Isoflurane depresses the intracellular Ca2+ transient and force development during a twitch, but its effects on crossbridge cycling rates are difficult to predict because of the transient nature of the twitch. Measurements of the effects of isoflurane on crossbridge cycling kinetics during tetanic contractions, which provide a steady state level of activation in intact cardiac muscle, have not been previously reported.
Ferret right ventricular papillary muscles were isolated, and superficial cells were microinjected with the bioluminescent photoprotein aequorin to monitor the intracellular Ca2+ concentration. The rate of tension redevelopment (kTR) was measured during steady state isometric activation (tetanic stimulation, frequency 20 Hz, 1 microM ryanodine, temperature = 30 degrees C) in the absence of isoflurane (2, 6, and 12 mM extracellular [Ca2+]) and in the presence of 0.5, 1.0, and 1.5 minimum alveolar concentration isoflurane (12 mM extracellular [Ca2+]).
Intracellular [Ca2+], isometric force, and kTR all increased when the extracellular [Ca2+] increased. Isoflurane (0.5, 1.0, and 1.5 minimum alveolar concentration) caused intracellular [Ca2+], isometric force, and kTR to decrease in a dose-dependent manner in the presence of 12 mM extracellular [Ca2+]. In the presence of increasing concentrations of isoflurane, the relation between intracellular [Ca2+] and force remained unchanged, whereas the relation between intracellular [Ca2+] and kTR was shifted toward higher [Ca2+].
These results indicate that isoflurane depresses myocardial crossbridge cycling rates. It appears that this effect is partially mediated by a decrease in the intracellular [Ca2+]. However, additional mechanisms must be considered to explain the shift of the relation between intracellular [Ca2+] and kTR toward higher [Ca2+].
MYOCARDIAL contraction results from a sequence of events that includes depolarization of the plasma membrane, release and sequestration of Ca2+by the sarcoplasmic reticulum (Ca2+transient), Ca2+binding to troponin C, and crossbridge cycling. Volatile anesthetics depress the contractility of isolated myocardium in a dose-dependent reversible manner. 1–4However, mainly because of the large number of potential sites of interaction, the mechanisms by which they exert this effect are still under active investigation. 5,6
Inotropic mechanisms can be classified according to their ability to alter (1) the intracellular Ca2+transient (“upstream” mechanisms), (2) the affinity of troponin C for Ca2+(“central” mechanisms), and (3) the response of the myofibrillar proteins to a given level of Ca2+(“downstream” mechanisms). 7Recent studies have exposed the ability of the volatile agents to depress contractility by upstream mechanisms. They are known to decrease the intracellular Ca2+transient 8–10and have been shown to affect Ca2+influx through surface membrane Ca2+channels and intracellular Ca2+release and sequestration by the sarcoplasmic reticulum. 11
There is also evidence that the volatile anesthetics act by altering calcium sensitivity (a central mechanism). It has been reported that halothane, enflurane, and isoflurane all appear to reduce myocardial Ca2+sensitivity at greater than 0.5 minimum alveolar concentration (MAC) in intact muscle. 10,12At 1 MAC, the reduction of myocardial calcium sensitivity was most pronounced in the presence of isoflurane and was estimated to play only a minor role in the negative inotropy of the volatile anesthetics. Similarly, Bosnjak et al. 8reported that isoflurane, but not halothane or enflurane, reduced the Ca2+sensitivity of intact cardiac muscle.
Little information is available about anesthetic effects on crossbridge cycling kinetics in the heart. Studies of anesthetic effects in myocardium with a porous surface membrane (skinned muscle) suggest that at high concentrations, volatile agents can affect crossbridge cycling. 13–15Extrapolation of these findings to intact myocardium is difficult because the skinning process can result in the loss of second messengers and enzymes that can affect the [Ca2+]–force relation and could alter crossbridge kinetics as well. 16,17
Therefore, we determined the effect of isoflurane on crossbridge kinetics in intact papillary muscles by measuring the rate of tension redevelopment (kTR) during the plateau phase of a tetanic contraction, when force and intracellular [Ca2+] have reached a steady state. 16,17
All experimental procedures were reviewed and approved by the Animal Care and Use Committee of the Mayo Foundation. Protocols were 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 (1.2 kg, Marshall Farms, North Rose, NY) were anesthetized with isoflurane in 100% oxygen, and the hearts were rapidly excised and placed in oxygenated physiologic salt solution. The aorta was then cannulated within 1 min, and the hearts were perfused with oxygenated physiologic salt solution. An appropriate papillary muscle was selected (cross-sectional area = 0.69 ± 0.20 mm2, length = 5.1 ± 1.0 mm, n = 8) and excised from the right ventricle.
