1 Department of Anaesthesiology and Critical Care, Centre Hospitalier Universitaire (CHU) Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris (AP-HP), Université Pierre et Marie Curie, Paris, France. 2 Department of Anaesthesiology and Critical Care, CHU Necker-Enfants malades, AP-HP, Université René Descartes, Paris, France. 3 Department of Physiology, CHU Bicêtre, AP-HP, Université Paris Sud, Le Kremlin-Bicêtre, Paris, France. 4 Service dAccueil des Urgences, CHU Pitié-Salpêtrière, AP-HP, Université Pierre et Marie Curie, Paris, France
Corresponding author. E-mail: olivier.langeron@psl.ap-hop-paris.fr
Accepted for publication: February 26, 2003
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Abstract |
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Methods. The effects of halothane and isoflurane (1 and 2 minimum alveolar concentration (MAC)) on rat diaphragm muscle strips were studied in vitro (KrebsHenseleit solution, 29°C, oxygen 95%/carbon dioxide 5%) in tetanus mode (50 Hz). From the forcevelocity curve and using A. F. Huxleys equations, we determined the main mechanical and energetic variables and calculated CB kinetics.
Results. At 1 and 2 MAC, isoflurane and halothane induced no significant inotropic effects. Whatever the concentrations tested, halothane and isoflurane did not significantly modify the CB number, the elementary force per CB, the attachment and detachment constants, the duration of the CB cycle and mean CB velocity.
Conclusion. In the rat diaphragm at therapeutic concentrations, halogenated anaesthetics do not significantly modify CB mechanical and kinetic properties.
Br J Anaesth 2003; 90: 75965
Keywords: anaesthetics volatile, halothane; anaesthetics volatile, isoflurane; muscle, skeletal, diaphragm; pharmacokinetics, cross-bridge kinetics; protein, actin; protein, myosin
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Introduction |
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In muscle cells, halogenated anaesthetics interfere with numerous cellular functions, including force generation. However, the effects of halogenated anaesthetics on the myosin CB kinetics remain a matter for debate.912 These effects are difficult to study in cardiac muscle because of the various sites of action of halogenated anaesthetics, including the calcium channel, the sarcoplasmic reticulum, and myofilaments.13 It is known that the marked negative inotropic effect of halogenated anaesthetics on the myocardium is mainly related to a decrease in the calcium transient, leading to difficulties in analysing CB kinetics. Indeed, any change in inotropy related to changes in calcium concentration induces some changes in CB, at least in their number. In contrast, halogenated anaesthetics do not induce significant inotropic effects on diaphragmatic muscle14 and A. F. Huxleys equations can be used to describe CB kinetics.68
Therefore, in the present study, we used isolated diaphragmatic muscle as a model to analyse the effects of halogenated anaesthestics on myosin CB kinetics and have investigated the effects of halothane and isoflurane on CB number, kinetics, and unitary force. We tested the hypothesis that halogenated anaesthetics significantly modify CB kinetics at therapeutic concentrations.
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Methods |
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Experimental protocol
Following brief anaesthesia with i.p. sodium pentobarbital, a muscle strip from the ventral part of the costal diaphragm was carefully dissected from the muscle in situ from 40 adult Wistar rats weighing 220260 g. The muscles were divided into four groups exposed to either 1 or 2 minimum alveolar concentration (MAC) of halothane or isoflurane. As reported previously,68 15 this diaphragm strip was immediately vertically suspended in a 200-ml jacketed reservoir with KrebsHenseleit bicarbonate buffer solution (118 mM NaCl, 4.5 mM KCl, 1.2 mM MgSO4, 1.1 mM KH2PO4, 25 mM NaHCO3, 2.5 mM CaCl2, and 4.5 mM glucose) prepared daily with highly purified water. The jacketed reservoir was kept at 29°C with a thermostatic water circulator and continuous monitoring of the solution temperature with a temperature probe. The bathing solution was bubbled with oxygen 95%/carbon dioxide 5%, resulting in a pH of 7.40, and the required anaesthetic.
