Volatile anesthetics such as isoflurane have been reported to have marked bronchodilatory potency in refractory status asthmaticus (1) and are recommended for asthmatic patients requiring general anesthesia. Experimental studies performed in vivo in dogs and rats (2–4) and clinical studies (5) have shown that volatile anesthetics improve respiratory mechanics by reducing pulmonary resistance, elastance, and inhomogeneous ventilation after lung constriction. Bronchodilation is attributed to indirect effects on the reflex neural pathway (6) and also to direct dose-dependent relaxant effects on airway smooth muscle (ASM) (7). Depressed ASM contractility mainly results from changes in Ca2+ signaling mediated by a decrease in the concentration of intracellular free calcium ([Ca2+]i) (8), reduced sensitivity of myofilaments to Ca2+ (9), or a combination of both. Force and shortening in ASM are ultimately regulated by crossbridge (CB) number and kinetics. However, the effects of volatile anesthetics on myosin CB number and CB cycling rates, i.e., ultimate contractile targets in ASM, have not been studied.
In addition to animal models of passive and active sensitization, animal models of spontaneous innate airway hyperresponsiveness (AHR) have also been developed for analyzing mechanisms associated with acquired AHR. Lung function tests have shown that, in response to various bronchoconstrictors, the Fisher F-344 rat strain exhibits more reactive airway responses than the Lewis strain (10). In addition, the in vivo AHR of Fisher rats is associated in vitro with changes in the intrinsic contractile properties of ASM compared with the Lewis strain, including changes in CB kinetics (11). Our aim was to determine the effects of isoflurane on mechanics and CB kinetics in isolated rat tracheal strips. We tested the hypothesis that ASM myosin molecular motor intrinsic mechanics and kinetics could be modified differently by isoflurane depending on the level of airway responsiveness in the two rat strains.
Animal care complied with the recommendations of the official edict of the Helsinki convention and those of the French Ministry of Agriculture and our institution, INSERM. These experiments were conducted in an authorized laboratory under the supervision of authorized researchers (BR, YL, CC). Experiments were performed on 2 inbred rat strains: Fisher (n = 10) and Lewis (n = 10) male rats 12 to 14 wk old. Animals were obtained from Charles River Laboratory (L’Arbresle, France).
After anesthesia with intraperitoneal pentobarbital sodium (60 mg/kg), animals were thoracotomized and cervicotomized. The trachea was carefully dissected and opened with a dorsal midline section through the cartilage to obtain 2 strips of 5 segments of the posterior membranous portion of trachea from each animal (n = 20 in each group). Each muscle strip was vertically suspended between 2 clips in a chamber containing a Krebs-Henseleit solution composed of 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. Throughout the experiment, the solution was bubbled with 95% oxygen and 5% carbon dioxide and maintained at 37°C, resulting in a pH of 7.40. The lower clip was stationary at the bottom of the bath, and the upper clip was linked to a special electromagnetic lever system as previously described (12). Tetanic supramaximal electrical field stimulation (30 V/cm, 50 Hz frequency, 3 ms pulse duration, 12 s train duration) was applied every 3 min by means of 2 electrodes arranged in parallel on either side of the muscle preparation.
All experiments were performed at 37°C and measurements were conducted after a 1-h equilibration period. The optimal initial muscle length (Lo) was determined as the resting muscle length corresponding to the maximum of the length-active isometric tension curve.
At the end of the study, cross-sectional area (mm2) was calculated from the ratio of muscle weight (mg) to muscle length at Lo (mm), assuming a tissue density of 1.
For each muscle strip, the following variables characterizing the electric field stimulated contraction phase were determined: peak isometric force (Fo, mN), normalized per cross-sectional area to obtain peak isometric tension (Po, mN/mm2) was measured from the fully isometric contraction; maximum unloaded shortening velocity of contraction (Vmax, Lo/s) was measured from the contraction abruptly clamped to zero-load just after the onset of electrical stimulation. The hyperbolic tension-velocity relationship was then derived from the velocity (V) of seven to 10 isotonic afterloaded contractions, plotted against the isotonic tension level (P), and from successive load increments from zero-load up to total isometric tension (Po). The tension-velocity curve was fitted according to Hill’s equation: (P + a)(V + b) = (Po + a)b, where −a and −b are the asymptotes of the hyperbola, as determined by multilinear regression. The curvature G of the tension-velocity relationship is equal to: G = Po/a = Vmax/b.
