INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE VOLATILE ANESTHETICS halothane, isoflurane, and sevoflurane depress myocardial contractility in vitro at clinically useful concentrations (3, 4, 9, 18, 23, 30). The negative inotropic effect results mainly from a decrease in the availability of myoplasmic Ca²⁺ for the activation of contraction (3, 4, 9, 18, 23), caused by a depressant effect on sarcolemmal L-type Ca²⁺ channels (5, 20, 25) and on sarcoplasmic reticulum function (9). Furthermore, these volatile anesthetics decrease the responsiveness of the contractile apparatus to Ca²⁺ (3, 4, 9, 18, 23). In addition, results, mostly from skinned fiber studies, suggest that volatile anesthetics directly inhibit cross-bridge function at a level "downstream" from Ca²⁺ binding to thin-filament regulatory proteins (19, 27, 28, 31, 33). In the present study, we have focused on direct effects of anesthetics on cross-bridge function in intact cardiac muscle by assessing dynamic stiffness during shortening and lengthening of isotonic twitch contractions. Instantaneous changes in dynamic stiffness are believed to arise from an elastic component associated with each individual attached cross bridge, hence dynamic stiffness is supposed to indicate the degree of cross-bridge attachment (13, 14). Compliance (= 1/stiffness) was measured by applying variable-frequency sinusoidal load oscillations maintained at the self-resonance frequency of the muscle-lever system to provide stiffness values throughout shortening and lengthening of isotonic twitch contractions of intact cardiac muscle. Dynamic stiffness was derived by relating total stiffness to values of passive stiffness at each muscle length during shortening and lengthening. The effects of halothane, isoflurane, and sevoflurane on isotonic contractility and dynamic stiffness were investigated in random order and underwent an intragroup comparison.

This study reports 1) a method to measure stiffness during shortening and lengthening of isotonically contracting cardiac muscle; 2) direct effects of halothane and isoflurane on cross bridges in intact cardiac muscle; and 3) the incidental finding that halothane and isoflurane increase resting stiffness of intact cardiac muscle.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study was approved by the Animal Care and Use Committee of the Mayo Foundation, with protocols completed in accordance with the 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; 16-19 wk of age) were anesthetized with pentobarbital sodium (100 mg/kg ip), and the heart was quickly removed through a left thoracotomy. During generous superfusion with oxygenated physiological 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 muscle chamber that contains a physiological salt solution made up in ultrapure water (Nanopure Infinity, Barnstead, Dubuque, IA) and with the following composition (in mM): 137.5 Na⁺, 5.0 K⁺, 2.25 Ca²⁺, 1.0 Mg²⁺, 127.0 Cl, 1.0 SO, 20.0 acetate¹, 10.0 glucose, and 5.0 MOPS, pH 7.40, bubbled with 100% O₂.

Suitable preparations were selected on the basis of the following criteria: length at which twitch active force is maximal (L_max)  3.5 mm, a mean cross-sectional area  1.2 mm², and a ratio of resting to total force in an isometric twitch at L_max  0.30.

The tendinous end of each muscle was tied with a thin braided polyester thread (size 9.0, Deknatel Surgical Suture, Fall River, ME) to the end of a stiff glass rod (7.5 cm, 40 mg), which was attached to the lever of a force-length servotransducer. The attachment was sealed with paraffin. This transducer system (Innovi) allows one simultaneously to 1) measure shortening up to 3 mm (resolution, 0.25 µm), 2) impose loads up to 299 mN, 3) measure force by feedback sensing (resolution, <0.1 mN), and 4) impose abrupt changes of load (load clamp) or of initial length (length clamps: quick release and quick stretch). The equivalent moving mass was 250 mg, and the static compliance was 0.28 µm/mN.