Preparations were field-stimulated by means of two platinum electrodes with rectangular pulses of 1 ms duration at 10 pulses min1 in twitch mode. Experiments were conducted after a 25 min stabilization period, at the initial muscle length at the apex of the length-active isometric tension curve (Lmax). Measurements of mechanical parameters were made in tetanic mode at 50 Hz (10 trains min1 of 300 ms duration, 1 ms rectangular pulses) as this assures a maximal tetanus without inducing high-frequency fatigue. At the end of the study, the cross-sectional area was calculated from the ratio of fresh muscle weight to muscle length at Lmax, assuming a muscle density of 1.06.
Mechanical variables
The electromagnetic lever system has been described previously.16 All analyses were made from digital records of force and length obtained with a computer. 68 15 Mechanical variables were calculated from 10 consecutive tetanic contractions preloaded at Lmax with increasing afterload from zero-load to fully isometric contraction. The first contraction was abruptly clamped to zero load just after the electrical stimulus, with critical damping in order to slow the first and rapid shortening overshoot resulting from the recoil of series passive elastic components,16 enabling determination of the maximum unloaded shortening velocity (Vmax). The second contraction was isotonic and loaded with preload only. The last contraction was fully isometric at Lmax. The hyperbolic forcevelocity relationship was derived from the peak shortening velocity (V) plotted against the isotonic load level normalized per cross-sectional area (F).68 15 Experimental data were fitted according to the equation of A. V. Hill:17
(F+a)(V+b)=(Fmax+a)b
where, a and b are the asymptotes of the hyperbola, Fmax is the calculated maximum isometric total force for V=0. The following parameters were derived from Hills hyperbola.17 The curvature of the hyperbola (G) of the FV relationship was
G=Vmax.b1=Fmax.a1.
The A. F. Huxleys equations were used to calculate the rate of total energy release (E), the isotonic force (PHux), and the rate of mechanical work (WM) as a function of V, as reported previously.68 15 E is given as:
E=.e.h.f1.(2l)1.(f1+g1)1.[g1+f1.V.
1(1e
/V)]
where is the CB number per mm2 at maximum PHux, f1 is the maximum value of the rate constant for CB attachment, and g1 and g2 are the peak values of the rate constants for CB detachment (Fig. 1). The instantaneous movement (x) of the myosin head relative to actin varies from h to 0. The CB step size (h) is defined by the translocation distance of the actin filament per ATP hydrolysis and produced by the swing of the myosin head; f1 and g1 correspond to x=h, and g2 corresponds to x
0; e is the free energy required to split one ATP molecule, l is the distance between two actin sites, and
=(f1+g1)h/2=b. Calculations of f1, g1, and g2 are given by the following equations:
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g1=2w.b.(e.h.G)1;
g2=2Vmax.h1.
The maximum value of total energy release occurs at Vmax. The minimum value of the rate of total energy release (E0) occurs under isometric conditions and is equal to the product axb and is also given by the following equation:
E0=.e.h.(2l)1.(f1.g1)(f1+g1)1.
The maximum turnover rate of myosin ATPase per site in isometric conditions (kcat) is given by the following equation:
kcat=E0.(e.)1=h.(2l)1.(f1.g1)(f1+g1)1.
Assuming that one molecule of ATP is split in each CB cycle, the total duration of the time cycle (tc), the duration of the power stoke (time stroke, ts), the duty ratio (ts/tc), and the mean velocity of each CB (0) were calculated as:
tc=1/kcat;
ts=Lmax.(.h.
.E0)1;
0=h/ts.
Isotonic tension (PHux) is given by the following equation:
PHux = .w.l1.f1.(f1+g1)1.[1 V.
1.(1e
/V).(1+(f1+g1)2. g22.V.(2
)1)]
where w is the mechanical work of a unitary CB. The elementary force per single CB in isometric conditions () is given by the following equation:
=PHux max.
1=w.l1.f1.(f1+g1)1.
The rate of mechanical work (WM) is given by:
WM=PHux.V.
