Sevoflurane depresses myocardial contractility by decreasing transsarcolemmal Ca2+ influx. In skinned muscle fibers, sevoflurane affects actin-myosin cross-bridge cycling, which might contribute to the negative inotropic effect. It is uncertain to what extent decreases in Ca2+ sensitivity of the contractile proteins play a role in the negative inotropic effect of sevoflurane in intact cardiac muscle tissue. The aim of this study was to assess whether sevoflurane decreases myofibrillar Ca2+ sensitivity in intact living cardiac fibers and to quantify the relative importance of changes in myofibrillar Ca2+ sensitivity versus changes in myoplasmic Ca2+ availability by sevoflurane.
The effects of sevoflurane 0-4.05% vol/vol (0-1.5 minimum alveolar concentration [MAC]) on isometric and isotonic variables of contractility and on the intracellular calcium transient were assessed in isolated ferret right ventricular papillary muscles microinjected with the Ca2+-regulated photoprotein aequorin. The intracellular calcium transient was analyzed in the context of a multicompartment model of intracellular Ca2+ buffers in mammalian ventricular myocardium.
Sevoflurane decreased contractility, time to peak force, time to half isometric relaxation, and the [Ca2+]i transient in a reversible, concentration-dependent manner. Increasing [Ca2+]o in the presence of sevoflurane to produce peak force equal to control increased intracellular Ca2+ transient higher than control.
Sevoflurane decreases myoplasmic Ca2+ availability and myofibrillar Ca2+ sensitivity in equal proportions except at 4.05% vol/vol (1.5 MAC), where Ca2+ availability is decreased more. These changes are at the basis of the negative inotropic effect of sevoflurane in mammalian ventricular myocardium.
IT is well established that the volatile anesthetic sevoflurane in clinical useful concentrations depresses myocardial contractility in vitro because of a decrease in transsarcolemmal Ca2+influx. 1–6There is evidence that sevoflurane might also decrease Ca2+sensitivity of the contractile proteins, 7,8as was shown for halothane, enflurane, and isoflurane in skinned 9,10and intact muscle fibers. 11The purpose of this study was to test the hypothesis that sevoflurane decreases myofibrillar Ca2+sensitivity in intact living cardiac fibers and to quantify the relative importance of changes in myofibrillar Ca2+sensitivity versus changes in myoplasmic Ca2+availability in the overall concentration-dependent negative inotropic effect of sevoflurane.
Materials and Methods
This study was approved by the Animal Care and Use Committee of the Mayo Foundation, Rochester, Minnesota, with protocols completed in accordance with National Institutes of Health guidelines and in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council). Adult male ferrets (weighing 1,100–1,500 g and aged 16–19 weeks) were anesthetized with sodium pentobarbital (100 mg/kg intraperitoneally), and the heart was quickly removed through a left thoracotomy. During generous superfusion with oxygenated physiologic solution (see below), suitable right ventricular papillary muscles were carefully excised from the beating heart. Papillary muscles were then mounted vertically in a temperature-controlled (30°C) muscle chamber that contains a physiologic salt solution made up in ultrapure water (Nanopure Infinity; Barnstead, Dubuque, IA) and with the following composition: Na+137.5 mm, K+5.0 mm, Ca2+2.25 mm, Mg2+1.0 mm, Cl−127.0 mm, SO42−1.0 mm, acetate−20.0 mm, glucose 10.0 mm, 3-(N-morpholino)propanesulfonic acid 5.0 mm, pH 7.40, bubbled with 100% O2. Experiments were conducted at 30°C and at a stimulus frequency of 0.25 Hz; isolated papillary muscle function is stable for many hours in these conditions. Suitable preparations were selected on the basis of the following criteria: length at which twitch active force is maximal (Lmax) more than or equal to 3.5 mm, a mean cross-sectional area less than or equal to 1.2 mm2, and a ratio of resting to total force in an isometric twitch at Lmaxless than or equal to 0.30. The tendinous end of each muscle was tied with a thin, braided polyester thread (size 6.0 Deknatel Surgical Suture, Fall River, ME) to the lever of a force-length servo transducer. 11The ventricular end of each muscle was held in a miniature Lucite (Dupont, Wilmington, DE) clip with a built-in platinum punctate electrode; two platinum wires were arranged longitudinally, one along each side of the muscle, and served as anode during punctate stimulation. Rectangular pulses of 5-ms duration were delivered by a Grass S88D (Astro-Med, Inc., West Warwick, RI) stimulator at a stimulus interval of 4 s. Stimuli at 10–20% above threshold (range, 4–12 V) were used to minimize the release of endogenous norepinephrine by the driving stimuli. The muscles were stimulated and made to contract in alternating series of four isometric and four isotonic twitches at preload only during a 1–2-h period of stabilization.