CB mechanics and kinetics were derived from the parameters of Hill’s hyperbola, using Huxley’s equations (13) applied to ASM (14). This formalism makes it possible to calculate the number of CB per mm2 at peak isometric tension (Ψ · 109 · mm−2), unitary CB force (π, in piconewtons, pN), and CB kinetics (i.e., maximum values of the rate constants for CB attachment f1 and detachment g1 and g2, maximum turnover of myosin adenosine triphosphatase activity kcat, and total CB cycle duration tc, which is equal to 1/kcat).
Original Huxley’s equations and detailed calculations of myosin CB number, force, and kinetics are given in the Appendix.
For each group, three protocols were investigated successively. First, the isotonic and isometric mechanical variables of tracheal strips, the tension-velocity relationship, and CB mechanics and kinetics were determined at baseline. Next, methacholine (10−6 M) was added to the bath. Initial length was readjusted after a 15-min equilibration period such that it was similar to baseline. Each variable was then measured after methacholine-induced contraction. Thereafter, isoflurane (2.9% vol./vol.) was added to the bath by means of a special calibrated anesthetic vaporizer (Fortec 3 for Iso; Cyprane Ltd., Keighley, UK), corresponding to a 2 MAC value in an adult rat at 37°C (15). The gas mixture was bubbled continuously with 95% oxygen and 5% carbon dioxide in the bath solution. Anesthetic concentration in the gas phase over the bath solution was monitored with a calibrated infrared analyzer (Artema MM 206; Taema, Antony, France). Evaporation of volatile anesthetics was prevented by a thin paraffin sheet over the jacketed reservoir. After a 15-min equilibration period, initial length was again readjusted so that it was similar to baseline.
Data are expressed as means ± sd and given as absolute values and as percentage changes from baseline values induced by methacholine and isoflurane. For the force-velocity curves, a correlation coefficient r between measured values of force and velocity and the model estimate was used to test the accuracy of the hyperbolic fit. Student’s unpaired t-test was used to analyze differences between strains. In addition, baseline, methacholine, and isoflurane conditions were compared by using repeated-measures analysis of variance. When the F test was significant, means were compared using a Bonferroni-corrected Student’s paired t-test. All P values were two-tailed and a P value < 0.05 was considered significant.
Mean animal weight was 358 ± 3 g in the Fisher rats and 391 ± 6 g in the Lewis rats (not significant). There was no significant difference in mean cross-sectional area (0.94 ± 0.08 versus 0.94 ± 0.08 mm2; not significant) and Lo (1.7 ± 0.1 versus 1.6 ± 0.1 mm; not significant) between the 2 groups. At baseline, Po did not significantly differ between strains, whereas Vmax was higher in Fisher than in Lewis rats (Fig. 1). The correlation coefficient of the force-velocity curve showed an accurate hyperbolic fit in both Fisher and Lewis rats (0.996 ± 0.003 versus 0.999 ± 0.001; not significant). Myosin CB number (Ψ · 109), the unitary CB force (π), total CB cycle (tc), and peak efficiency (Effmax) did not significantly differ between strains (Figs. 1 and 2). Durations of attachment step (1/f1) and detachment step (1/g2) were significantly shorter in Fisher than in Lewis rats (Fig. 2).
Methacholine increased Po in both Fisher and Lewis strains (Fig. 3A) (P < 0.05). Isoflurane totally reversed the effect of methacholine on Po in the Lewis rats whereas the reversal was only partial in the Fisher rats (Fig. 3A). Thus, after methacholine then isoflurane exposure, Po was significantly higher in Fisher than in Lewis strains (24.6 ± 5.0 versus 15.3 ± 3.0 mN · mm−2; P < 0.05).
Methacholine significantly increased Vmax in the Lewis rats whereas no significant changes were observed in the Fisher rats (Fig. 3B). After exposure to isoflurane, Vmax decreased significantly in both groups (Fig. 3B). In the Lewis rats, this value did not significantly differ from baseline, whereas it was significantly less than baseline in the Fisher rats (Fig. 3B). However, in all conditions, Vmax was significantly higher in Fisher than in Lewis (each P < 0.05).
In both strains, methacholine significantly increased the total number of active CBs (Ψ · 109) (Fig. 3C), and this effect did not significantly differ between strains. Compared with baseline, isoflurane completely reversed the effect of methacholine on CB number in the Lewis group but only partially in the Fisher group (Fig. 3C), although there was no significant difference in Ψ · 109 between strains.
