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ANESTHETIC PHARMACOLOGY

Halothane, Isoflurane, and Sevoflurane Increase the Kinetics of Ca²⁺-Induced Conformational Change of Recombinant Human Cardiac Troponin C

Dirk Breukelmann, MD^*, and Philippe R. Housmans, MD, PhD

From the *Department of Anesthesiology and Intensive Care, University of Muenster, Muenster, Germany; and Mayo Clinic and Foundation, Rochester, Minnesota.

Address correspondence and reprint requests to Dirk Breukelmann, MD, University of Muenster, Klinik und Poliklinik fuer Anaesthesiologie und Operative Intensivmedizin, Albert-Schweitzer-Strasse 33, 48149 Muenster, Germany. Address e-mail to breukel{at}uni-muenster.de.

	Abstract

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BACKGROUND: Halothane, isoflurane, and sevoflurane exert negative inotropic side effects, generally mediated via a reduced availability of intracellular calcium. Other possible mechanisms include modified intracellular calcium handling, impaired actomyosin cross-bridge cycling, and/or alteration of calcium-induced conformational changes of the regulatory troponin complex.

METHODS: We investigated the effect of halothane, isoflurane, and sevoflurane on calcium-dependent kinetics of isolated human recombinant cardiac troponin C labeled with IAANS (HrcTnC_IAANS) using stopped-flow and calcium titration techniques.

RESULTS: Calcium concentration at half-maximal fluorescence intensity (K_d) in the control group was 2.1 ± 0.1 mM. Volatile anesthetics increased calcium sensitivity in a concentration-dependent fashion sevoflurane (K_d 1.5–1.7 mM, P = 0.001) > halothane (K_d 1.7–1.9 mM, P < 0.01) > isoflurane (K_d 1.8–1.9 mM, P < 0.05). The rate constant of conformational changes after rapid dissociation of calcium from HrcTnC_IAANS (k_off(c)) was moderately prolonged at 4°C by halothane and isoflurane > sevoflurane.

CONCLUSION: These mechanisms may counteract the effects of lower calcium availability, and can be responsible for abbreviated, and possibly incomplete, relaxation of cardiac muscle fibers in the presence of volatile anesthetics.

	Introduction

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Volatile anesthetics such as halothane, isoflurane, and sevoflurane decrease myocardial contractility of the mammalian heart. This can have potentially life-threatening effects in patients with myocardial disease, and may also result in significant pharmacodynamic interactions with cardiovascular drugs.

Halothane, isoflurane, and sevoflurane are the most common volatile anesthetics used in clinical anesthesia today. It has been demonstrated that these anesthetics reduce intracellular Ca²⁺ availability during contraction (1,2), and also Ca²⁺ sensitivity, defined as the maximum amount of force generated at a given intracellular Ca²⁺ concentration (2–5). Whereas the diminished intracellular Ca²⁺ availability has been related to a decrease in intracellular L-type channel Ca²⁺ influx (6) and decreased sarcoplasmic reticulum Ca²⁺ content (3), the exact locus of action to affect Ca²⁺ sensitivity is not known. Possible mechanisms include, but are not limited to, the binding characteristics of Ca²⁺ to troponin C (TnC),¹ interactions between regulatory troponin subunits, troponin–tropomyosin–actin interactions, and changes in actomyosin cross-bridge kinetics (7).

Human cardiac troponin consists of three subunits: troponin T is the tropomyosin-binding site, troponin I functions as the inhibitory subunit, and TnC has been identified as the Ca²⁺-binding regulatory subunit. Conformational changes of the troponin complex ultimately affect the probability of actomyosin binding, and thus regulate cross-bridge cycling in the cardiac muscle. Those effects are induced by Ca²⁺-binding and -release to and from the Ca²⁺-specific binding site II of the cardiac TnC molecule, whereas the remaining two high-affinity binding sites of the cardiac TnC molecule are saturated with Mg²⁺ in physiological conditions.

