Isoform-dependent Effects of Halothane in Human Skinned Striated Fibers

The study protocol was approved by our University Studies Ethics Committee, and written informed consent was obtained from all patients before participating in the study. Left ventricular muscle strips were dissected from the endocardial surface from eight patients undergoing cardiac surgery for valvular heart disease. Left ventricular function, as assessed by cardiac catheterization (left ventricular size, wall thickness, ejection fraction, cardiac output) was within the normal range in all patients. The myocardial fragments removed were immediately placed in cardioplegic solution at room temperature and rapidly transported to the laboratory. Skeletal biopsy specimens were taken from the vastus lateralis muscle from ten patients undergoing elective surgery of the lower limbs. None of the patients were confined to bed before surgery. Bundles containing several hundred fibers were attached at their extremities to a wooden stick to maintain their resting length and immediately placed in a relaxing solution at room temperature and rapidly transported to the laboratory.

Chemically skinned cardiac and skeletal fibers were prepared as previously described. [11,15]Chemical skinning with EGTA renders the muscle fiber sarcolemma freely permeable to external solutes. Segments of cardiac and skeletal muscle, containing several hundred fibers, were dissected free and immediately placed in a relaxing solution at 4 degrees Celsius for 24 h. The skinning solution was replaced with fresh solution after 1, 4, and 12 h. After 24 h, the segments were transferred to a skinning storage solution that was identical to the relaxing solution except for the addition of 50% glycerol and stored at -20 degrees C until used (1 or 2 weeks). This technique is identical to that used by a number of other laboratories. [16,17]No change in skinned fiber properties could be noticed after 2 or 3 weeks of storage.

Bundles of cardiac muscle (which will be called fibers hereafter) or single skeletal fibers were isolated from the main fascicle under a 40-power Swift Model 31-400-00 binocular microscope (Tokyo, Japan). Each cardiac or skeletal fiber was mounted horizontally between two clamps in a muscle bath (0.8 ml) filled with a relaxing solution. One clamp was attached to a Grass Model FT-03 force displacement transducer (Quincy, MA). The muscle contracture was amplified and recorded on a Gould 2200 S device (Valley View, OH). The preparation was bathed for 15-20 min in a relaxing solution containing the nonionic detergent Brij 58 (2%), which irreversibly eliminates the capability of the SR to sequester Calcium sup ++ and to release it under appropriate stimulation, but does not affect the contractile proteins. [14]The length and diameter of the preparations were measured under a 400-power Olympus lens. Each skeletal fiber was straightened by adjusting the position of the transducer, and the resting tension was then applied by stretching the fiber by 20% of its initial length. For cardiac fibers, the initial straightening was made to a length at which an increase in resting tension was first detected, and the preparation was then stretched a further 20% of the initial length of the bundle, as previously described by Maughan et al. [18]Finally, the functional destruction of SR was confirmed by studying Calcium sup ++ release from SR with 40 mM caffeine after loading the SR with a known concentration of Calcium sup ++ (pCa 6.8) in the presence of adenosine triphosphate. Only preparations with no significant contracture to caffeine (i.e., no functional SR) were included in the study.

For all experiments described, the length of the fibers was kept constant to avoid sarcomere length-dependent changes in Calcium sup ++ sensitivity. All experiments were performed at room temperature (20 +/-1 degree C).

The concentrations of the different components in the solutions were calculated using program 3 of Fabiato and Fabiato [19]to keep the ionic strength at 200 mM. The stability constants of Orentlicher et al. [14]were used in the calculations: K^CaEGTA1.919 x 10⁶/M, K sub CaATP 5.0 x 10³/M, K^MgEGTA40/M, and K^MgATP1.0 x 10 sup 4/M. Composition of solutions has been previously published. [11,15].