At any given load, the mechanical efficiency (Eff) of the muscle is defined as the ratio of WM to E, and Effmax is the maximum value of Eff.
Stroke size (h) of 11 nm has been determined by means of optical tweezers18 and is supported by the three-dimensional structure of the crystallized myosin head.19 The distance l is equal to 36 nm.20 The free energy required to split one ATP molecule per contraction site is 5.1x1020 J; w is 0.75 e and the value of w is 3.8x1020 J.20
Administration of anaesthetics
Halothane and isoflurane were added to the carbon dioxide/oxygen mixture with a specific calibrated vaporizer (Fluotec 3 for halothane and Fortec 3 for isoflurane; Cyprane Ltd, Keighley, UK). The gas mixture bubbled continuously in the bathing solution. To minimize evaporation of volatile anaesthetics, the jacketed reservoir was almost completely sealed with a thin paraffin sheet. The concentration of volatile anaesthetics in the gas phase was continuously monitored with a calibrated infrared analyser (Artema MM 206 SD; Taema, Antony, France). Halothane concentrations used were 0.6 and 1.2% and isoflurane concentrations used were 0.8 and 1.6%. These concentrations are equivalent to 1 and 2 MAC of halothane and isoflurane in the adult rat at 29°C.21 Halothane and isoflurane were administered for a 15 min equilibration period before any measurements being made.
Statistical analysis
Data are expressed as mean (SD). Comparisons of baseline values between groups were performed using analysis of variance. The effects of anaesthetic agents were assessed using a paired Students t-test. All P values were two-tailed, and a P value <0.05 was considered significant. Statistical analysis was performed using NCSS 6.0 software (Statistical Solutions Ltd, Cork, Ireland).
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Results |
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Discussion |
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Conflicting effects of volatile anaesthetics on diaphragmatic muscle have been observed in in vivo experiments.25 26 Because of the changes induced by anaesthetics on arterial blood pressure and diaphragmatic blood flow, central nervous system, and neuromuscular transmission, the precise effects of halogenated anaesthetics on intrinsic diaphragmatic contraction are very difficult to assess in vivo. In an in vitro study under isometric conditions, Kawaga et al.14 have suggested that halothane and sevoflurane decrease force of the hamster diaphragm only during fatigue. In our study in isolated rat diaphragm, we confirmed the lack of significant effect of halogenated anaesthetics. Using the same experimental model, we recently observed that isoflurane, but not halothane, induced very moderate negative inotropic effects and only at 2 MAC.27 However, the magnitude of these effects was not markedly different from those observed in the present study (halothane 2 MAC: 7 vs 3% of baseline AF; isoflurane 2 MAC: 7 vs 7% of baseline AF). However, the absence of significant inotropic effect of halogenated anaesthetics in our present study enables a precise assessment of their effects on CB kinetics.
Little information is available regarding anaesthetic effects on CB kinetics. Using skinned preparation of cardiac muscle, several authors have suggested that halogenated anaesthetics affect CB kinetics.10 11 However, the skinning process can result in the loss of enzymes and second messengers that modulate the calcium-force relationship and/or CB kinetics. Using sinusoidal length oscillation in rabbit papillary muscle during barium contracture, Shibata et al.28 reported that halogenated anaesthetics did not modify CB kinetics. Using superimposed sinusoidal oscillations in ferret cardiac papillary muscles, Bartunek et al.29 reported that halothane and isoflurane decreased dynamic and passive stiffness, thought to be related to CB kinetics, and weakly bound CB or passive elastic elements, respectively. Using tetanic papillary muscle, Hannon et al.9 reported that isoflurane depressed CB cycling rate. However, it should be emphasized that: (1) a simple two-state model of CB cycling was used during the plateau phase of an isometric tetanic contraction; (2) very high concentrations of calcium (up to 12 mM) were used. In our opinion, the use of cardiac muscle in these two studies9 29 represents a methodological flow in the analysis of CB kinetics. Indeed, in cardiac muscle, halogenated anaesthetics are known to markedly modify intracellular calcium transients through a decrease in calcium entry through sarcolemmal calcium channel, a decrease in calcium released from the sarcoplasmic reticulum, and a decrease in myofilament calcium sensitivity. Hannon et al.9 compared the effects of isoflurane to calcium on force-calcium and force redevelopment (kTR)-calcium and Bartunek et al. 29 increased calcium concentration to obtain equivalent peak shortening, making it difficult to separate the effects on myofilament calcium sensitivity (mainly calcium affinity of troponin C) and those on CB kinetics per se. It is known that calcium modifies myofilament calcium sensitivity (cooperativity concept)30 and may therefore modify CB kinetics.31 As halogenated anaesthetics do not significantly modify intracellular calcium transients in diaphragm muscle,27 there is a unique opportunity to directly assess CB kinetics when exposed to halogenated anaesthetics. Moreover, our experimental model enables a study of muscle contraction over the entire continuum of load, from isotony to isometry. In our study, at therapeutic concentrations, halothane and isoflurane did not significantly modify the number of CB, the elementary force per CB, their attachment and detachment constants, the durations of CB cycle, or mean CB velocity.