Therefore, a possible mechanism by which volatile anesthetics reduce cardiac cross-bridge-binding upstream in the regulatory chain could be via a direct effect on the Ca²⁺-dependent properties of cardiac TnC.

We have examined conformational changes of isolated human recombinant cardiac TnC labeled with sulfhydryl-specific fluorescent probe molecule 2-(4'-iodo-acet-amido-anilino)-naphthalene-6-sulfonic acid (HrcTnC_IAANS) (8) on binding and release of Ca²⁺ to and from the Ca²⁺-specific site II during exposure to different concentrations of halothane, isoflurane, and sevoflurane. HrcTnC_IAANS has been shown to change its fluorescence properties on calcium-induced conformational changes of cardiac troponin when bound to cysteine 35 and 84 of the native TnC molecule. The labeled protein undergoes a small decrease in fluorescence due to Ca²⁺ or Mg²⁺ binding to its two high-affinity sites, and a large increase in fluorescence on Ca²⁺ binding to the low-affinity Ca²⁺-specific site. HrcTnC_IAANS exhibits a Ca²⁺-sensitive fluorescence peak at excitation 340 nm and emission 445 nm (9).

We hypothesized that volatile anesthetics reduce the affinity of HrcTnC_IAANS site II Ca²⁺-binding and/or kinetics of subsequent conformational changes by exerting direct effects on the isolated protein.

	METHODS

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Material
All reagents were purchased from Fisher Scientific (Fair Lawn, NJ) except for CaCl₂ (BDH Laboratory Supply, Poole, England), halothane (Halocarbon Laboratories, River Edge, NJ), isoflurane, and sevoflurane (Abbot Laboratories, North Chicago, IL). HrcTnC_IAANS was a generous gift of Dr. James D. Potter, University of Miami. Solutions were made up in ultra-pure water with a resistivity >17.8 M · cm (Nanopure Infinity UF Series 898, Barnstead/Thermolyne, Dubuque, IA) to minimize ion contamination. Solutions were kept at 4°C for a maximum of 5 days to avoid degradation. Since pH affects Ca²⁺ binding characteristics of myocardial TnC, 3-(N-morpholino) propanesulfonic acid (MOPS) pH buffer capacity was carefully chosen, so that Ca²⁺ binding to [ethylenebis-(oxyethylenenitrilo)]-tetraacetic acid (EGTA) would not change pH over a range of pCa 3.5–9.0 (data not shown).

Protein Purification and IAANS Labeling
IAANS labeling of human recombinant cardiac TnC (HrcTnC_IAANS) was performed using standard methods (8). IAANS binding stoichiometry was determined to be 1.08 moles per mole protein using absorbance characteristics specific to IAANS. Since the wild type TnC possesses two cysteine residues (Cys35 and Cys84), about 50% of the potential IAANS binding sites can be considered labeled.

HrcTnC_IAANS was extensively dialyzed against Ca²⁺-free protein buffer containing MOPS 120 mM, KCl 90 mM, pH 7.0 in purified water, aliquoted, and stored at –80°C until use.

Anesthetic-Saturated Buffers
To obtain anesthetic-saturated buffer, each anesthetic was allowed to equilibrate with titration buffer for at least 2 h at room temperature. Different volumes of anesthetic-saturated buffer were mixed with titration buffer to expose HrcTnC_IAANS to increasing anesthetic concentrations.

Gas Chromatography
Gas chromatographic analysis was performed in a Hewlett Packard 5880A Gas Chromatograph, using either a Porapak Q 80/100 column (Hewlett Packard, Avondale, PA) with a Nickel-63 electron capture detector, for halothane and isoflurane, or a fused silica column (007-624 Series, Quadrex Corp. Woodbridge, CT) combined with a flame ionization detector for sevoflurane. Anesthetic concentrations were determined after extracting the volatile compound of the buffer in hexane, which was then injected into the gas chromatograph.