To assess the effects of halothane, the test solutions were equilibrated by continuous bubbling with the anesthetic agent for 20 min. Halothane was mixed with 100% nitrogen by means of a calibrated vaporizer (Fluotec Mark III, Cyprane Keighley). The anesthetic concentrations in the gas phase were monitored with an infrared calibrated analyzer (Capnomac, Datex, Finland). The anesthetic concentrations used were 0.75 and 2.25 vol% halothane. These concentrations in gas phase are roughly equivalent to 1 and 3 minimum alveolar concentration multiples of halothane in humans at 37 degrees C. The anesthetic concentrations obtained in the experimental chamber were measured by gas-liquid chromatography to determine the amount of anesthetic present in the solutions. A Varian 1400 gas chromatograph equipped with flame ionization detector and a Porapack Q 3.17 mm by 150 cm column (Palo Alto, CA) was used for determination of anesthetic concentrations. [20]A 60-ml flask containing 100 micro liter of the solution equilibrated for 20 min with the anesthetic was maintained at 60 degrees C (above the boiling point for halothane) for 20 min before injecting 1 ml gas into the apparatus, previously calibrated with known concentrations of halothane (head space technique). The anesthetic concentrations measured in the experimental solution after 20 min of continuous bubbling were as follows: 0.44+/-0.03 and 1.26+/-0.08 mM for 0.75 and 2.25 vol% halothane (concentrations in the gas phase given by the calibrated analyzer), respectively.

For each skinned cardiac or skeletal fiber, a pCa-tension curve was obtained under control conditions by stepwise exposure of the preparation to solutions with increasing Calcium sup ++ concentrations and measurements of developed tension (Figure 1). Calcium sup ++ concentrations ranged from pCa 6.4 ([Calcium sup ++] = 0.3 micro Meter) to pCa 4.8 ([Calcium sup ++] = 15.8 micro Meter) where pCa = -log¹⁰[Calcium sup ++]. Intermediate tensions were expressed as a percentage of the maximal tension. Data were fitted using nonlinear regression analysis (Enzfitter, Elsevier Biosoft, Cambridge, UK) to the modified form of Hill's equation: [Ca sup ++]^nH/(K^50nH+ [Ca sup ++]^nH), where F is the relative tension, nH (Hill coefficient) is a measure of the slope of the relationship, and K⁵⁰is the Calcium sup ++ concentration (expressed in micro Meter) that yields 50% of the maximal Calcium sup ++ -activated force.

Under all experimental conditions, halothane was tested at 0.75 and 2.25 vol% on the same cardiac fibers. Hence, pCa-tension curves were obtained for the two concentrations studied in a random order in each fiber. A final pCa-tension curve was obtained with solutions free of anesthetic. Because maximal activated tension decreased regularly during the study and represented roughly 80-85% of the initial developed force at the end of the overall experiment, tension values were normalized to their maximal value at each anesthetic concentration, and then plotted to allow analysis of the sensitivity of the preparations to Calcium sup ++ in the presence of 0.75 and 2.25 vol% halothane. The mean values of the two control curves were used to assess the effects of halothane. To minimize the decline in maximal tension at < 15%, the effects of each concentration of halothane on skeletal contractile proteins were studied in different fibers. Hence, after determination of the first control curve, each skinned fiber was exposed to 0.75 or 2.25 vol% halothane, and finally, the experiment was completed with a second control curve.

In a second series of experiments, changes of tension at maximal Calcium sup ++ -activated force were examined using a pCa 4.8 solution. Each fiber was exposed to test solutions equilibrated with 0.75 or 2.25 vol% halothane. Each test was immediately preceded and followed by determination of maximal Calcium sup ++ -activated tension with the control test solution (i.e., free of anesthetic) so that no significant differences between controls were observed. Isometric tension development from baseline to steady state was compared between test solutions and the mean of the two control measurements. Results were expressed as a percentage of these corresponding control values.

In skeletal fibers, a final series of experiments examined changes of tension at half-maximal activation using solutions with concentrations of Calcium sup ++ that were close to the calculated K⁵⁰, obtained at the beginning of the study. Each fiber was exposed to test solution equilibrated with 0.75 vol% halothane. Each test was bracketed by determination of half-maximal activated tension with the control solution, free of halothane. No significant difference between controls was observed. Results were expressed as percentages of these corresponding control values.