The ventricular end of each muscle was held in a miniature Lucite clip (Dupont, Wilmington, DE) with a built-in platinum punctate electrode. Two platinum wires were arranged longitudinally, one along each side of the muscle, and serve as anode during punctate stimulation. A Grass S88D stimulator (Astro-Med, West Warwick, RI) delivered rectangular pulses of 5-ms duration. Stimuli at 10-20% above threshold (range, 5-12 V) were used to minimize the release of endogenous norepinephrine by the driving stimuli.

Throughout the stabilization period, the muscles were stimulated and made to contract in alternating series of four isometric and four isotonic twitches for 1-1.5 h at 0.25 Hz at the temperature of the bathing solution of 30°C. The solution was changed at least once during this stabilization period. When muscles had reached steady state, initial muscle length was set at L_max. Thereafter, the temperature was set to 25°C. The muscles were then allowed to stabilize for another 60 min, first in the same manner as above and then in isotonic twitches only. Muscles contracted isotonically at the preload of L_max throughout the experiment except for the test contractions (see below). To minimize effects of release of endogenous catecholamines in ferret cardiac muscle, -adrenoceptor blockade was achieved by the administration of (±)-bupranolol hydrochloride (10⁷ M).

On-line measurement of compliance in isotonic conditions. The isotonically contracting muscles (Fig. 1, top) were subjected to variable-frequency sinusoidal load oscillations (37-182 Hz, 0.195-0.56 mN peak to peak) and were maintained at the self-resonant frequency of the muscle-lever system by means of an automatic gain-control frequency modulation feedback circuit. Compliance and equivalent moving mass of the muscle-lever system constitute a mechanical tuned circuit. The muscle-lever system was made self-resonant, whereby velocity and force oscillations were held in phase throughout contraction and relaxation. An electronic feedback loop provided the necessary positive and negative feedback to meet the basic conditions for sustained oscillation as the instantaneous resonant frequency (f = /2, where  is angular frequency) varies during muscle activity. The lever displacement signal was differentiated to yield a velocity signal that was fed back to the force-generation circuit. An automatic gain-control circuit stabilized the amplitude of the velocity oscillations at a constant peak-to-peak level so as to result in length (L) oscillation amplitudes of 0.0012-0.0065 L/L_max, which are small enough not to actually disrupt cross-bridge binding. A digital circuit computed 1/² after each period. Instantaneous compliance was computed from the angular frequency  and equivalent moving mass (m) as C_m = 1/(m · ²) (Fig. 1, middle). The elastic component of stiffness was derived as 1/C_m (Fig. 1, bottom). The viscous component of stiffness is the ratio of the peak amplitudes of force and velocity oscillations, but its study was not pursued here.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Isotonic shortening with superimposed sinusoidal oscillations (top), compliance (middle), and stiffness (bottom) as a function of time during shortening and lengthening of an isotonic twitch contraction, with superimposed sinusoidal load oscillations, in a representative muscle, in control conditions, at the beginning of the experiment at preload 3 mN. Vertical arrow (top) indicates the time of the electric stimulus. During rapid isotonic relaxation, compliance signals were unreliable as the frequency of lengthening and of oscillations overlapped (dotted lines). Muscle characteristics are as follows: length (L) at maximal twitch force (Lmax), 5.71 mm; mean cross-sectional area, 0.67 mm2; preload at Lmax, 7 mN.

Calculation of dynamic stiffness, passive stiffness, and active tension during shortening. Muscles were made to contract isotonically at different preloads from twice the preload of L_max (e.g., 16, 14, 12 mN) to 2 mN in 1-mN steps (first series of experiments) or in 2-, 3-, or 4-mN steps (second series of experiments). Passive stiffness (PS), the stiffness before the onset of the twitch (Fig. 2B), was plotted as a function of the corresponding resting length (RL) (Fig. 2A). The passive stiffness-resting length relation was exponential (Fig. 2C) and gave an excellent fit to the equation

(1)

