Effects of halothane and isoflurane on the contraction, relaxation and energetics of rat diaphragmatic muscle

B. Bouhemad1, O. Langeron1, G. Orliaguet2, P. Coriat1 and B. Riou*,1,3

1 Laboratory of Experimental Anaesthesiology, Department of Anaesthesiology and Critical Care, CHU Pitié-Salpêtrière, Assitance Publique-Hôpitaux de Paris (AP-HP), Université Pierre et Marie Curie, 2 Department of Anaesthesiology and Critical Care, CHU Necker-Enfants Malades, AP-HP, and 3 Department of Emergency Medicine and Surgery, CHU Pitié-Salpêtrière, AP-HP, Université Pierre et Marie Curie, Paris, France*Corresponding author: Departement d’Anesthésie-Réanimation, CHU Pitié-Salpêtrière, 47 Boulevard de l’Hôpital, F-75651 Paris Cedex 13, France

Accepted for publication: May 3, 2002


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. The inotropic effects of halogenated anaesthetics on diaphragmatic muscle remain a matter of debate. Their effects on its relaxation are poorly understood, although diaphragmatic relaxation is recognized as an important physiological process that may interfere with diaphragmatic performance, fatigue and arterial blood flow.

Methods. The effects of halothane and isoflurane (1 or 2x minimum alveolar concentration [1 or 2 MAC]) on contraction and relaxation of rat diaphragm muscle strips (n=40) were studied in vitro from force–velocity curves obtained at various loads from isotonic to isometric conditions. From these curves we determined the peak power output and the curvature. Data are mean (SD) percentage of baseline values.

Results. At 1 MAC, isoflurane and halothane induced no significant inotropic and lusitropic effects. At 2 MAC, isoflurane induced a negative inotropic effect (active force, 93(5)% of baseline). Halothane and isoflurane induced a significant decrease in the peak power output at 2 MAC (88(8) and 86(9)% of baseline; P<0.05), without significant changes in the curvature of the force–velocity curve. At 2 MAC isoflurane under high loads and halothane under low loads induced moderate negative lusitropic effects.

Conclusion. Halothane and isoflurane induced very moderate inotropic and lusitropic effects, suggesting that the decrease in diaphragm function observed in vivo is not related to a direct effect on diaphragmatic contractility.

Br J Anaesth 2002; 89: 479–85

Keywords: anaesthetics volatile, halothane; anaesthetics volatile, isoflurane; muscle skeletal, diaphragm; muscle skeletal, contractility


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
When anaesthetics are administered during spontaneous breathing, or during recovery from anaesthesia, the respiratory effects of volatile anaesthetics may be of clinical importance, particularly when diaphragmatic function is compromised. The direct effects of halogenated anaesthetics on the intrinsic mechanical properties of the diaphragm remain a matter of debate. These effects are difficult to study in vivo because assessment of intrinsic mechanical properties of any muscle requires simultaneous measurements of force, length and velocity,1 which are difficult to measure simultaneously in vivo. Even if such measurements could be obtained, interference with arterial blood flow, arterial blood gases, abdominal pressure and thus diaphragmatic load, and neuromuscular transmission would still be difficult to eliminate.24 Even in vitro studies have provided conflicting results with5 and without6 impairment of diaphragmatic contractility by volatile anaesthetics. Although it is well-established that volatile anaesthetics only have modest effects on the contractility of skeletal muscle, their effects have been reported to be more pronounced in the diaphragm.4 Moreover, although diaphragmatic relaxation is now recognized as an important physiological process that may interfere with diaphragmatic performance, fatigue and arterial blood flow,7 the effects of halogenated anaesthetics on this function remain poorly understood.

We therefore conducted an in vitro study of the effects of halothane and isoflurane on isolated rat diaphragm. The experimental model used enabled us to determine their effects, not only on contraction (inotropy), but also on relaxation (lusitropy) and energetics.8


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animal care conformed to the recommendations of the Helsinki Declaration and the study was performed in accordance with the regulations of the official regulations issued by the French Ministry of Agriculture.