Methacholine significantly increased the CB unitary force (π) in both groups, and there was no significant difference between groups (Fig. 3D). Isoflurane completely reversed the effect of methacholine in the Fisher rats. In contrast, in the Lewis rats, isoflurane did not significantly modify π compared with the methacholine value (Fig. 3D). However, after isoflurane exposure, the absolute values of π did not differ significantly between strains.
Methacholine significantly increased the duration of the attachment step (1/f1) in the Fisher rats, whereas no significant difference was noted in the Lewis rats (Fig. 4A). In both groups, isoflurane significantly increased 1/f1, but the effect was more pronounced in Lewis than in Fisher rats (Fig. 4A). Thus, after isoflurane exposure the absolute value of 1/f1 was significantly higher in Lewis than in Fisher rats (250 ± 83 ms in Lewis versus 152 ± 27 ms in Fisher; P < 0.05) and these values were significantly higher than in baseline conditions (Fig. 4A).
Methacholine significantly decreased 1/g2 in the Lewis rats, whereas no significant change was noted in the Fisher rats (Fig. 4B). In both groups, isoflurane induced a significant increase in 1/g2 as compared with the methacholine value. Thus, after methacholine and isoflurane exposure, 1/g2 did not significantly differ from baseline in the Lewis rats (P = 0.19), whereas 1/g2 was significantly higher than baseline in the Fisher group (Fig. 4B). In all these conditions, the absolute value of 1/g2 was significantly higher in Lewis than in Fisher strains (each P < 0.05).
In both strains, methacholine significantly increased the total duration of the CB cycle (tc) (Fig. 4C). Thus, after methacholine exposure, there was no significant difference in tc between Fisher and Lewis rats (P = 0.29). As compared with baseline, isoflurane increased the total CB cycle duration in Lewis rats by nearly twofold, whereas no significant change was observed in the Fisher rats (Fig. 4C). After methacholine and isoflurane exposure, tc was significantly shorter in Fisher than in Lewis rats (5.3 ± 1.8 s versus 6.6 ± 2.6 s; P < 0.05). In both strains, methacholine significantly increased Effmax (Fig. 4D). Isoflurane induced a slight but significant decrease in Effmax in the Fisher group, whereas it did not significantly change Effmax in the Lewis group. Thus, isoflurane completely reversed the effect of methacholine in the Fisher rats but not in the Lewis rats (Fig. 4D). After isoflurane exposure, the absolute values of Effmax did not differ between strains (P = 0.95).
The main results of our study are as follows.
- i) Isoflurane altered the intrinsic mechanics of isolated ASM preparation in both isometric and isotonic conditions in two rat strains with different airway responsiveness levels.
- ii) In addition, isoflurane induced modifications in CB mechanics and CB kinetics that did not simply consist of a reversal of changes in the CB cycle induced by methacholine. Thus, isoflurane either completely or partially counteracted methacholine-induced contraction or, in some cases, enhanced the effects of methacholine on the mechanical properties of whole muscle and the actomyosin CB.
- iii) Finally, myosin molecular mechanics and energetics were modified differently depending on the airway responsiveness level.
In smooth muscle, new insights into events governing the actomyosin adenosine triphosphatase cycle and myosin molecular motor mechanics have been provided by radiographic crystallographic studies on three-dimensional molecular structures, in vitro motility assays, and optical tweezers. In addition, theoretical models of contraction based on Huxley’s CB theory, the most commonly accepted model of muscle contraction, have been proposed in smooth muscle (14,16). Huxley’s equations can be adapted to ASM to determine myosin molecular mechanics including total CB number, single CB force, maximum rate of attachment and detachment steps, and the total duration of the CB cycle (14). This formalism thus represents an attractive method for explaining how volatile anesthetics modulated myosin molecular mechanics in strains characterized by different levels of airway responsiveness.
Although it is clear that volatile anesthetics relax ASM, at least in part, by reducing the force (7) or shortening the capacities of the muscle (17), their effects on actomyosin CB remain poorly documented. According to Huxley’s equations, the peak isometric tension is the product of CB number and CB unitary force generated during contraction (13). In both strains, we found that the decrease in total isometric tension after isoflurane exposure resulted mainly from a decrease in the total number of active CBs (Fig. 3C), associated in the Fisher AHR model with a slight decrease in the unitary force produced per CB. Interestingly, volatile anesthetics decrease the concentration of cytosolic Ca2+ during contraction (18) by reducing Ca2+, K+, and Cl− (19) currents and membrane potential (19). In addition, halogenated anesthetics decrease the sensitivity of the contractile apparatus to Ca2+ (9), probably via an increase in smooth muscle protein phosphatase activity (20). These changes in intracellular Ca2+ signaling are expected to reduce actomyosin CB activation and are thus likely to explain the reduction in active CB interactions during contraction in Fisher and Lewis rats.