All skinned skeletal fibers were tested with increasing concentrations of Strontium sup ++ as described by Takagi et al. [21]and validated in our laboratory. [15]For each fiber, a pSr-tension curve (where pSr = -log¹⁰[Strontium sup ++]) was obtained in a similar experimental procedure as pCa-tension curves. The concentration of Strontium sup ++ for half-maximal activation (K^Sr50) was computed. Using this analysis, two populations were clearly identified: type I (slow twitch) fibers contracted with low concentrations of Strontium sup ++ (K sub Sr50 = 1.35+/-0.30 micro Meter [mean+/-SD]), and type II (fast twitch) fibers, which contracted with only high concentrations of Strontium sup ++ (K^Sr50= 7.20+/-1.25 micro Meter [mean+/-SD]).

Results were expressed as mean+/-standard error of the mean. For pCa-tension curves in myocardial fibers, comparisons of K⁵⁰(the Calcium sup ++ concentration giving half-maximal tension) and the Hill coefficient between control values, 0.75, and 2.25 vol% were made by repeated-measures analysis of variance (multiple comparisons used Fisher's protected least significant difference testing). In other experimental conditions, the effects of halothane were analyzed with Wilcoxon (paired data) or Mann Whitney U tests (unpaired data). Values of P < 0.05 were considered significant.

Characteristics of the muscle fibers for myocardium, type I and type II skeletal muscle are shown in Table 1. The pCa-tension curves were determined in eight myocardial fibers with 0.75 and 2.25 vol% halothane used in a random order. Figure 1(top) shows an example of the changes in tension in response to increasing Calcium sup ++ concentrations observed with solutions free of anesthetic (Figure 1(A and C)) and with activating solutions equilibrated with the two concentrations of halothane (Figure 1(B)). Tension changes after Calcium sup ++ changes were plotted to allow analysis of the sensitivity of the preparations to Calcium sup ++ in the absence and in the presence of halothane. At higher anesthetic concentration, a higher concentration of Calcium sup ++ was necessary to generate the same amount of tension as in control (Figure 2, top). Halothane thus shifted the pCa-tension curves to the right in a dose-dependent fashion. This was attested by the significant increase in K⁵⁰values with increasing halothane concentrations with no significant change in the Hill coefficient (Table 2).

The pCa-tension curves in skeletal fibers were determined using muscle biopsy specimens obtained from ten patients. The effects of halothane on Calcium sup ++ sensitivity were dependent on muscle fiber types. Figure 1(middle) shows a typical example of the changes in tension of type I skeletal fiber in response to increasing Calcium sup ++ concentrations in control conditions (Figure 1(A and C)) and in the presence of 0.75 vol% halothane (Figure 1(B)). Higher concentrations of Calcium sup ++ were needed to generate tension in the presence of both 0.75 and 2.25 vol.% halothane. The difference between results obtained at 0.75 vol% and 2.25 vol% was not statistically significant (Table 2). In contrast, normalized pCa-tension curves in type II skeletal fibers were not modified by halothane at both concentrations studied (Figure 1and Figure 2, bottom). Hence, no significant changes were observed between K⁵⁰and Hill coefficients of type II fibers obtained in control conditions and in the presence of halothane (Table 2).

The effect of halothane on maximal activating tension was determined in 16 cardiac bundles, 20 type I and 18 type II skeletal fibers. Fibers were equilibrated in a maximally activating solution at pCa 4.8 and subsequently in the presence of 0.75 or 2.25 vol% halothane. Maximal activated tension decreased significantly in a dose-dependent fashion with increasing concentrations of halothane in the three types of fibers (Table 3). However, the decrease in maximal activated tension obtained in type II skeletal fibers was clearly smaller than in both type I and myocardial fibers (Table 3). A typical set of results obtained with skinned skeletal fibers is shown in Figure 3. The force traces were obtained when the preparation was maximally activated at pCa 4.8, initially in the absence of halothane. After a substantial rise in force, the type I fiber was exposed to 0.75 vol% halothane (Figure 3(A)), which caused a prolonged and stable decrease in force immediately reversible on switching from halothane-equilibrated solution to control solution of identical pCa. In contrast, the same experiment performed with 2.25 vol% halothane caused only a moderate decrease in tension of type II fibers (Figure 3(B)).