Detection of the Intracellular Ca2+Transient
Aequorin (Friday Harbor Laboratories, Friday Harbor, WA) was introduced into superficial cells of the papillary muscles with a glass micropipette (1.5-mm OD, thin walled; World Precision Instruments, Sarasota, FL). 18The light emitted by aequorin was detected with a photomultiplier (9235QA; Electron Tubes Inc., Rockaway, NJ) and a current-to-voltage converter (Melles Griot, Irvine, CA). Digital signal averaging was used to obtain a satisfactory signal-to-noise ratio. Light measurements were converted into units of fractional luminescence (L/Lmax) by dividing them by the peak light intensity (Lmax) recorded during the optical conditions of the experiment when all of the remaining aequorin was exposed to a saturating concentration of calcium. 19To obtain Lmax, the cell membranes of the preparation were lysed with 4% Triton X-100 (Sigma, St. Louis, MO), and the light emitted was integrated electronically. 18Lmaxwas corrected for consumption of aequorin by subtracting the amount of light emitted during each tetanus. 16The amount of aequorin consumed during the course of an entire experiment averaged less than 2% of the total injected (mean = 1.4 ± 1.1%). The intracellular [Ca2+] was determined from the fractional luminescence by referring to an in vitro calibration curve. 18,19The entire protocol was conducted on seven papillary muscle preparations and all but the measurements at 1.5 MAC on one other.
Experimental Setup for Force and Force Redevelopment Measurements
Muscles were mounted in a chamber that contained 70 ml of physiologic salt solution maintained at a temperature of 30°C. The muscle was clamped at its lower end to a small plastic block. Electrical stimulation was accomplished with square-wave unipolar pulses of 5-ms duration delivered through platinum field electrodes at 20% above threshold intensity. To reduce the compliance of the preparation, the chorda tendinea was lashed to a thin glass strand (100-μm diameter, fiber optic) attached to the arm of a servo-controlled electromagnetic muscle lever operated in the isometric mode (Model 300B; Cambridge Technology, Inc., Cambridge, MA).
Muscle length was adjusted to a point at which the tension developed was optimal. The diameter and length of the muscle from the clamp to the chorda tendinea were measured in situ . The bathing solution was continuously bubbled with 100% oxygen, buffered with 3-[N -morpholino] propanesulfonic acid (MOPS) at pH 7.4, and contained 140 mm Na+, 5 mm K+, 2.0 mm Ca2+, 1 mm Mg2+, 103.5 mm Cl−, 1 mm SO42−, 5 mm MOPS, 10 mm glucose, and 20 mm acetate.
Appropriate conditions for tetanic contractions were produced by exposing the muscles to1 μm ryanodine (Calbiochem, San Diego, CA). 20Muscles were allowed to equilibrate for 30 min after exposure to ryanodine while being stimulated at 0.25 Hz. Fused tetani were produced with 20 Hz stimulation for 6 s. Tetani were induced at 2-min intervals. To achieve low, intermediate, and high levels of activation, the muscles were exposed to 2, 6, and 12 mm extracellular [Ca2+].
The constant for kTRwas determined with a length-change protocol that results in the dissociation of crossbridges. In this protocol, muscle length is quickly (< 1 ms) reduced by 20% to allow the muscle to shorten for a brief period (50 ms) and then rapidly returned to the initial length. Crossbridges then reattach, and tension redevelops at a rate determined by a least squares fit to a single exponential curve [T = T0(1 − e−kt)]. A computer program written in graphical software language (LabView, National Instruments, Austin, TX) was used to control the experiments. Signals were recorded by sampling at 1,000 Hz and digitizing with a 12-bit data acquisition board (AT-MIO16-L9, National Instruments).