Equilibrium Titration
Steady-state fluorescence (excitation 330 nm, emission 445 nm) was determined in a constantly stirred, temperature-controlled quartz cuvette (pathlength 1 cm, total volume 4.5 mL) at 25°C in a SLM Aminco 8100 Spectrofluorimeter (Software Version 4.00) at a sampling rate of 1 Hz. Fifty microliters protein buffer (MOPS 120 mM, KCl 90 mM, HrcTnC_IAANS 2000 µg/mL, pH 7.0) was added to a total volume of 1950 µL titration buffer (MOPS 120 mM, KCl 90 mM, EGTA 2 mM, pH 7.0) and allowed to equilibrate for stable baseline fluorescence. Different anesthetic concentrations in the solution were achieved by varying the portion of anesthetic-saturated buffer.

Equation 1 is the Hill equation to fit experimental data to sigmoid curve where K_d is the Ca²⁺ concentration with half-maximal fluorescence Y and n is the Hill coefficient, steepness of the sigmoid curve.

The expected changes in anesthetic concentrations due to gas–liquid equilibration in the cuvette were calculated on the basis of temperature-corrected anesthetic partition coefficients () (10) under the assumption that the total amount of anesthetic is added into the liquid phase and remains constant in the liquid–gas system [Eq. (2)]. Anesthetic concentrations were determined by gas chromatography before and at the end of the experiment.

Statistical analysis for differences between control and anesthetic groups was performed using Kruskal–Wallis and Wilcoxon's tests, with P < 0.05 being considered significant (11).

Stopped-Flow Spectrofluorimetry
The effect of Ca²⁺ dissociation from the single regulatory site II on the kinetics of conformational changes of HrcTnC was assessed in a fluorescence stopped-flow analyzer (Applied Photophysics SX.18 MV, Leatherhead, Surrey, UK) at 4°C and 20°C. HrcTnC_IAANS saturated with Ca²⁺ (HrcTnC_IAANS 15 µg/mL, pCa 3.6 in K⁺ 140 mM, Cl^– 90 mM, MOPS 120 mM, pH 7.0) in the presence or absence of 1 mM Mg²⁺ was rapidly (0.9 ms) mixed 1:1 with EGTA 9.75 mM (same buffer). Fluorescence (excitation 330 nm, emission >395 nm) between 1 and 100 ms was recorded and fitted to a first-order exponential function to yield the rate constant of conformational change k_off(c). Different anesthetic concentrations were obtained by varying the fraction of anesthetic saturated buffer (same composition) in both solutions before mixing. Glass syringes were used to minimize evaporation.

Anesthetic concentration in each syringe was determined by gas chromatographic analysis. Effects of the respective anesthetic were analyzed according to the anesthetic concentration for both Mg²⁺ 1 mM and Mg²⁺-free groups individually.

Statistical analysis was performed using Wilcoxon's ranked sum test to test for differences of anesthetic group versus control and effect of anesthetic concentration. P < 0.05 was considered significant (11).

	RESULTS

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IAANS Fluorescence Titration
Expected Change in Anesthetic Concentration
To assess the consistency of anesthetic concentration during the experiment, expected changes in anesthetic concentration at 25°C in the liquid phase due to equilibration were calculated using a temperature-corrected (293 K) partition coefficient (water/air) of 1.50 (halothane), 0.94 (isoflurane), and 0.60 (sevoflurane) (10) under the assumption that the volatile anesthetic is initially introduced into the liquid phase of a gas-tight system [Eq. (2)]. For the experimental system used in this study (2.0 mL liquid and 0.4 mL gas phase), the expected equilibrium concentrations in the liquid phase were calculated to be 0.81 (halothane), 0.73 (isoflurane), and 0.63 (sevoflurane) of the initial concentration (12).

Anesthetic concentrations in the liquid phase at the end of the calcium titration (expressed as a fraction of the concentration immediately after injection and mixing ± sd) were determined to be 0.82 ± 0.13 (halothane, n = 18), 0.69 ± 0.10 (isoflurane, n = 25), and 0.75 ± 0.11 (sevoflurane, n = 21).