After brief ether anaesthesia, adult Wistar rats (220– 260 g) were killed and a muscle strip from the ventral part of the costal diaphragm was carefully dissected from the muscle in situ. As previously reported,9 this diaphragm strip was immediately suspended vertically in a 200-ml jacketed reservoir with Krebs–Henseleit bicarbonate buffer solution (NaCl 118 mM, KCl 4.5 mM, MgSO4 1.2 mM, KH2PO4 1.1 mM, NaHCO3 25 mM, CaCl2 2.5 mM, and glucose 4.5 mM) prepared daily with highly purified water. The jacketed reservoir was maintained at 29°C with a thermostatic water circulator and a temperature probe. The bathing solution was bubbled with 95% oxygen–5% carbon dioxide, resulting in a pH of 7.40.

Preparations were field stimulated by means of two platinum electrodes with rectangular pulses of 1 ms duration at a rate of 10 pulses min–1 in the 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 using tetanic stimulation at 50 Hz (10 trains min–1 of 300 ms duration, 1 ms rectangular pulses) to obtain maximal contractions. Cross-sectional area was calculated from the ratio of muscle weight to muscle length, assuming a muscle density of 1.06.

The electromagnetic lever system has been described previously.10 All analyses were made from digital records of force and length obtained with a computer, as previously described.810 Variables characterizing contraction (Fig. 1A) were the shortening velocity (Vc), extent of shortening ({Delta}L), peak of the positive force derivative normalized per cross-sectional area (+dF.dt–1) and active force normalized per cross-sectional area (AF). Variables characterizing relaxation (Fig. 1A) were the lengthening velocity (Vr) and peak of the negative force derivative normalized per cross-sectional area (–dF.dt–1). Mechanical variables were calculated from 10 consecutive tetanic contractions preloaded at Lmax with increasing afterload from zero load to fully isometric contraction (Fig. 1B). 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, enabling determination of the maximum unloaded shortening velocity (Vmax). The second contraction was isotonic and loaded with preload only. The maximum extent of shortening ({Delta}Lmax), and the maximum lengthening velocity (Vrmax) were determined from this contraction. The last contraction was fully isometric at Lmax. The maximum active force (AFmax) and the peak of the positive (+dF.dt–1max) and negative (–dF.dt–1max) force derivatives were determined from this fully isometric contraction. Vmax and AFmax described the contraction phase (inotropy), while Vrmax and –dF.dt–1max described the relaxation phase. Nevertheless, since changes in the contraction phase induce co-ordinated changes in the relaxation phase, relaxation parameters alone cannot adequately assess lusitropy, and therefore, variations in contraction and relaxation must be considered simultaneously to quantify drug-induced changes in lusitropy.9 Thus, we calculated the ratios Vrmax/{Delta}Lmax and –dF.dt–1max/AFmax, which assessed lusitropy in isotonic and isometric conditions, respectively. Moreover, relaxation is highly dependent on load level, and thus must also be studied over the entire load continuum (from preload to fully isometric contraction).7 To characterize the isotonic relaxation process, the relationship between Vr and {Delta}L was determined over the load continuum.11 Indeed, {Delta}L is the main determinant of Vr. The relationship between Vr and {Delta}L is almost linear up to 80–90% of {Delta}Lmax, but then Vr increases to a lesser degree as {Delta}L increases up to {Delta}Lmax. To characterize the isometric relaxation process, the relationship between –dF.dt–1 and AF was determined over the load continuum (from 10 to 100% AFmax).12 In muscle isometrically relaxing at initial muscle length, the relationship between –dF.dt–1 and AF is almost linear up to 80% of AFmax, and then –dF.dt–1 decreases to a lesser degree as AF increases up to AFmax.



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Fig 1 Determination of mechanical variables of contraction and relaxation. Muscle shortening length plotted vs time (top panels), and force plotted vs time (lower panels). (A) Tetanus 1 was isotonic, and tetanus 2 was fully isometric. Shortening (Vc) and lengthening (Vr) velocities, maximal extent of shortening ({Delta}L), peaks of positive (+dF.dt–1max) and negative (–dF.dt–1max) force and active force (AFmax) were determined from these typical isotonic and isometric tetanic stimuli. (B) Ten consecutive contractions at different afterload levels from zero load to isometric load. Lmax is the initial muscle length corresponding to the apex of the length–force curve.