Administration of Anesthetic
In all experiments, an inline calibrated anesthetic vaporizer was used to add the appropriate concentration of isoflurane to the preparation. Concentrations of volatile anesthetics in the gas over the bathing solution were monitored by Raman spectroscopy (Ohmeda Rascal II; Ohmeda, Madison, WI) and were also verified in the bathing solution by gas chromatography (5880A; Hewlett-Packard, Palo Alto, CA). 21The 1-MAC value for isoflurane (Abbott Laboratories, North Chicago, IL) in the ferret at 30°C (1.16%) was calculated from the 1-MAC value at 37°C (1.5% gas phase) 22because the MAC values at different temperatures are essentially constant when expressed as aqueous phase concentrations. 23
A simple two-state model of crossbridge cycling was used to interpret the mechanical data obtained in these experiments. 24–27In this model, crossbridges exist in two states: non–force-generating (A), and force-generating (B). The transitions between the two states are described by two apparent rate constants, one for the transition to the force-generating state (fapp), and one for the transition to the non–force–generating state (gapp). This model treats crossbridge formation as a reversible first-order chemical reaction. In theory, the total number of active crossbridges per half sarcomere and gapp are fixed, and fappincreases during activation. 27
During steady state conditions, as in a tetanus, the rate of formation of crossbridges is equal to the rate of dissociation of crossbridges and the equilibrium between force-generating (B) and non–force-generating (A) crossbridges is described by:
During these circumstances, if all crossbridges start in the non–force-producing state, then the rate of kTRwill reflect the sum of the forward and reverse rate constants:
In addition, according to this model, the force is proportional to the ratio:
where C is a constant that describes the total number of active crossbridges and the average force per crossbridge. Therefore, the derived relation between kTRand normalized force derived from the two-state model is:
where Fnis the normalized force and kTRmaxis the maximum value of kTR.
Results are reported as the mean ± SD. Force was normalized for muscle cross-sectional area. The length and diameter were measured at Lmax, and the muscle was assumed to approximate a cylinder. Linear or nonlinear regression was used to fit the data. Statistical significance (P < 0.05) was determined by a paired t test or one-way repeated-measures analysis of variance with the Dunnett test for multiple comparisons versus control.
Figure 1shows a representative recording of muscle length and tension during a tetanic contraction in a right ventricular papillary muscle. A high level of steady force has been achieved before decreasing the muscle length by 20%. When the muscle length falls, the tension rapidly decreases to zero and the muscle starts to shorten. The muscle length is then reextended to Lmax, breaking any crossbridges that may have formed during shortening. The tension therefore starts out at near the resting level and quickly redevelops as crossbridges reform.
We found that intracellular [Ca2+], isometric force, and kTRall increased as the extracellular [Ca2+] was increased (table 1). Figure 2shows a recording of aequorin luminescence and isometric force at 2, 6, and 12 mm extracellular [Ca2+]. There was a progressive increase in the steady level of intracellular [Ca2+] as the extracellular [Ca2+] was elevated. Superimposable records could be obtained from successive tetani so that several consecutive tetani could be averaged to improve the signal-to-noise ratio. 16After a suitable number of tetani (4–7) had been recorded for intracellular [Ca2+] measurement, the kTRprotocol was imposed during the subsequent tetanus. Although the light-gathering apparatus used in these experiments is designed to minimize movement artifact, we did not attempt to measure intracellular [Ca2+] during the length change to eliminate this possibility.
Isoflurane caused kTRto decrease when the muscles were tetanized in 12 mm extracellular [Ca2+] (table 1). Figure 3shows the effect of extracellular [Ca2+] and isoflurane on kTR. kTRwas clearly faster when the extracellular [Ca2+] was increased from 2 to 12 mm. The kTRwas 2.2 s−1in 2 mm extracellular [Ca2+] and increased to 6.9 s−1when the extracellular [Ca2+] was increased to 12 mm. In this example, 1 MAC isoflurane slowed kTRto 3.5 s−1(in the continuing presence of 12 mm extracellular [Ca2+]).
Isoflurane caused the intracellular [Ca2+] to decrease in a concentration-dependent manner in the presence of 12 mm extracellular [Ca2+] (table 1). Figure 4shows the effect of 0.5, 1.0, and 1.5 MAC isoflurane on kTRand intracellular [Ca2+]. The relation between intracellular [Ca2+] and kTRin the absence of anesthetic (obtained by changing the extracellular [Ca2+]) is also shown. The figure shows that the relation between intracellular [Ca2+] and kTRis shifted toward higher [Ca2+] in the presence of isoflurane. On the other hand, increasing isoflurane from 0.5 to 1.5 MAC did not appear to alter the relation between intracellular [Ca2+] and force in the presence of 12 mm extracellular [Ca2+], as shown in figure 5.
The relation between isometric force and kTRis altered in the presence of isoflurane (figure 6). The data points obtained in the presence of anesthetic fall below the curve obtained in its absence. During these circumstances, gappis decreased in the presence of anesthetic from 2.7 ± 0.8 to 1.7 ± 0.9 s−1(P < 0.05).