At the end of the stabilization period, electrical stimulation was stopped, and multiple superficial cells were microinjected with the Ca2+-regulated photoprotein aequorin (Friday Harbor Laboratories, University of Washington, Friday Harbor, WA) to allow for subsequent detection of the intracellular Ca2+transient as previously described. 11It was usually necessary to average luminescence and force signals of 64–256 twitches to obtain a satisfactory signal-to-noise ratio in aequorin luminescence signals. For all muscles (n = 32) combined, the peak aequorin signal was 10.18 ± 9.99 (mean ± SD) times the root mean square of the baseline “noise” value (range, 59.32–2.83).
We quantified peak systolic aequorin luminescence and time to peak aequorin luminescence. The decline of the aequorin signal was quantified by measuring the time from the stimulus to the time when aequorin luminescence had decreased to 25% of its peak value (tL25), and the slope of the logarithm of aequorin luminescence from 50% peak to 10% peak (k50/10). The measurement of k50/10is based on the fact that the decline of the aequorin signals in isometric (but not isotonic 12) twitches follows an exponential decline, and that aequorin luminescence is approximately a 2.5 power function of [Ca2+]i.
The methods of delivery of sevoflurane were the same as for other volatile anesthetics. 13In brief, oxygen flowed through the calibrated sevoflurane vaporizer and was allowed to mix in a 3-l reservoir bag. An occlusive roller pump (Masterflex; Cole-Parmer, Chicago, IL) delivered a continuous gas flow to the bubbler in the organ bath. The muscle chamber was covered with a tightly sealing Parafilm (American Can Company, Greenwich, CT) except for a narrow slit for the muscle clip and transducer hook. The concentration of sevoflurane was measured continuously between the reservoir bag and the roller pump with an anesthetic agent monitor (Ohmeda 5330, Madison, WI).Gas chromatography (Hewlett-Packard 5880A, Palo Alto, CA) measurements showed that 1% (vol/vol) sevoflurane corresponded to 0.18 mm in fluid at 30°C. The concentration of sevoflurane in fluid and the calculated partial pressure of sevoflurane in fluid followed closely imposed changes of anesthetic vapor concentration in the gas phase. After the sevoflurane administration was discontinued, its concentration in liquid declined rapidly and was always undetectable at 20 min.
Sevoflurane minimum alveolar concentration (MAC) in the ferret was calculated to be 2.7% (vol/vol) from the following data. The MAC values for isoflurane and halothane in the ferret are 1.52% and 1.01% (vol/vol). 14The ratio of isoflurane MAC to halothane MAC in the ferret is 1.5, similar to that found in humans, dogs, and horses. 15Sevoflurane MAC values for humans, dogs, and horses are 2.05, 2.36, and 2.31, respectively. 16–18The halothane MAC values for humans, dogs, and horses are reported to be 0.75, 0.86 and 0.88, respectively. 15We calculated the MAC value for sevoflurane in the ferret assuming that the relative potency ratio of sevoflurane to halothane is close to that in humans (2.73), dogs (2.74), and horses (2.63) as well, which brings us to an estimated MAC of 2.7% (vol/vol) in the ferret used in this study.