Figure 2C shows an example of the exponential relationship between resting length and passive stiffness (see Eq. 1); units are given in volts from the original data. This particular relationship (Eq. 1) reflects the fact that, in our muscle transducer, the length signal in volts increases as muscle length becomes shorter. In other experimental setups, the relation could be different. The resting length-passive stiffness plot was performed in each muscle, in control conditions, in each anesthetic concentration, in anesthetic washout, and in Ca²⁺ back-titration experiments. Total stiffness during contraction is the sum of passive stiffness, caused by passive elements of the muscle, and dynamic stiffness, generated by attached cross bridges (see DISCUSSION). Dynamic stiffness during muscle shortening increases because of cross-bridge attachment as passive stiffness decreases because of unloading of tension in passive elements.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Total (TS) and dynamic stiffness (DS) during isotonic twitches at different preloads. A: muscle shortening (horizontal deflection) as a function of time (vertical axis) at 12- to 2-mN preloads in 1-mN steps. B: corresponding traces of TS obtained from analysis of sinusoidal load oscillations. Eight twitches were averaged at each preload. C: plot of corresponding passive stiffness (PS) vs. resting length (RL) values for 12- to 2-mN preloads in 1-mN steps () and the single 3-parameter exponential decay regression line PS = y0 + a * eb*RL. Amplitude values are in volts as the original data were used for nonlinear regressions. D: time course of DS during isotonic shortening as derived from subtracting PS (y0 + a * eb*RL) from TS throughout shortening and lengthening. E: time course of isotonic shortening as shown in A. Vertical arrow indicates the time of the electric stimulus. B, D, and E are on the same time scale. Signals of 8 twitches were averaged. Muscle characteristics are as follows: Lmax, 4.6 mm; mean cross-sectional area, 0.41 mm2; preload at Lmax, 6 mN.

(2)

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Top left: measured TS (8 twitches averaged), calculated PS, and DS, according to equations PS = y0 + a * eb*RL and DS = TS  PS, are superimposed as a function of time. Middle left: calculated passive (PT) and active tension (AT) according to equations PT = c + d * PS and AT = TT  PT, where TT is total tension, are superimposed as a function of time. Bottom left: isotonic contraction at Lmax (8 twitches averaged) during which TS was measured. Vertical arrow indicates time of electric stimulus. Right: exploded view of the boxes on the left for DS (top), AT (middle), and isotonic shortening around the time of peak shortening (bottom). Muscle characteristics are as follows: Lmax, 4.6 mm; mean cross-sectional area, 0.41 mm2; preload at Lmax, 6 mN.

(3)

(4)

Methods of delivery of volatile anesthetics. The methods of delivery of anesthetics were the same as previously described (2, 22). In brief, oxygen flowed through the calibrated vaporizer for halothane, isoflurane, or sevoflurane and was allowed to mix with the respective anesthetic in a 3-liter 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, Greenwich, CT), except for a narrow slit for the muscle clip and the glass rod. The concentration of the anesthetic was measured continuously between the reservoir bag and the roller pump with an anesthetic agent monitor (Ohmeda 5330, Madison, WI). Gas chromatography (model 5880A, Hewlett-Packard, Palo Alto, CA) measurements showed that the concentrations of halothane, isoflurane, and sevoflurane and their calculated partial pressure in fluid followed closely imposed changes of anesthetic vapor concentration in the gas phase. After the administration of anesthetic was discontinued, anesthetic concentration was always undetectable after a few minutes.