Finally, the effects of halothane on half-maximal activated tension were determined in nine type I, and eight type II skeletal fibers. At half-maximum tension, the effects of halothane were the consequence of the effects on maximal activated tension as well as on calcium sensitivity. As a result, 0.75 vol% halothane dramatically decreased half-maximal activated tension in type I fibers, whereas the decrease in type II fibers was not different from the decrease observed using the pCa 4.8 solution (Table 3). These effects were immediately reversible when switching from anesthetic-equilibrated solution to control solution of identical pCa.

We have used three human skinned fiber systems to investigate the possible role of cardiac and skeletal muscle-specific myofibrils isoforms in the mechanism of halothane depression of contractility. Our results demonstrate that the volatile anesthetic decreases both Calcium sup ++ sensitivity and maximal force in human slow (ventricular and type I skeletal) skinned fibers and has no effects in fast skinned fibers. Because most of the skeletal muscles in humans are mixtures of fast and slow fibers, [22]the absence of effects of halothane on fast fibers may contribute to the absence of direct negative inotropic action of this agent on skeletal muscle.

The Calcium sup ++ sensitivity of the cardiac and skeletal contractile apparatus expressed as K⁵⁰and Hill coefficients closely overlaps that found by other studies in humans using saponin pretreatment of trabeculae from normal heart [23]and EGTA + Brij skinned fibers obtained from lateral gastrocnemius muscle. [16]Type I fibers had a lower Calcium sup ++ threshold for tension development than type II fibers, and both had steeper calcium-tension relationships than myocardial fibers. Our technique characterizes the fiber type of skinned mammalian muscle fibers based on their relative sensitivities to strontium. It was assumed that type I fibers had high sensitivities to strontium, and that type II fibers had low sensitivities to strontium. [21]This method has been validated by correlating it with standard histochemical staining in a large number of muscle fibers. [15].

Our study uses a skinned fiber preparation because it allows rapid application and removal of particular agents, which influences the myoplasmic Calcium sup ++ concentrations. Such a preparation has been widely used to study contractile apparatus itself. [14,16]The technique uncouples T tubules from SR structures with EGTA and then uses Brij 58 to destroy the SR membranes. However, the skinned fiber techniques were demonstrated to be highly sensitive to experimental conditions such as temperature, intracellular pH, or changes in surrounding substrate concentrations. [24]The technique allows the diffusion outside the cell of low molecular-weight proteins, and also may induce inadvertent proteolysis, alterations in myosin light chain phosphorylation, and changes in cross-bridge kinetics. [25]This could partly explain the finding that the myofilament sensitivity to Calcium sup ++ appears to be lower in skinned preparation than in intact muscle.

Although the effects of volatile anesthetics on calcium sensitivity and maximal force of contractile proteins have been investigated extensively in the literature, few authors have compared the responsiveness of both cardiac and skeletal muscles using similar experimental conditions. Our results differ in part from those reported by Su et al. [26,27]in mechanically disrupted rabbit ventricular and skeletal fibers. They found that halothane slightly decreased maximal Calcium sup ++ -activated tension both in papillary muscle and soleus muscle (type I, slow twitch skeletal muscle), whereas in adductor magnus (type II, fast twitch skeletal muscle), halothane produced no change. However, they could not demonstrate any significant effect on ventricular and soleus Calcium sup ++ sensitivity except at high concentrations (greater or equal 3 vol% halothane). In addition, they observed an increase in Calcium sup ++ sensitivity with halothane in type II skeletal fibers. More recently, Blanck et al., using chemically skinned rabbit soleus (slow twitch) and cardiac fibers with EGTA alone, found no effect of halothane on the contractile apparatus. [17]These authors concluded from their observations on skinned fibers and isolated troponin C that halothane does not alter the affinity of cardiac troponin C for Calcium sup ++ and that the myofibrils are not an important site for the negative inotropic effect of halothane. In fact, the simple removal of the sarcolemmal diffusion barriers with a mechanical technique or EGTA pretreatment alone is a different experimental condition than that used in our study. [28]Because the volatile anesthetic effects observed in the current study after a similar treatment with EGTA plus Brij 58 differ between type I and cardiac muscle compared to type II skeletal muscle, it is likely that the technique used to skin the membrane does not account for the effects observed in the study. Because type I fibers share many subcellular structural features with cardiac fibers, it is likely that these effects reflect direct influences on the contractile apparatus of slow skinned fibers.