Equation 2 is used to calculate expected anesthetic concentration in the liquid phase after gas:liquid equilibrium where n_total is the total amount of substance injected into liquid phase; vol_liq is the volume of liquid phase; vol_gas is the volume of gas phase; is the liquid:gas partition coefficient; and n_liq is the amount of substance in liquid phase after equilibration.

Site II Ca²⁺ Affinity
Least square fitting and statistical analysis showed that K_d in the control group was 2.1 ± 0.1 mM; the Hill coefficient (n) for the cooperativity of the metal–ligand interaction was 2.7 ± 0.6 (n = 7). Halothane at concentrations of 0.2 ± 0.1 mM decreased K_d to 1.9 ± 0.2 mM (n = 7, P < 0.05 compared with control). Halothane 1.0 ± 0.2 mM led to a further decrease of K_d to 1.7 ± 0.1 mM (n = 9, P < 0.01 to control) and also increased the Hill coefficient n to 3.7 ± 0.4 (n = 9, P < 0.01 to control). A further excessive increase of halothane concentration to 2.5 ± 0.3 mM showed K_d at 1.7 ± 0.1 (n = 8, P < 0.001 to control). However, at that concentration the Hill coefficient decreased to 3.1 ± 0.3 (n = 8, n.s. compared with control). Isoflurane decreased K_d at concentrations of 0.1 ± 0.02 mM, 0.6 ± 0.1 mM, and 1.2 ± 0.3 mM to 1.8 ± 0.1 mM (P < 0.01, n = 8), 1.8 ± 0.1 mM (P < 0.01, n = 10), and 1.9 ± 0.2 mM (P < 0.05, n = 9), respectively. Isoflurane did not affect the Hill coefficient at any concentration. Sevoflurane decreased K_d to 1.7 ± 0.2 mM at 0.4 ± 0.1 mM (P < 0.001, n = 9) and 1.5 ± 0.1 mM at 0.6 ± 0.1 mM (P = 0.001, n = 9), without significant effect on the Hill coefficient (Figs. 1 and 2).

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of halothane, isoflurane, and sevoflurane on half-maximal Ca2+-binding to human recombinant cardiac troponin C labeled with IAANS (HrcTnCIAANS).

View larger version (21K): [in this window] [in a new window]   Figure 2. Effect of halothane, isoflurane, and sevoflurane on cooperativity of Ca2+-binding to human recombinant cardiac troponin C labeled with IAANS (HrcTnCIAANS).

	DISCUSSION

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Ca²⁺ binding to site II of cardiac TnC protein in vivo triggers conformational changes of cardiac TnC, initiating a cascade of interactions within the regulatory protein complex, ultimately leading to an increased probability of actin–myosin interaction, and thus, muscle contraction. The negative inotropic effects of volatile anesthetics routinely used today are well described, although the underlying mechanisms are not yet completely understood. In addition to effects on Ca²⁺ influx via L-type Ca²⁺ channels (13), sarcoplasmic calcium handling (14), depressed cross-bridge cycling kinetics (15,16), and passive stiffness (17), it has been shown that volatile anesthetics decrease myofibrillar Ca²⁺ sensitivity in skinned (4,18), and intact (19,20) muscle preparations in particular, by affecting cross-bridge cycling rates. Experiments including actin filament motility over myosin monomers did not demonstrate any direct interaction of halothane or isoflurane with cross-bridge kinetics (21).

However, the exact mechanisms of the latter effect remain unclear. One of the possible mechanisms playing an important role in the process of force generation in cardiac muscle upstream of the cross-bridge level is the Ca²⁺ affinity of the regulatory protein complex, which is most likely to be determined by the Ca²⁺ binding characteristics of TnC.

In this in vitro study, halothane, isoflurane, and sevoflurane lead to a decrease of K_d (e.g., a left shift of the troponin-Ca²⁺ binding curve), whereas the Hill coefficient n, describing the steepness of the sigmoid curve, was increased (halothane) or remained largely unaffected. The effect was concentration-dependent, reaching a maximum impact at a concentration of 1 mM (1.7 vol %, halothane) and 0.1 mM (0.3 vol %, isoflurane) with no further decrease of K_d at higher concentrations. For sevoflurane K_d decreased in a concentration-dependent manner up to the highest concentration 0.6 mM (2.5 vol %) used.