Do our results apply to CB in all striated muscle, including cardiac muscle? Although the myofilament structure and regulation of contraction by calcium are similar in cardiac and skeletal muscles, there are important differences in myofilament activation.32 In cardiac muscle cycling, CB attachment enhances the affinity of troponin C for calcium, in contrast to skeletal muscle. Conversely, the effects of rigor CB binding on thin filament are similar in cardiac and skeletal muscles.32 Several different myosin heavy chain (MHC) isoforms are expressed in striated muscle that directly influence CB kinetics. In adult rat diaphragm muscle, the MHC isoforms are mainly the slow (MHCslow), fast type IIa (MHC2A), fast type IIb (MHC2B), and fast type IIx (MHC2X)33 whereas in cardiac muscle the MHC isoforms are the fast alpha (MHC) and slow beta (MHCß).34 Nevertheless, the skeletal MHCslow and the cardiac MHCß represent the same isoform.34 MHC isoforms are thought to be linked to maximum unloaded shortening velocity, ATP consumption rate, and the rate constant for force generation whereas the link with maximum force remains controversial.34 Therefore, we cannot exclude the hypothesis that the effects of halogenated anaesthetics may differ in cardiac CB, especially in the rat where most MHC are of the fast alpha type, not evaluated in our study. Nevertheless, the predominant MHC isoform in cardiac muscle from various species, including humans, is the slow beta type (or skeletal MHCslow). Moreover, the most important differences in the effects of halogenated anaesthetics between cardiac and diaphragmatic muscles may possibly be a result of differences in proteins that regulate CB kinetics and not to CB per se.
The following points must be considered in the assessment of the relevance of our results. First, the study was conducted at 29°C, because of low stability of the model at 37°C. Secondly, the design and methodology of our study do not allow analysis of CB kinetics at the single-fibre level. In muscle strips, series compliance and muscle fibre heterogeneity (fast vs slow MHC) may affect mechanical properties and CB kinetics. However, it should be pointed out that A. F. Huxleys theoretical data1 were fitted by means of A. V. Hills data obtained from muscle strips and not from isolated fibres.17 Thus, the model accurately fits the mechanical properties of a muscle whose fibre composition includes different MHCs and the FV relationship reflects the relative contribution of each fibre and CB characteristics reflect the average value of the myosin molecular motors. Lastly, our assessment of CB kinetics was indirect. Further studies using either optical tweezers18 or an actinmyosin motility assay35 should be performed to confirm our results. Nevertheless, the fact that our experimental tissue was an intact muscle preparation may be considered an advantage over these more basic models.18 35
In conclusion, in isolated rat diaphragm muscle, halothane and isoflurane did not modify CB kinetics, suggesting that the negative inotropic effects of halogenated anaesthetics on striated muscles are a result of their effects on other targets, likely to be calcium transients and/or myofilament calcium sensitivity.
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Acknowledgement |
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References |
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