 
Muscle energetic parameters were determined from Huxley’s equations.13 The force–velocity curve was derived from the peak shortening velocity (Vc) plotted against the total force (TF=resting force+active force) normalized per cross-sectional area.14 Both measurements were obtained from 10 tetanic contractions, from zero load to fully isometric contraction. The total force–velocity (TFVc) curve was fitted according to the Hill equation:15

(TF+a)(Vc+b)=(TFmax+a)b

where –a and –b are the asymptotes of the hyperbola, TFmax is the calculated maximum isometric total force for Vc=0. The following energetic parameters were derived from the Hill hyperbola: the curvature of the hyperbola (G) and the non-normalized maximum power output (Emax).14 G is linked to myothermal efficiency and cross-bridge kinetics. The more curved the Hill hyperbola (i.e. the higher value of G), the higher the muscle efficiency.16

Diaphragmatic strips were divided into five groups (n=8 in each group), as follows: (i) time-matched control, (ii) 1 minimum alveolar concentration (1 MAC) halothane, (iii) 2 MAC halothane, (iv) 1 MAC isoflurane and (v) 2 MAC isoflurane. 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 layer. The concentration of volatile anaesthetics in the gas phase was continuously monitored with a calibrated infrared analyser (Artema MM 206 SD; Taema, Antony, France). The halothane concentrations used were 0.6 and 1.2%, and isoflurane concentrations were 0.8 and 1.6%. These concentrations are equivalent to 1 and 2 MAC of halothane and isoflurane, respectively, in the adult rat at 29°C.17 Halothane and isoflurane were administered for a 15-min equilibration period before measurements were made.

Data are expressed as mean (SD). Comparisons of two means were performed using the Student’s t test and comparison of several means was performed using analysis of variance and Newman–Keuls test. The energetic parameters were derived from the Hill equation15 using multilinear regression and the least-square method, as previously described.14 All P-values were two-tailed, and a P-value <0.05 was considered significant.


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 Abstract
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 Methods
 Results
 Discussion
 References
 
Mean cross-sectional area was 1.07 (0.25) mm2, and mean Lmax was 8.7 (1.0) mm. The baseline values of mechanical and energetic parameters were not significantly different between groups (Table 1).


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Table 1 Baseline values of mechanical parameters. {Delta}L=maximal extent of shortening; Vmax=maximal unloaded shortening velocity; Vrmax=maximal lengthening velocity; AFmax=maximal isometric active force normalized per cross-sectional area; +dF.dt–1max=peak of the positive force derivative normalized per cross-sectional area; –dF.dt–1max=peak of the negative force derivative normalized per cross-sectional area; Emax=peak power output; G=curvature of the force–velocity curve. Data are presented as means (SD). There were no significant differences between groups
 
Under low load (Vmax, {Delta}L, Vrmax) at 1 and 2 MAC, halothane and isoflurane induced no significant inotropic or lusitropic effects (Table 2). Under high load, isoflurane at 2 MAC induced significant negative inotropic (AFmax) and lusitropic (–dF.dt–1max) effects (Table 2). Conversely, at 1 MAC, halothane and isoflurane induced no significant effects.


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Table 2 Effects of halothane and isoflurane on diaphragmatic muscle. {Delta}L=maximal extent of shortening; Vmax=maximal unloaded shortening velocity; Vrmax=maximal lengthening velocity; AFmax=maximal isometric active force normalized per cross-sectional area; +dF.dt–1max=peak of the positive force derivative normalized per cross-sectional area; –dF.dt–1max=peak of the negative force derivative normalized per cross-sectional area; Emax=peak power output; G=curvature of the force–velocity curve. Data are mean percentage of baseline values (SD). *P<0.05 vs control group
 
Isoflurane induced no significant changes in the Vrmax/{Delta}Lmax ratio (Table 2). Halothane 2 MAC induced a significant decrease of the Vrmax/{Delta}Lmax ratio. Because {Delta}L is the main determinant of Vr, we also studied the relationship, Vr/{Delta}L, over the load continuum. Halothane and isoflurane did not significantly modify the initial linear part of the Vr/{Delta}L relationship (Fig. 2). Isoflurane at 2 MAC induced a significant change in the –dF.dt–1max/AFmax ratio. Because AF is the main determinant of –dF.dt–1, we also studied the relationship –dF.dt–1/AF over the load continuum. Under baseline conditions, an increase in load accelerated –dF.dt–1 in a linear manner of up to around 80% of AFmax. However, under high loading conditions, –dF.dt–1 decreased as AF increased. Halothane and isoflurane did not significantly modify the initial linear part of the –dF.dt–1/AF relationship (Fig. 3).