All volatile compounds investigated tended to increase the rate-constant of Ca²⁺ dissociation-induced conformational change k_off(c) of HrcTnC_IAANS at lower temperatures, although it has to be noted that the changes observed are rather small with considerable concentrations of anesthetics used. At 20°C halothane, more than isoflurane, decreased k_off(c) at lower concentrations and showed a progressive increase with higher anesthetic concentrations. Sevoflurane did not affect the rate-constant of conformational change.

On the basis of the law-of-mass action, increased Ca²⁺ sensitivity (K_d) and altered rate of conformational change after Ca²⁺ dissociation from HrcTnC (off-rate) suggest an increase in the rate of conformational change on Ca²⁺ binding to TnC (on-rate). This is consistent with experimental observations of anesthetic effects on cardiac muscle contractility showing decreased time to peak shortening and time to half-maximal relaxation for sevoflurane (2), little effect of isoflurane, and prolonged contraction and relaxation on exposure to halothane (1).

This result is in contrast with the findings of Blanck et al. (22) who were not able to demonstrate any effects of halothane on TnC-Ca²⁺ binding kinetic using intrinsic tyrosine fluorescence, ultraviolet circular dichroism, and 5,5'-dithiobis(2-nitrobenzoic acid) binding techniques. This might be explained by the different sensitivities of the experimental procedures used. One advantage of using a previously IAANS-labeled TnC protein is that the molecular structure of the protein-IAANS complex remains unaltered during the entire experimental procedure. It has been shown that the IAANS-labeled protein does not significantly differ in physiological Ca²⁺ binding, undergoes Ca²⁺-induced increases in helix, and also forms complexes with other troponin subunits similar to unlabeled cTnC (8). There is, however, a possibility that volatile anesthetics do interact specifically with IAANS labeling sites, and thus, interfere with Ca²⁺-dependent conformational change-induced fluorescent signals.

Skinned muscle experiments have suggested that halothane and isoflurane decrease Ca²⁺ sensitivity and maximal force in human cardiac fibers (4,18). Since skinned muscle experiments focus on a disrupted, although vital, part of the contractile machinery, possible important interactions with enzymes, second-messengers, or even parts of the interacting filaments may remain undetected.

Physiologically, increased Ca²⁺ sensitivity counteracts negative inotropic side effects of halothane, isoflurane, and sevoflurane, which are to a great extent due to decreased intracellular Ca²⁺ availability (1–3). Sevoflurane exhibited the most pronounced increase in Ca²⁺ sensitivity in our study which may, in part, account for the relatively lower cardio-depressant activity compared with other volatile anesthetics. When mechanisms mediating negative inotropic effects are absent or weak, a sevoflurane-dependent increase in Ca²⁺ sensitivity might even explain an overall positive inotropic response to sevoflurane exposure, as described by Vivien et al. (23).

	CONCLUSION

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Volatile anesthetics such as halothane, isoflurane, and sevoflurane specifically affect Ca²⁺ interactions with low-affinity Ca²⁺ binding sites II of HrcTnC_IAANS leading to increased Ca²⁺ sensitivity. This effect can be based on changes in Ca²⁺ binding kinetics of HrcTnC_IAANS, altered rate of conformational change, or both. The changes described in this study are small, but can at least partly be responsible for the distinctive concentration-dependent effects caused by halothane, isoflurane, and sevoflurane in intact ventricular myocardium.

	Footnotes

 
¹Breukelmann D, Potter JD, Housmans PR. Effects of isoflurane and sevoflurane on Ca²⁺ binding of recombinant human cardiac troponin C. Anesthesiology 2001;95:A616.

Accepted for publication October 16, 2006.

	REFERENCES

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