Experimental design. Two experimental protocols were used to examine the effects of anesthetics on stiffness in cardiac muscle in which each muscle served as its own control. Anesthetics were studied in random order. Table 1 summarizes the characteristics of the muscles used in each of the two experimental series (n = 7 muscles per series). Muscles twitched isotonically at the preload of L_max, except during the test contractions at different preloads throughout the experiments. In the first series of experiments, muscles (n = 7) were exposed to halothane, isoflurane, and sevoflurane in random order and in a concentration of 0.0, 0.5, 1.0, and 1.5 minimum alveolar concentration (MAC) each. MAC is an anesthetic half-maximal effective dose as defined by Eger et al. (10). One MAC is the concentration of anesthetic at which 50% of the animals respond to a standardized supramaximal stimulus with "gross purposeful muscular movements of body or extremities" (10). It is a measure of anesthetic potency. One-MAC halothane, isoflurane, and sevoflurane in the ferret corresponds to 1% (vol/vol), 1.5% (vol/vol), and 2.7% (vol/vol) of the respective anesthetic (2, 26). Effects of each anesthetic on isotonic contraction at the preload of L_max were monitored until a steady state was reached, and this was usually the case after 10-15 min of equilibration in each concentration. The muscles were then subjected to continuous load oscillations at the instantaneous and varying self-resonance frequency, and test contractions at different preloads were recorded. Subsequently, the concentration of the anesthetic was increased. Two records were taken at every preload: one with a single waveform and one with eight waveforms averaged. The latter was used for quantification of variables of interest. The procedures required for recording test contractions for analysis and stiffness lasted ~10 min, such that total exposure to a particular anesthetic concentration was ~25 min. After the highest concentration of anesthetic, the anesthetic was turned off, the reservoir bag was emptied, and the gas-delivering system was flushed with oxygen. The muscle was allowed to recover for 1 h before new control measurements were taken, and the administration of the next anesthetic was commenced.

Statistics. In the first series of experiments, measurements during anesthetic exposure were compared with those obtained in the immediately preceding control period, or between anesthetics of the same clinically effective concentration, by means of repeated-measures analysis of variance, followed by Bonferroni corrected paired t-test for comparisons vs. control or for pairwise comparisons. When data were not normally distributed, we used Friedman tests followed by Dunnett's test for comparisons vs. control or Student-Newman-Keuls for pairwise comparisons. In the second series of experiments, Student's paired t-tests were performed to assess differences for a particular variable in control conditions and in 1-MAC anesthetic and elevated [Ca²⁺]_o. Differences were considered significant at the P < 0.05 level. Data are reported as means ± SD, except in Fig. 6, in which SE is plotted for reasons of clarity.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Muscle length and passive stiffness during resting conditions. In the first series of experiments, the effects of halothane, isoflurane, and sevoflurane were examined in random order in seven muscles in concentrations of 0.0, 0.5, 1.0, and 1.5 MAC and after a 30-min anesthetic washout. Resting length and passive stiffness increased over time during the experiment. Changes in resting length and passive stiffness in a typical muscle experiment are displayed in Fig. 4, and a summary of data is presented in Table 2. Volatile anesthetics increased resting length and stiffness. After a 30-min anesthetic washout, resting length and stiffness recovered partially with even more recovery after a 60-min anesthetic washout (control measurement before the next anesthetic). The tendency in recovery in resting length and passive stiffness during 30-min anesthetic washout seems obvious in all anesthetics but reached significance only in halothane and isoflurane for passive stiffness values. Increasing [Ca²⁺] in the bathing solution did not abolish the anesthetic's effects on resting length and passive stiffness (not shown). Halothane increased the rate constant b of the exponential passive stiffness-resting length relation, the effect of which is to decrease passive stiffness for a given resting length. The rate constant b was normalized for L_max for statistical comparison and was significantly different between control and 1.5-MAC halothane (P = 0.049). The correlation coefficients (r) of the exponential passive stiffness-resting length relation were 0.9996 ± 0.0003 (mean ± SD; n = 168; range 0.9986-0.9999). Resting stiffness was linearly related to passive tension with excellent correlation (r = 0.9953 ± 0.0035; mean ± SD; n = 168; range 0.9873-0.9998) for the linear passive tension-passive stiffness relation.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   RL () and PS () in a typical muscle during the time course of an experiment when muscle was exposed consecutively to 0.5-, 1.0-, and 1.5-minimum alveolar concentration (MAC) sevoflurane (sev), isoflurane (iso), and halothane (hal) with extended periods of recovery between anesthetics. RL and PS increased, recovered after 30-min, and recovered more so after 60-min anesthetic washout when control measurements before onset of the next anesthetic were recorded. Muscle characteristics are as follows: Lmax, 4.29 mm; mean cross-sectional area, 0.54 mm2; preload at Lmax, 8 mN.