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Fig 2 Effects of (A) 1 MAC halothane (n=8) and (B) 1 MAC isoflurane (n=8) on the relationship between lengthening velocity (Vr) and extent of shortening ({Delta}L) in diaphragmatic muscles. {Delta}L is the main determinant of Vr, and the initial part of Vr/{Delta}L relationship is almost linear up to 80–90% of {Delta}Lmax; then Vr increases to a lesser degree as {Delta}L increases up to {Delta}Lmax. Data are means (SD).

 


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Fig 3 Effects of (A) 1 MAC halothane (n=8) and (B) 1 MAC isoflurane (n=8) on the relationship between the peak of the negative force derivative (–dF.dt–1) and active force (AF) in diaphragmatic muscles. AF is the main determinant of Vr; and the initial part of –dF.dt–1/AF relationship is almost linear up to 80–90% of AFmax; then –dF.dt–1 decreases to a lesser degree as AF increases up to AFmax. Data are means (SD).

 
The force–velocity curves were correctly depicted by the Hill hyperbola, as shown by the high correlation coefficients (0.998 (0.002)). Halothane and isoflurane at 2 MAC induced a moderate but significant decrease in Emax (Table 2). Halothane and isoflurane did not significantly modify the curvature, G, of the force–velocity curve (Table 2, Fig. 4).



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Fig 4 Effects of 1 MAC of halothane (n=8) and isoflurane (n=8) on (A) the total force (F)–velocity (V) relationship and (B) the normalized FV relationship. Emax=maximum power output; G=curvature of the FV curve; NS=not significant.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The main findings of this study were that (i) at 2 MAC, isoflurane under high loads (isometric conditions) induced a negative inotropic effect; (ii) at 2 MAC, isoflurane under high loads and halothane under low loads (isotonic conditions) induced a negative lusitropic effect; (iii) halothane and isoflurane at 2 MAC decreased the maximal power output, Emax, whereas they did not modify the curvature, G, of the force–velocity curve.

In vitro, halogenated anaesthetics have a moderate positive inotropic effect on skeletal muscle,18 but the diaphragm, unlike other skeletal muscles, requires extracellular calcium for its normal contractile function.19 Conflicting effects of volatile anaesthetics have been observed in in vivo experiments. Indeed some studies have suggested that halogenated anaesthetics induce diaphragmatic contractile dysfunction,4 20 21 whereas other studies did not.6 22 Because of the anaesthetic-induced changes in 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 and colleagues5 have suggested that halothane and sevoflurane decrease the force of the hamster diaphragm only during fatigue. In our study in isolated rat diaphragm, we confirmed the lack of significant effect of halothane under isometric conditions. When the effects of anaesthetics on myocardial muscle are compared as force development (isometric condition) vs shortening (isotonic condition), anaesthetics typically depress force development more than velocity of shortening.23 Our study agrees with these results because we found a moderate negative inotropic effect only under isometric, and not isotonic, conditions.