Our results obtained on human type II skinned fibers are consistent with those reported by Ohta et al. in saponin pretreatment of pig gracilis muscle. In their experiment performed only on fast fibers (type II), the pCa-tension relationship of normal muscle was not altered by halothane. [29]Our results obtained on human skinned cardiac are are also in agreement with previous studies demonstrating a decrease in maximal activated tension ranging between 5% and 15% for clinically relevant anesthetic concentrations in skinned cardiac fibers from rats, [10]hamsters, [30]and rabbits. [31]The volatile anesthetics also decreased myocardial Calcium sup ++ sensitivity in a dose-dependent and reversible fashion. The effects of the three anesthetics used (halothane, enflurane, and isoflurane) were identical for equianesthetic concentrations expressed in minimum alveolar concentration multiples. [10]However, with regard to our results and to our previous study on myocardial proteins, [11]the major shift in Calcium sup ++ sensitivity appears to occur with 0.75 vol%, with very little (although significant) further shift being present with a threefold increase in concentration. This behavior suggests some sort of saturable phenomenon.

In several respects, the conditions of our experiments were different from those encountered in the anesthetized human. To preserve the viability of the preparations, it was necessary to work at a temperature lower than 37C, as is commonly done with skinned preparations. Because there could be temperature-dependent differences (i.e., greater depression at a lower temperature) in the negative inotropic effect of the volatile anesthetics, [32]and because minimum alveolar concentration values decrease with decreasing body temperature, [33]our data may overestimate the effects of anesthetics on Calcium sup ++ sensitivity.

To date, the exact cellular mechanism by which halothane decreases Calcium sup ++ sensitivity and maximal force of the contractile proteins is not known. According to previous studies, the effects on Calcium sup ++ sensitivity may involve the troponin-tropomyosin system [10,27,31](but not directly tropinin C [17]). Our results suggest that troponin I may not be a direct target of halothane, because cardiac and skeletal (type I and type II) isoforms are clearly different. Alternatively, the decrease in Calcium sup ++ sensitivity could be directly related to the decrease in maximum Calcium sup ++ -activated force, as previous studies have shown that myosin cross-bridges binding enhances Calcium sup ++ binding to tropinin C and may thus play a key role in activation of the thin filament. Combined decreases in maximum tension and Calcium sup ++ sensitivity also are observed with agents such as 2,3-butanedione monoxime, which affects the biochemical states of the cross-bridges during the working cycle, resulting in a reduction in the number of cross-bridges in a force-generating state. [34,35]With halothane, the decrease in maximal force also could involve processes of attachment and detachment of actomyosin cross-bridges, leading to a decrease in the number of cross-bridges involved in force generation [36]as well as the amount of force developed by individual cross-bridges. [37]Additional studies are needed to define the exact cellular mechanism by which halothane interferes with contractile proteins.

In conclusion, the current study demonstrates that halothane decreases both calcium sensitivity and maximal force of contractile proteins in human cardiac and type I skeletal skinned muscle fibers and has no significant effect in type II skeletal skinned muscle fibers. Such significant effects depending on contractile protein isoforms have not been described previously. If these results can be extrapolated to in vivo conditions, they may partly explain the difference in the overall inotropic action of halothane between cardiac and skeletal muscle.