Dynamic stiffness, passive stiffness, active tension, and passive tension during isotonic shortening. Figure 2, D and E, shows that, as preload decreased, the amplitude of shortening increased, dynamic stiffness decreased, and time to peak shortening and time to late stiffness maximum decreased. Dynamic stiffness increased rapidly at the onset and during the first one-third of shortening. There was a dip in dynamic stiffness at peak shortening, followed by a secondary rise in early relaxation, and finally a fast decrease in stiffness (Fig. 2D, Fig. 3, top, and Fig. 5B, middle). This pattern of stiffness (dip and rise) was obvious in control conditions in all seven muscles of the first series of experiments but only in five of seven muscles in the second series of experiments (Ca²⁺ back-titration). The increase in dynamic stiffness after peak shortening occurred after the muscle had lengthened by 0.96 ± 0.04% of L_max (mean ± SD, n = 7), with no effect of preload on this value (linear regression slopes not significantly different from zero). Halothane and isoflurane 1.0 and 1.5 MAC decreased the amount of lengthening at late stiffness peak. There was no systematic relation between stiffness and velocity of shortening (not shown). Stiffness values were not valid during maximal velocity of lengthening because compliance signals were not reliable as the frequency of lengthening and oscillations overlapped.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Isotonic shortening (A) and DS (B) as a function of time in control conditions and during exposure to 1-MAC sevoflurane, isoflurane, and halothane in a representative muscle at 10-mN preload. DS shows an early stiffness maximum, which occurs before peak shortening and a late stiffness maximum (LSM), which occurs after peak shortening. One-MAC sevoflurane, isoflurane, and halothane decreased isotonic shortening, DS, time to peak shortening, and time to LSM. Vertical arrow in A indicates the time of the electric stimulus. C: DS is plotted against isotonic shortening. During early shortening, isoflurane and sevoflurane had no effect on stiffness-shortening relationship. Anesthetics decreased stiffness during late and peak shortening and increased stiffness during relaxation at any given muscle length (vertical dashed line in middle panel). Eight twitches were averaged in each panel. Muscle characteristics are as follows: Lmax, 4.29 mm; mean cross-sectional area, 0.64 mm2; preload at Lmax, 8 mN.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effects of halothane, isoflurane, and sevoflurane on peak isotonic shortening (DL) (A), DS at DL (SDL) (B), LSM (C), and time to LSM at muscle length set to Lmax (i.e., muscle length at which developed force is maximal) (D). * Significant difference by repeated-measures analysis of variance, comparisons vs. corresponding control by Bonferroni-corrected paired t-test or Friedman test, and comparisons vs. control by Dunnett's test (for effects of halothane on SDL and effects of sevoflurane on time to LSM), P < 0.05. Significant difference by repeated-measures analysis of variance and pairwise comparisons between equipotent concentrations of anesthetics by Bonferroni-corrected paired t-test or Friedman test and pairwise comparisons between anesthetics with Student-Newman-Keuls test (for SDL and LSM in 1.5 MAC): # halothane vs. sevoflurane, $ isoflurane vs. sevoflurane, & halothane vs. isoflurane, P < 0.05. Values are means ± SE and are from the same muscles.