In our study, halothane and isoflurane at 2 MAC decreased the maximal power output, Emax, whereas they did not modify the curvature, G, of the force–velocity curve (Table 3). G is linked to myothermal economy and cross-bridge kinetics.14 16 The more curved the hyperbola (i.e. the higher value of G), the higher the muscle efficiency. It has been shown that fatigue increases mechanical efficiency in rat diaphragm.24 Rat diaphragm is composed of both faster fatigue-sensitive and slower fatigue-resistant fibre types, and fatigue displaces the characteristics of the muscle towards the slower fatigue-resistant fibres.24 In our study, halothane and isoflurane did not modify the G curvature and these findings support a lack of effect of volatile anaesthetics on fast fatigue-sensitive fibres.25

In skeletal muscle, relaxation consists of an isotonic lengthening, during which the muscle returns to its initial length (isotonic relaxation), and thereafter an isometric decay, during which the muscle returns to its initial resting force (isometric relaxation).26 In separately studying isotonic and isometric relaxation, the experimental model used in the present study enabled determination of the effects of halogenated anaesthetics on the main subcellular pathways responsible for relaxation. In striated muscle, sarcomere length modulates myofilament calcium sensitivity and thus relaxation. Under isotonic conditions, which correspond to significant sarcomere shortening, the sensitivity of troponin C to calcium is low and relaxation proceeds more rapidly than contraction, apparently due to rapid calcium uptake by the sarcoplasmic reticulum.11 Only halothane at 2 MAC induced a significant decrease in the Vrmax/{Delta}Lmax ratio, suggesting an increase in resting cytosolic calcium using halothane. Isometric relaxation was studied by the –dF.dt–1/AF relationship. The initial part of this relationship up to 80% of AFmax is determined by passive mechanisms,12 including the effects of external load and restoring forces, and was not altered by halothane or isoflurane (Fig. 3). Under isometric (i.e. high load) conditions, which correspond to greater sarcomere length, the affinity of troponin C to calcium is higher, and relaxation is mainly determined by the dissociation of calcium from troponin C, rather than calcium uptake by the sarcoplasmic reticulum.11 Isoflurane at 2 MAC induced a significant change in the –dF.dt–1max/AFmax ratio, suggesting an interaction with contractile proteins. Our results agree with the effects observed on the myocardial and skeletal muscles because we found (1) an impairment of isotonic relaxation by halothane, supporting a greater effect of halothane on calcium leakage from the sarcoplasmic reticulum27 and a higher resting cytosolic calcium; (2) an impairment of isometric relaxation by isoflurane as recently showed by Kunst and colleagues28 on skeletal muscle, supporting a greater interaction between contractile proteins and isoflurane than halothane.29

The assessment of the effects of halogenated anaesthetics on isometric and isotonic relaxation was not only motivated by the study of main intracellular components involved in relaxation, i.e. sarcoplasmic reticulum and calcium myofilament sensitivity. Indeed, relaxation of the diaphragm is now recognized as an important active physiological process that requires energy and whose alteration may produce clinically relevant consequences.7 An incomplete relaxation prevents the return of the diaphragm to its optimal length between each inspiration (i.e. at the apex of the length–active isometric tension curve) and thus optimal force generation. Secondly, during extreme activity, blood flow to the diaphragm is reduced or even abolished during contraction, so relaxation is critical to allow adequate blood flow.30 Very few studies have assessed the effects of halogenated anaesthetics on diaphragmatic relaxation. Veber and colleagues22 reported a prolonged relaxation rate with isoflurane (1.5 MAC) in the rat, but relaxation was studied only in quasi-isometric conditions and not on the load continuum.7 Moreover, the effects on relaxation were not assessed by taking into account the simultaneous effects of contraction.7 9 In our study, we observed that halogenated anaesthetics weakly interfere with diaphragmatic relaxation, and only at a high concentration (2 MAC) not usually encountered during spontaneous ventilation in anaesthetized patients.

The following points must be considered when assessing the relevance of our results. First, because this study was conducted in vitro, it dealt only with intrinsic diaphragmatic contractility. Observed changes in diaphragmatic function after anaesthetic administration may also depend on modifications in diaphragmatic arterial blood flow, central nervous system respiratory drive, modification of diaphragmatic shape and neuromuscular transmission. Second, the study was conducted at 29°C because the stability of the model is low at 37°C. In addition, the temperature had to be lowered to prevent core hypoxia.31

In conclusion, in isolated rat diaphragmatic muscle, halothane and isoflurane induced very moderate inotropic and lusitropic effects, which were only significant at 2 MAC, and myothermal efficiency was not significantly affected. These results suggest that the decrease in diaphragm function observed in vivo with halogenated anaesthetics is not related to a direct effect on diaphragmatic contractility.


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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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