All ferret papillary muscles were pretreated with bupranolol HCl 5 × 10−7m before the onset of the experiment to abolish any β-adrenergic effects. All experiments were conducted with the initial muscle length set at Lmax.
Two experimental protocols were used to examine the mechanism of the inotropic effect of sevoflurane. Each muscle served as its own control. Muscles contracted isometrically throughout the experiments.
In group 1 muscles (n = 8), sevoflurane was applied in concentrations of 0.7%, 1.35%, 2.7%, and 4.05% (vol/vol). These concentrations correspond to 0.25, 0.5, 1.0, and 1.5 MAC in the ferret (see above). As soon as contractility had reached a steady state (which was usually the case after 10–12 min of equilibration with a particular sevoflurane concentration), signals of force and aequorin luminescence were averaged on a digital storage oscilloscope (Nicolet 4094C, Madison, WI). Averaged signals were stored on 5.25-inch floppy disks and transferred to a desktop computer by software programs written in WFBASIC (Blue Feather Software, New Glarus, WI), which also measures all variables of contraction, relaxation, and aequorin luminescence. One to three records with 64 averaged twitches each were taken at control, at each sevoflurane concentration, and after sevoflurane washout, and were averaged to further improve signal-to-noise ratio of the aequorin luminescence signals, if necessary, before quantification of variables. Variables of contraction and relaxation were determined from isometric twitches at the preload of Lmax: peak developed force, time to peak force, and time from peak force to half-isometric relaxation.
In group 2 muscles (n = 24), we determined whether sevoflurane alters myofibrillar Ca2+sensitivity. After measurement of control variables of the isometric twitch, group 2a, 2b, and 2c muscles were exposed to 1.35% (0.5 MAC), 2.7% (1.0 MAC), or 4.05% (1.5 MAC) sevoflurane, respectively. When aequorin luminescence and peak isometric force had reached steady state, extracellular Ca2+was rapidly increased by adding small aliquots of a concentrated CaCl2solution (0.25 m) to the bathing solution, until the amplitude of peak developed force was equal to that in the control twitch. In three experiments, peak force slightly exceeded that of the control twitch after titration with CaCl2. In these instances, [Ca2+]owas decreased by the addition of 10–40 μl of EGTA 0.2 m, pH 7.0, to precisely match peak force to that in the control twitch. Free [Ca2+]oin the Ca2+back-titrated twitch was calculated with Fabiato’s program. 19Signals of force and aequorin luminescence were averaged and stored in the same way as in group 1. The protocol of Ca2+back-titration allowed us to compare aequorin luminescence signals in the absence (control) and presence of sevoflurane at equal peak developed force. If the magnitude of the intracellular Ca2+transient in the presence of sevoflurane (at equal peak force as in control) is different from that in the control twitch, there is likely to have been a change in myofibrillar Ca2+sensitivity.
To assess the relative effects of sevoflurane on intracellular Ca2+availability versus myofibrillar Ca2+sensitivity, the isometric Ca2+back-titration experiments were analyzed with the use of a multicompartment computer model 11comprising the following compartments: free [Ca2+]i, Ca2+bound to troponin C (TnC) and its force dependence, Ca2+bound to calmodulin, and sarcoplasmic reticulum (SR) Ca2+release and uptake. The model assumes that peak Ca2+occupancy of TnC is the same at equal developed force with or without sevoflurane. For the sake of simplicity, all sources of myoplasmic Ca2+delivery (transsarcolemmal Ca2+current, Na+–Ca2+exchange, and SR Ca2+release) were lumped into one myoplasmic Ca2+delivery term (SR release) to gain one value, which is referred to as myoplasmic Ca2+availability. Myoplasmic Ca2+availability is expressed as percent of the value of the nonanesthetic control. Changes in myofibrillar Ca2+sensitivity were expressed as changes in off-rate of Ca2+from TnC at the Ca2+-specific site II (koff(TnC.Ca); the on-rate is very fast and limited only by diffusion), 20with the understanding that various mechanisms can generate changes in Ca2+sensitivity (see Discussion). A detailed description of the analysis has been described elsewhere. 11Myofibrillar Ca2+sensitivity was defined as 1/koff(TnC.Ca)and was expressed as percent of the value in the nonanesthetic control.