Ca²+ back-titration experiment. To determine whether anesthetics change dynamic stiffness by a mechanism different from that exerted by anesthetic-induced changes in [Ca²⁺]_i, Ca²⁺ back-titration experiments were performed. Shortening and dynamic stiffness were measured in control and during exposure to 1-MAC isoflurane, both in 2.25 mM [Ca²⁺]_o. [Ca²⁺]_o was then rapidly increased until peak shortening in isoflurane was the same as in the control twitch. Figure 7 shows the control and back-titrated twitch. Dynamic stiffness was higher in isoflurane than in control for the same extent of shortening. Table 3 summarizes results of the Ca²⁺ back-titration experiments to 1-MAC halothane, isoflurane, and sevoflurane. In one muscle exposed to 1-MAC halothane, shortening amplitude could not be raised to control values by increasing [Ca²⁺]_o. This muscle was excluded from further analysis. In 1-MAC halothane and high [Ca²⁺]_o, dynamic stiffness at peak shortening was higher than dynamic stiffness in a twitch with the same peak shortening amplitude in control conditions without halothane. In 1-MAC isoflurane and high [Ca²⁺]_o, the late stiffness peak was higher than in control twitches. Sevoflurane, isoflurane, and halothane decreased time to peak shortening from control values. Increasing [Ca²⁺]_o in halothane increased time to peak shortening, whereas raising [Ca²⁺]_o in isoflurane or sevoflurane further decreased time to peak shortening.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Shortening and DS of isotonic twitches during a typical Ca2+ back-titration experiment for 1-MAC isoflurane. Traces of shortening (top) and of DS (middle) as a function of time in control conditions and in 1-MAC isoflurane and elevated extracellular Ca2+ concentration ([Ca2+]o) (4.54 mM) are superimposed. Bottom: DS plotted vs. shortening in control conditions and in 1-MAC isoflurane and elevated [Ca2+]o. At equal peak shortening, DS peak during early relaxation was higher in the presence of isoflurane and elevated [Ca2+]o. Vertical arrow under the length trace indicates the time of electric stimulus. Eight twitches were averaged. Muscle characteristics are as follows: Lmax, 4.29 mm; mean cross-sectional area, 0.47 mm2; preload at Lmax, 8 mN.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We used small-amplitude, frequency-modulated sinusoidal load oscillations to derive dynamic stiffness of intact cardiac muscle throughout isotonic twitch contractions. We examined the effects of halothane, isoflurane, and sevoflurane on dynamic stiffness during isotonic contraction to determine their effects on cross-bridge function in intact cardiac muscle. A change in cross-bridge function was evident in 1-MAC halothane and 1-MAC isoflurane. Passive stiffness increased during exposure to halothane and isoflurane.

In skinned rat cardiac fibers, volatile anesthetics impaired cross-bridge performance directly. Halothane, isoflurane, and enflurane decreased maximal calcium-activated force, when [Ca²⁺] was sufficiently high to saturate Ca²⁺ binding sites of troponin C (TnC) (28, 33). Yet these anesthetics decreased active stiffness and force per cross bridge when stiffness was measured with quick length changes (27). In another study in skinned rat cardiac fibers, halothane and, to a lesser extent, sevoflurane decreased the rate of cross-bridge attachment, the steady-state fraction of cross bridges in the force-generating state, and mean force per cross bridge (31). Halothane decreased stiffness, calculated from 1-kHz sinusoidal length oscillations, more effectively than did sevoflurane. Yet results from skinned fibers cannot easily be extrapolated to intact heart muscle because the skinning process removes surface membrane regulation of the contractile apparatus and many intracellular constituents (35). The effects of isoflurane on cross-bridge kinetics were recently studied during isometric tetani in intact cardiac muscle. The rate constant of cross-bridge attachment was estimated from the rate of force redevelopment (k_TR) after quick release stretch during the plateau of an isometric tetanus. Isoflurane decreased k_TR and shifted the k_TR-[Ca²⁺]_i relationship to higher [Ca²⁺]_i, an observation that strongly suggests that isoflurane decreases cross-bridge cycling kinetics (19). This effect was only partially mediated by a decrease in [Ca²⁺]_i.

