This study addresses the question of how temperature affects L-type Ca²⁺currents and their modification by anesthetic gases in human atrial cardiomyocytes. The main findings are that the amplitudes, inactivation kinetics, and inhibition of I^Ca,Lby halothane and sevoflurane differed strikingly between 36°C and 21°C. However, the percent inhibition of peak I^Ca,Lshowed an almost linear relationship to the concentrations of the volatile anesthetics in the bathing medium. This relationship was the same at either temperature. These results indicate that the concentration of an anesthetic in the aqueous phase, but not in the gas phase, determines its percent inhibition of I^Ca,L, independently of the temperature.

The solutions were prepared by gassing with air enriched with the volatile anesthetics at a gas concentration corresponding to 1–3 MAC in patients at 37°C. When the resulting concentrations in the aqueous phase were measured with head space gas chromatography, a temperature dependence was confirmed that has been reported in previous studies for halothane and sevoflurane in various types of solutions.9,17,25,26Similar findings have also been reported for xenon.27The solubility of the anesthetics decreased strongly with increasing temperatures. It has been previously suggested that concentrations of anesthetics in solutions used for experimental in vitro  studies should be given in moles per liter rather than in volume percent in the gas phase.2,17The relevance of this suggestion becomes clear in light of the temperature dependency of the solubility and the fact that most in vitro  studies are performed at room temperature. The present study is, to our knowledge, the first one that explores the effects of temperature on I^Ca,Lin human atrial cardiomyocytes. The current amplitudes were more than doubled by a rise in the temperature from 21°C to 36°C. On the other hand, the inactivation kinetics of I^Ca,Limportant for the duration of the plateau phase of the action potential were markedly accelerated by an increased temperature. For example (fig. 2A), 25 ms after the peak of I^Ca,L, current amplitudes at 36°C had receded below a level observed at 21°C at the same time point, despite the fact that the maximal current (observed 25 ms previously) had shown a much larger amplitude. Therefore, it is obvious that the usual approach to perform experiments at room temperature does not sufficiently take into account the temperature effects on ion currents in the heart. As long as inhibitory effects on current amplitudes are in the experimental focus, studies at room temperature may be sufficiently relevant, in the light of the present results. If, however, data on current kinetics are to be interpreted in a physiologic context, the experiments should be carried out at physiologic temperatures, although this is technically demanding. In our experience, the stability of the cells over time decreases dramatically with higher temperatures. This means that considerably more attempts must be made before sufficient data for a valid analysis are sampled.

Moderate hypothermia is known to exert a positive inotropic effect on the heart that is independent of the negative chronotropic one.28The enhanced contractility is explained by an increased Ca²⁺sensitivity of the myofilaments, whereas the temperature-dependent modifications of I^Ca,Ldo not fit to the positive inotropic effects of hypothermia.

Xenon had no affected I^Ca,Lin our previous studies when it was tested at room temperature.22In the present study, it once more failed to produce any significant modification of I^Ca,Leven when studied at 36°C and analyzed in terms of peak amplitude and inactivation kinetics. Hence, xenon seems to represent an anesthetic with the favorable property of being inert to cardiac I^Ca,L.

An anesthetic-induced change in the fast component of inactivation kinetics was not observed in our study, in agreement with previous results by Pancrazio.3However, halothane and sevoflurane had opposite effects on the slow component τ^slowof the time-dependent inactivation of I^Ca,Lat 36°C. Halothane significantly accelerated the slow component of inactivation at 2 MAC, consistent with other work,3whereas sevoflurane delayed the slow component of inactivation. This observation is not in line with findings in single bullfrog atrial myocytes by Hirota et al. , who demonstrated that sevoflurane (5.0 vol%) reduced the time constant of I^Ca,Linactivation by 25% at room temperature.29However, this effect was analyzed with a monoexponential fit of the decay of I^Ca,L. The calculation of τ^slowin our study was mainly performed because the current decay of I^Ca,Lcannot be properly fitted to a single exponential function. The fast component τ^fastis far more decisive for the process of inactivation and determines the total amount of transsarcolemmal moved Ca²⁺(i.e. , the current-time integral) over the time of an action potential. We do not think that effects on τ^slowcan be interpreted in terms of specific interactions of the two anesthetics with the L-type Ca²⁺channel.

Halothane may act on L-type Ca²⁺channels by a direct binding of the anesthetic to the protein at its dihydropyridine binding site.30In general, binding of anesthetics to ion channels may be the principal mechanisms relevant for their narcotic action.31–33In our study, decreased availability of the anesthetic gases in the bath solution with rising temperature would be in accordance to the attenuated inhibition of I^Ca,Lif the anesthetics acted from the extracellular side. However, it is not known whether temperature influences the fractional amount of anesthetics that are taken up from the bath into the membrane of cardiac cells. Thus, our study design cannot exclude that the gases act on Ca²⁺channels after accumulation within the cell membrane and that the temperature effects are related to alterations in the rate of membrane accumulation and binding to channel proteins.

From a clinical point of view, it is a common experience that the required concentrations for anesthesia by gases decrease in the presence of reduced body temperatures in animals and humans.34–38For example, hypothermia decreases the required concentration of isoflurane in children by 5.1% per each degree Celsius that the body temperature is lowered.38The present study suggests as one possible explanation for this phenomenon that smaller concentrations of the anesthetics in the gaseous phase are effective modulators of ion channels when the temperature is lower as a result of increased solubility.

The authors thank Manfred Möller, Ph.D. (Assistant Professor, Department of Hygiene and Environment medicine, University Hospital, RWTH Aachen, Aachen, Germany) for analysis of gas concentrations with gas chromatography. The authors acknowledge the excellent cooperation of the staff of the Department of Thoracic and Cardiovascular Surgery, University Hospital, RWTH Aachen.