Muscle characteristics between muscle groups were compared with one-way analysis of variance. Concentration–response relations between sevoflurane concentration and variables of contractility and aequorin luminescence were tested for differences with repeated-measures analysis of variance; pairwise comparisons versus control were conducted with Bonferroni-corrected paired t tests. In Ca2+back-titration experiments, aequorin luminescence in sevoflurane and high [Ca2+]owere compared with control by the Student paired t test. Because absolute values of aequorin luminescence varied from muscle to muscle, percentage values of aequorin luminescence and relative changes are reported in which each muscle serves as its own control. 11Relative changes in Ca2+sensitivity and availability derived at different sevoflurane concentrations were compared using one-way analysis of variance followed by pairwise comparison versus control with Bonferroni-corrected t test. Differences between relative values of Ca2+sensitivity and availability in a particular sevoflurane concentration were compared with the Student paired t test. Values were reported as mean ± SD. Differences were considered significant at the P less than 0.05 level.
Effects of Sevoflurane on Contractility and Intracellular Ca2+Transient
Group 1 muscle characteristics in control conditions at Lmaxare shown in table 1. Figure 1illustrates a representative example of a concentration–response experiment to incremental concentrations of sevoflurane on aequorin luminescence and force in isometric twitches. Table 2and figure 2summarize the values of variables of contractility and of aequorin luminescence during control and cumulative concentration–response experiments to sevoflurane. Sevoflurane caused a concentration-dependent decrease of force and aequorin luminescence over the concentration range studied (figs. 1 and 2, left). Sevoflurane shortened the duration of the isometric twitch (time to peak force) and isometric relaxation half time in a concentration-dependent manner (fig. 2, right). Sevoflurane did not change time to peak light (fig. 2, right) and did not affect the decline of aequorin luminescence measured by tL25and the slope k50/10of the logarithm of aequorin luminescence during decline from 50% to 10% of peak.
Effects of Sevoflurane on Myofibrillar Ca2+Sensitivity
There were no statistically significant differences for Lmax, mean cross-sectional area, resting force or preload at Lmax, total force, and the ratio of resting to total force among the three muscle groups 2a, 2b, and 2c used in this experimental protocol (table 1). A typical Ca2+back-titration experiment is shown in figure 3. Aequorin luminescence and isometric force were measured in control (fig. 3, left) and during exposure to sevoflurane 2.7% (1 MAC;fig. 3, middle), both in [Ca2+]o2.25 mm. Extracellular [Ca2+] was then rapidly increased with small aliquots of a concentrated CaCl2solution (0.25 m) until peak developed force in sevoflurane and high [Ca2+]owas equal to that in the control twitch. At equal peak developed force, aequorin luminescence was higher in the presence of sevoflurane and elevated [Ca2+]o(fig. 3, right) than in its absence. The resulting [Ca2+]ovalues at the end of the procedure were 3.00 ± 0.09, 3.70 ± 0.29, and 4.59 ± 0.42 mm in 0.5, 1.0, and 1.5 MAC sevoflurane, respectively. Figure 4summarizes the results of the Ca2+back-titration experiments in each of three sevoflurane concentrations.