Cross-bridge performance in intact muscle can be investigated by stiffness measurements (13, 16, 32). Stiffness (the ratio of force changes over length changes) of cardiac muscle consists of two components: passive stiffness arising from extracellular collagen and the cytoskeletal network, and dynamic or active stiffness, which mainly represents series elastic elements within cross bridges. Dynamic stiffness is proportional to the number of cross bridges attached at any one moment (13, 14). Shibata et al. (32) derived dynamic stiffness from sinusoidal length oscillations in rabbit papillary muscle in Ba²⁺ contracture. The frequency at which stiffness went through a minimum value was thought to reflect cross-bridge kinetics. Halothane, isoflurane, and enflurane did not alter the frequency at which minimum stiffness occurred, implying no apparent effect on cross-bridge kinetics. In light of the results in tetanized muscle (19) and in skinned fiber studies (27, 28, 31, 33), the Ba²⁺ contracture method is not sufficiently sensitive to detect small effects on cross bridges or may significantly alter the physiological state of the muscle.

The present study examines and compares the effects of three commonly used volatile anesthetics, halothane, isoflurane, and sevoflurane, on dynamic stiffness of intact cardiac muscle in which, unlike skinned fibers, endocardium, connective tissue, and cell membrane are completely intact and functional. In contrast to tetani or Ba²⁺ contracture studies, the present study investigates contractility and cross-bridge performance in single twitches, the physiological mode of contraction of the heart. We measured stiffness throughout isotonic shortening in single twitches of intact cardiac muscle from the varying frequency of frequency-modulated sinusoidal load oscillations imposed on the muscle lever system, which was kept at its instantaneous resonance frequency by feedback.

Interpretation of stiffness in isotonic twitch contractions. With respect to the interpretation of stiffness during shortening, two main facts have to be considered: 1) stiffness is the sum of passive stiffness and dynamic stiffness throughout the isotonic twitch; and 2) in isotonic contractions, stiffness of an attached cross bridge might vary during its cycle. Therefore, measured stiffness might not simply reflect the number of attached cross bridges, as it does in isometric conditions.

(b*RL)

Effects of anesthetics on isotonic contraction and dynamic stiffness. Halothane, isoflurane, and sevoflurane caused a concentration-dependent decrease in peak shortening and dynamic stiffness, with halothane exerting the most depressant effect and sevoflurane the least. Volatile anesthetics decrease contractility and stiffness because of decreased Ca²⁺ activation of the contractile apparatus due to smaller Ca²⁺ transients and decreased Ca²⁺ responsiveness (2-4, 9, 18, 23). Stiffness increases with Ca²⁺ activation and reflects the recruitment of cross bridges during Ca²⁺ activation and/or change in cross-bridge turnover kinetics (7, 8, 13, 17). To investigate whether impaired cross-bridge performance plays a role in the overall negative inotropic effect of anesthetics in intact cardiac muscle, [Ca²⁺]_o was increased during exposure to 1-MAC anesthetic until peak shortening in anesthetic and elevated [Ca²⁺]_o equaled that in control conditions. The underlying assumption is that, at equal peak shortening, the Ca²⁺ binding sites responsible for Ca²⁺ regulation of the contractile system are occupied to an equal extent.

Effects of volatile anesthetics on resting length and passive stiffness. Resting length and passive stiffness increased during exposure to halothane, isoflurane, and sevoflurane and recovered partially toward control values during anesthetic washout. The change in resting length and resting stiffness ran parallel, and it is tempting to assume that this effect of volatile anesthetics is based on the same mechanism. On the other hand, passive stiffness was smaller at each resting length in the presence of halothane than in control.

Conclusion. Part of the negative inotropic effect of halothane and isoflurane in clinically useful concentrations in isotonic twitch contractions in intact cardiac muscle is exerted at the level of cross bridges and might contribute significantly to their depressant action on the heart. The effects seem not to be mediated by the effects on Ca²⁺ binding sites of the regulatory proteins of the contractile apparatus. Halothane and isoflurane increased passive stiffness, which might reflect an effect on weakly bound cross bridges or an effect on passive elastic elements, or both.