Furthermore, our study showed that isoflurane influenced actomyosin interaction kinetics in ASM, strongly suggesting that volatile anesthetics also modulated CB performance at a level beyond CB activation. Actin-myosin attachment occurs after adenosine triphosphate hydrolysis and is thought to play an important role in regulating CB kinetics. In addition, the attachment step leads to the creation of a CB generating force that is assumed to modulate bronchial tone. At baseline, attachment step duration (1/f1) was shorter in Fisher rats than in Lewis rats (Fig. 2A). It has been proposed that differences in CB kinetics contribute to the AHR phenotype in Fisher rats (11). Interestingly, isoflurane substantially prolonged the duration of the attachment step by nearly 30%–40% compared with baseline in both strains (Fig. 4A). As a consequence, the potential number of CB entering the CB cycle was expected to be decreased after isoflurane exposure. In addition, isoflurane increased the duration of the detachment step in both strains, although to a larger extent in Fisher than in Lewis strains (Fig. 4B). Given that the rate constant of CB detachment (g2) limited Vmax (11), the longer 1/g2 reported in our study after isoflurane exposure could help explain the decrease in Vmax. Importantly, slower CB attachment and detachment steps were associated with a dramatic twofold increase in the overall duration of the CB cycle; therefore each active CB was expected to cycle twofold more slowly after isoflurane exposure than at baseline (Fig. 4C). Together, these effects were likely to reduce the tone of ASM and favor bronchodilation.
Isoflurane affected CB kinetics and peak efficiency differently in each strain. Despite extensive studies, the mechanisms accounting for interstrain mechanical differences at baseline have still not been fully determined. The number of intracellular Ca2+ homeostasis changes may depend on the type of volatile anesthetic and the level of airway reactivity (17). Variations in regulatory mechanisms and differences in myosin isoform may help explain differences in mechanical behavior between the two strains (21). The Fisher strain is associated with a shift in myosin isoform expression towards a larger amount of myosin heavy chain with a 7-amino acid insert isoform in the head region (22). This insert, located near the ATPase site, results in increased actin-activated ATPase activity (22) and a more rapid rate of actin filament movement in the in vitro motility assay (23). Such modifications have been related to AHR in vivo (24). Further studies are needed to investigate whether such mechanisms contribute to the interstrain mechanical differences observed after administration of isoflurane.
The following limitations must be considered when assessing the relevance of our results. First, this study was performed in isolated ASM and may not reflect what is happening in vivo. In vivo, volatile anesthetics can also partly modulate central nervous system activity or regulatory mechanisms involved in the control of airway caliber. Second, because ASM contraction depends on transsarcolemmal calcium movement and because volatile anesthetics markedly interfere with these calcium movements, we cannot infer from our study that isoflurane has a direct effect on CB kinetics (25). Further studies using either optical tweezers or actin-myosin motility assays should be performed to assess the direct effect of volatile anesthetics on CBs. Third, rat ASM may differ from that of humans, and species differences cannot be excluded.
In conclusion, isoflurane-induced changes in isotonic and isometric contractile properties in isolated rat ASM were associated with a reduced number of active CB and prolonged duration of the attachment and detachment steps and overall duration of the CB cycle. CB cycling rates in ASM were modulated differently depending on the rat strain and the level of airway responsiveness. Taken as a whole, our results provide new insights into the intracellular effects of volatile anesthetics on ASM and suggest that the beneficial effects of isoflurane on bronchoconstriction might result at least in part from modulation of myosin CB kinetics.
A. F. Huxley’s equations (13) give the total rate of energy release (
), Huxley isotonic tension (PHux), total rate of mechanical energy (
) and mechanical efficiency (Eff) as a function of velocity according to the following equations:
where e is the free energy required to split one adenosine triphosphate molecule (e = 5.1 × 10−20 J), w is the maximum mechanical work of a single CB (w = 0.75 e = 3.8 × 10−20 J) (13), θ is the length between two actin binding sites (θ = 36 nm), h is the molecular step size (h = 11 nm), and Φ = (f1+ g1) h/2 = b (14). The maximum value of Eff is termed Effmax. CB mechanics and kinetics are given by the following equations: (11,14)
g1 = 2 w b / e h G,
g2 = 2 Vmax/h,
and the total duration of the CB cycle tc = 1/kcat.
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