We determined the effect of isoflurane on crossbridge kinetics in intact papillary muscles by measuring the rate of kTRduring the plateau phase of a tetanic contraction, when force and intracellular [Ca2+] have reached a steady state. 16,17The results of these experiments indicate that isoflurane slows myocardial crossbridge kinetics in the heart. This is the first report of such an effect on crossbridge cycling (kTR) in intact myocardium.
Previous studies have focused on mechanical measurements in skinned cardiac muscle at room temperature or lower. 13,14Skinned preparations are useful because they have their surface membranes disrupted, rendering them permeable to small ions and molecules. In this way, the ionic composition of the solution bathing the myofibrils can be precisely controlled and the calcium concentration altered to produce varying levels of activation. Thus, a clearer picture of the effects of an intervention directly on the function of the myofibrillar apparatus can be obtained.
On the other hand, soluble second messengers, enzymes, and perhaps other unknown small molecules would be expected to be lost in the skinning process, and this fact might alter the conclusions of such studies. 28The skinning process clearly alters Ca2+sensitivity and the steepness of the [Ca2+]–force relation. 16,17The effects of interventions on processes such as phosphorylation and dephosphorylation would be lost unless the proper enzymes were included. Therefore, it is very important to examine the effects of the halogenated volatile anesthetics on crossbridge kinetics in intact myocardium.
The link between [Ca2+] and kTRhas been well documented in skinned skeletal muscle, where kTRis known to increase exponentially as the level of activation by calcium increases. 27,29The effect of Ca2+on kTRin intact and skinned cardiac muscle is slightly more controversial. Hancock et al. 30,31reported that kTRin intact ferret cardiac muscle was unaffected by altering the extracellular or intracellular [Ca2+], but they used a different protocol to control the muscle length. In their protocol, muscle length was decreased by 3% but was not returned to Lmax. Other investigators have found that calcium increases kTRin skinned rat trabeculae, 32skinned rat myocytes, 33and, most recently, in intact rat trabeculae. 34Our results, obtained using a similar length-change protocol, indicate that kTRin intact cardiac muscle varies with the level of activation as achieved by altering the level of extracellular [Ca2+] (fig. 4).
The length-step protocol used in these experiments is essentially the same as that used by other investigators in skinned muscle. 25–27,29,32The results obtained in this investigation are not influenced by length-dependent mechanisms such as Starling’s law because the muscle was returned to Lmaxbefore the measurement started. In a similar manner, it is unlikely that the length-change protocol altered the function of the surface membrane or the sarcoplasmic reticulum.
Volatile anesthetics are known to alter the intracellular [Ca2+] during a twitch. 8–10,35For isoflurane, this effect appears to be primarily caused by an inhibition of Ca2+entry through surface membrane Ca2+channels. Therefore, it seemed likely to us that isoflurane would slow crossbridge transitions in normal muscle with an intact surface membrane primarily by decreasing the intracellular [Ca2+]. However, if isoflurane decreased the rate of crossbridge cycling only by this mechanism, then we would expect to find that the relation between intracellular [Ca2+] and kTRwould not be altered in the presence of anesthetic. On the contrary, we found that isoflurane shifts the relation between intracellular [Ca2+] and kTRtoward higher [Ca2+] (fig. 4). This indicates that isoflurane may influence crossbridge cycling by another mechanism.
The observation that kTRwas changed to a greater extent than isometric force in the presence of isoflurane (fig. 5) is consistent with this interpretation and suggests that both fappand gappprobably decrease in the presence of isoflurane. The crossbridge model can illustrate this concept. According to the model, kTRis the sum of fappand gapp(equation 2), and force is proportional to the ratio of fappand kTR(equation 3). If only fappdecreases with decreasing activation, then the points obtained in the presence of isoflurane should fall along the curve obtained by changing the extracellular [Ca2+]. However, if only gappdecreased in the presence of isoflurane, the force would be expected to increase, since each crossbridge would be expected to remain attached for a longer time.
Surprisingly, isoflurane did not appear to alter the relation between intracellular [Ca2+] and isometric force during the conditions of the experiment. One might expect to see a change in calcium sensitivity because it has been reported that crossbridge attachment can influence calcium binding to troponin C. However, the conditions used in these experiments are different from those used by other investigators to show that isoflurane decreases calcium sensitivity. 8,12
In summary, these results show that isoflurane alters myocardial crossbridge kinetics in intact heart muscle. The depression of crossbridge cycling seems to be caused, in part, by an indirect effect of the volatile anesthetics on intracellular Ca2+, but a more direct effect at the level of the crossbridge is the most likely explanation for the decrease in crossbridge detachment rate (gapp).