Figure 5shows a typical Ca2+back-titration experiment (fig. 5, top) and part of the analysis procedure (fig. 5, middle and bottom). First, aequorin luminescence signals that would match the amplitude and time course of experimental gained aequorin luminescence in sevoflurane in high [Ca2+]o(fig. 5, top right) were simulated by varying SR release and koff(TnC.Ca)in small steps. Ca2+availability and Ca2+sensitivity of the Ca2+back-titrated twitch were derived by the SR release and koff(TnC.Ca)values of the simulated aequorin luminescence signals that produced (1) the best fit to the measured Ca2+transient in high [Ca2+]oplus sevoflurane by least squares differences, and (2) equal peak Ca2+occupancies of TnC in control and in sevoflurane plus high [Ca2+]oat equal peak force (fig. 5, bottom). This procedure yielded a value of myofibrillar Ca2+sensitivity in sevoflurane. The effect of sevoflurane in control [Ca2+]oon myoplasmic delivery was found by searching the least squares fit to the experimentally measured aequorin luminescence signal (fig. 5, top, middle) by varying SR release only and using the koff(TnC.Ca)value found earlier for the aequorin luminescence signal in the back-titrated twitch.
Figure 6summarizes the analyses for all Ca2+back-titration experiments and shows the relative effects of sevoflurane on myoplasmic Ca2+availability and on myofibrillar Ca2+sensitivity. The results of the analysis are as follows: (1) sevoflurane decreases myoplasmic Ca2+availability in a concentration-dependent manner; (2) sevoflurane decreases myofibrillar sensitivity; (3) the decrease of myofibrillar Ca2+sensitivity is already fully present at 2.7% (vol/vol; 1 MAC) sevoflurane; and (4) sevoflurane decreases myoplasmic Ca2+availability and myofibrillar Ca2+sensitivity to the same relative extent, except at 4.05% (vol/vol; 1.5 MAC), where sevoflurane decreases Ca2+availability more than Ca2+sensitivity.
Studies in isolated heart, intact cardiac muscle tissue preparations, and single ventricular myocytes demonstrated a concentration-dependent decrease of indices of contractility by sevoflurane in various species such as rat, dog, guinea pig, and humans. 1–6,21–24As for other volatile anesthetics, the negative inotropy of sevoflurane might be associated with (1) effects on transsarcolemmal Ca2+flux; (2) alteration of SR function; (3) a decrease of free intracellular [Ca2+] level during systole; and (4) modification of the responsiveness of the contractile proteins to activation by Ca2+. The current study is the first to show the effects of sevoflurane on the intracellular Ca2+transient and to assess whether sevoflurane alters myofibrillar Ca2+sensitivity in intact ventricular myocardium. Measurements of free intracellular Ca2+, using the bioluminescent protein aequorin, and of contractile force show a concomitant concentration-dependent decrease in these variables, indicating that the negative inotropy of sevoflurane is related to a decreased intracellular Ca2+availability.
Indeed, the negative inotropic effect of sevoflurane has been mainly attributed to depression of transsarcolemmal Ca2+influx, 1–5whereas the effects of sevoflurane on the SR seem to be modest. 1,2,22Sevoflurane interacts with the L-type Ca2+channel in cultured neonatal rat ventricular myocytes since the L-type Ca2+channel agonist Bay K 8644 significantly prevented the sevoflurane-depressed contractile amplitude. 5The sevoflurane-induced depression of myocardial contractility was accompanied by a shortening of duration of action potential in canine ventricular muscle strips and results from significant blockade of transsarcolemmal Ca2+current. 3,4In guinea pig papillary muscle, sevoflurane decreased contractile force and duration of action potential in a concentration-dependent fashion. 2Sevoflurane depressed contractile force at rested state and at low stimulation frequencies, whereas it did not suppress potentiated state contractions and also depressed contractile force after ryanodine. 2A comprehensive study on the mechanical and electrophysiologic effects of sevoflurane was conducted by Park et al. 1In guinea pig myocardium, sevoflurane decreased maximum rate of force development at low stimulation rates but not at high stimulation rates. Sevoflurane decreased the initial rate of force development less than the rate of “late” force development, and selectively decreased late peak force in high K+Tyrode solution without changing early peak force. Taken together, these observations led to the conclusion that sevoflurane has almost no effect on SR Ca2+release. Sevoflurane increased duration of action potential, decreased peak L-type Ca2+current, and suppressed the delayed K+current, which appears to underlie the increased duration of action potential. 1
A decrease in myofibrillar Ca2+sensitivity by sevoflurane may contribute to the overall negative inotropic effect of sevoflurane in cardiac ventricular muscle. Ca2+sensitivity is defined as the contractile response (usually measured as force) of the myofibrils to a given myoplasmic Ca2+concentration. Changes in any of the following processes can lead to a change of myofibrillar Ca2+sensitivity: (1) the affinity of TnC for Ca2+(Ca2+-specific binding site II); (2) interactions of TnC with TnI and TnT; (3) the state of phosphorylation of TnI that changes the Ca2+affinity for TnC 25; (4) signal transduction via tropomyosin to actin; (5) the interaction of actin with myosin heads via the formation of cross-bridges; (6) regulation of contraction via myosin light chains 26; and/or (7) possibly other mechanisms. The affinity of TnC for Ca2+is determined by the off-rate of TnC’s Ca2+-specific binding site II, as the on-rate is very fast and only limited by diffusion. 20Halothane did not change 27or slightly increased the Ca2+affinity of isolated cardiac TnC in vitro 28and decreased koff(TnC.Ca)in human recombinant cardiac TnC. 29These effects cannot account for the decrease in myofibrillar Ca2+sensitivity by halothane. The effects of sevoflurane on Ca2+binding to TnC have not yet been investigated.
In rat skinned cardiac fibers, sevoflurane decreased the rate of force redevelopment after a release-stretch cycle (ktr). 7When interpreted in a two-state cross-bridge model, this finding suggests a decrease in the cross-bridge apparent attachment rate (fapp) with no changes in the cross-bridge detachment rate (gapp). This would keep fewer cross-bridges in the force-generating state, so that less force is generated, even if the Ca2+transient were not changed. This would manifest itself as a decreased myofibrillar Ca2+sensitivity in intact fibers as observed in this study.
In intact cardiac muscle, there is indirect evidence for a decrease in myofibrillar Ca2+sensitivity by sevoflurane. 8The negative inotropic effect of sevoflurane was more pronounced in isometric conditions, where the native myofibrillar Ca2+sensitivity is high, than in unloaded contractions, where the native myofibrillar Ca2+sensitivity is low. 8Sevoflurane abbreviated both time to peak force and isometric relaxation half-time. The acceleration of isometric relaxation was not a consequence of the concomitant decrease in peak force, as isometric relaxation half-time was unchanged in control conditions over the range of extracellular Ca2+concentrations of 0.45–2.25 mm. 8Isometric relaxation in cardiac muscle is controlled by the contractile proteins themselves, whereas cell relengthening rates in isolated myocytes (similar to isotonic lengthening) are limited by the rate of decrease of the [Ca2+]itransient. 30,31The acceleration of isometric relaxation by sevoflurane in intact cardiac muscle might result from a decrease in Ca2+sensitivity. This is in contrast to findings by Hanouz et al., 22who reported that sevoflurane and isoflurane did not induce a lusitropic effect under high load and concluded that these anesthetics might not modify myofilament Ca2+sensitivity. The differences may result from species differences (rat vs. ferret).
To assess whether sevoflurane alters myofibrillar Ca2+sensitivity in intact fibers, peak aequorin luminescence was compared with and without sevoflurane at equal peak force in Ca2+back-titration experiments. The higher Ca2+transient in sevoflurane and increased extracellular [Ca2+] than in control indicates a decreased Ca2+sensitivity. To quantify the relative changes in myofibrillar Ca2+sensitivity versus changes in myoplasmic Ca2+availability, we analyzed the Ca2+transients in the context of a multicompartment computer model that considered SR Ca2+release, Ca2+uptake, and binding to the principal intracellular Ca2+buffers, TnC and calmodulin. 11Published values for rate constants of association and dissociation of Ca2+to the intracellular buffers TnC and calmodulin and a Michaelis-Menten kinetic scheme for SR Ca2+uptake were used. The values of Vmax(maximal rate of Ca2+uptake by the SR) and of Km([Ca2+]oat which Ca2+uptake rate equals Vmax/2) were not allowed to vary. 11However, sevoflurane might slightly increase the rate of Ca2+removal from the cytoplasm, as reflected by a faster and earlier isotonic relaxation when compared with amplitude-matched twitches in low [Ca2+]o. 8This would cause a faster decline of the Ca2+transient. If this were the case, the effect would be very small, a 6-ms shorter signal (measured at the midpoint of the decline) for a 20% increase in SR Ca2+uptake rate (Vmax) in experimental conditions of this study. Measurements of tL25and k50/10were not affected by sevoflurane, yet these variables may not be able to resolve small changes in the decline of the Ca2+transient.
Sevoflurane up to a concentration of 2.7% (vol/vol; 1 MAC) changes Ca2+sensitivity and Ca2+availability to the relative same extent. At 4.05% (vol/vol; 1.5 MAC), Ca2+availability was significantly more decreased than Ca2+sensitivity. This pattern of relation between decrease in Ca2+sensitivity and Ca2+availability is similar to that observed for isoflurane. 11By contrast, halothane decreased myofibrillar Ca2+sensitivity less than Ca2+availability over the entire concentration range (0–1.5 MAC). 11Sevoflurane decreased myoplasmic Ca2+availability to the same extent as isoflurane yet less than halothane (P < 0.01, one-way analysis of variance and Bonferroni-corrected t tests). Yet the decrease in myofibrillar Ca2+sensitivity was not concentration-dependent. Anesthetic-induced decreases in myofibrillar Ca2+sensitivity were already maximal at 0.5 MAC halothane and 1 MAC isoflurane and sevoflurane. This suggests a saturable process, the nature of which remains to be defined. In skinned rat cardiac fibers, 2 MAC sevoflurane decreased ktrand fappmore than 1 MAC sevoflurane, observations that suggest a concentration-dependent effect. 7Yet, because of the loss of certain natively present constituents and of membrane regulation of contractility, results obtained in skinned muscle fiber preparations are difficult to extrapolate to intact myocardium.
Halothane, isoflurane, 11and sevoflurane (current study) at equipotent concentrations decreased Ca2+sensitivity in intact muscle to the relative same extent (one-way analysis of variance, P > 0.1). This is consistent with observations that there were no differences between effects of volatile anesthetics on pCa–force relations and maximal activated force in skinned rat cardiac fibers 9and in human skinned cardiac fibers. 10However, sevoflurane and halothane exerted differential effects on cross-bridge cycling parameters. Sevoflurane did not decrease the fraction of attached cross-bridges (αfs) and gapp, whereas halothane did. Sevoflurane 1 MAC decreased ktrand fappless than 1 MAC halothane, and 2 MAC sevoflurane decreased ktrand fappmore than 2 MAC halothane. 7Studies of cross-bridge kinetics have shown differences between anesthetics that were not resolved from examination of pCa–force relations of skinned fibers and from analysis of myofibrillar Ca2+sensitivity in intact fibers. Therefore, one might conclude that various sites of contractile proteins are affected by volatile anesthetics.
The results of this study must be interpreted in the context of the experimental conditions in which they were obtained. Results obtained here at 30°C and a stimulus interval of 4 s may differ from those that one could obtain at the more physiologic conditions of the animal, 37–38°C and 200 beats/min.
In summary, in intact ferret papillary muscle, the negative inotropic effect of sevoflurane is caused by a decrease of myoplasmic Ca2+availability and of myofibrillar Ca2+sensitivity in equal proportions, except at 1.5 MAC, where myoplasmic Ca2+availability decreases more. These changes are at the basis of the negative inotropic effect of sevoflurane in mammalian ventricular myocardium.
The authors thank Laurel Wanek, B.A., Mayo Foundation, Rochester, Minnesota, for outstanding support in this project.