INTRODUCTION

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IT IS WELL KNOWN that the commonly used vapor anesthetic agents have significant negative inotropic effects on the heart (32). This poses a substantial problem in human anesthesia, since these agents are often administered to patients with preexisting cardiac disease. It has been suggested that the vapor agents may act to depress the heart in the following ways: 1) by influencing upstream the influx during activation of Ca²⁺ through the L-type calcium channels in the sarcolemma; 2) by altering upstream the Ca²⁺ uptake and release process during activation of the sarcoplasmic reticulum (SR), the principal intracellular Ca²⁺ storage and release organelle in the muscle cell; and 3) by modifying downstream the so-called calcium responsiveness of the contractile proteins during activation (8). The problem is to sort out which of these factors, and possibly others, plays the dominant role in the negative inotropic action of a particular vapor agent. Because the vapor anesthetics may act on specific targets in neurons rather than nonspecifically as commonly thought (15), these agents may also act on targets in the heart more specifically than previously believed. Therefore, to better understand the mechanisms of negative inotropy in the heart, it is important now to determine precisely where and how these agents act.

We have recently completed a study of the negative inotropic action of the benchmark vapor agent halothane in the heart (21). We used intact trabeculae from the rat heart loaded iontophoretically with the fluorescent dye fura 2 to report intracellular [Ca²⁺] ([Ca²⁺]_i) during fixed end twitch contractions. Our results indicated that at a low dose [<1 minimum alveolar concentration (MAC)] halothane markedly decreased peak twitch force mainly through a reduction of the Ca²⁺ responsiveness of the contractile system. At a high dose (near 2 MAC), its effect was most likely through both reduction in the [Ca²⁺]_i transient and reduction of the Ca²⁺ responsiveness of the contractile system. Even at the high dose of halothane, the SR Ca²⁺ content was not substantially reduced, as assayed using the rapid-cooling response (21). These results are not in complete agreement with current ideas about how halothane acts to depress the heart.

It seems to be generally believed that isoflurane has actions to depress heart contractility that are somewhat unique. In particular, compared with halothane and enflurane, isoflurane seems to exert a lesser depression on the [Ca²⁺]_i transient following stimulation, whereas it has a larger effect on inhibiting myofibrillar calcium responsiveness (8). Because our new results with halothane indicated actions not completely in agreement with current views (21), it became necessary to test another agent with supposedly different mechanisms of action to determine whether our results were mainly different with respect to halothane or showed differences with respect also to another agent, isoflurane, commonly believed to act differently from halothane.

The goal of this study was, therefore, to investigate the relative depressant contributions on upstream and downstream variables by isoflurane and to compare these results with our previous ones from halothane. We find that isoflurane depresses the peak twitch force more than the peak [Ca²⁺]_i transient. It also acts to diminish myofibrillar Ca²⁺ responsiveness but less so than does halothane at equipotent doses. Another striking new finding is that isoflurane nearly completely blocks the liberation of Ca²⁺ from the SR in response to rapid cooling, using fura 2 to report [Ca²⁺]_i, whereas halothane does not.

	METHODS

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A recent paper (20) presents many important details of the methods used in this work. The following condensed version provides essential information either not provided in the recent paper or of particular importance here.

Dissection procedure. Male Lewis-Brown-Norway rats (150-175 g) were used in these experiments (Harlan, Indianapolis, IN). Hearts were quickly removed and placed in a cold modified Krebs solution (MKS). The hearts were then perfused retrogradely through the aorta using a Langendorff apparatus for 10 min at room temperature with the same MKS containing (in mM) 108 NaCl, 6 KCl, 1.2 MgCl₂, 2 CaCl₂, 24 NaHCO₃, 10 sodium pyruvate, and 4 glucose, in addition to 5 U/1,000 ml insulin; and bubbled with 95% O₂-5% CO₂, final pH 7.4. Finally, the hearts were flushed with dissection solution, which is the standard MKS plus 15 mM KCl (final [KCl] = 21 mM).

Trabeculae similar to those described previously were isolated from the right ventricle (11, 29). The trabeculae were attached at one end of a force transducer, and the other end was tied to a stainless steel wire connected to a micropositioner. In an important step in the mounting procedure, cyanoacrylate tissue adhesive was used to fix firmly the two ends of the preparation to the force transducer wire and the micropositioner wire. We have previously shown that this method of attachment of a papillary muscle very much decreases extra end compliance as demonstrated by flash photography and use of markers and spot-follower apparatus (23). Consequently, internal movement associated with extra compliance at the ends of the preparation is minimized, thereby ensuring a contraction as close to isometric as possible.

Bath setup and electrical stimulation. The preparation was placed into a temperature-regulated chamber. The chamber had a glass coverslip floor and was positioned on the stage of an inverted microscope (Nikon Diaphot 300) fitted with a fluorescence system [Photon Technology International (PTI) Deltascan 4000]. Preparations were superfused at a rate of 12 ml/min with MKS. The MKS solutions were vigorously bubbled with 95% O₂-5% CO₂. The temperature in the bath was maintained at 30 ± 0.2°C, unless otherwise stated. The preparations were stimulated at 0.5 Hz with 5-ms pulses via platinum plate electrodes in the bath connected to a stimulator (Grass S88). The stimulus strength was adjusted to 1.5 times threshold, and tension was continuously monitored on both a chart recorder and one channel of a digital oscilloscope. The optimal tension response with respect to length was obtained. Resting sarcomere length at peak maximal active force was always in the range of 2.18-2.30 µm. These values were measured from hard copy striation patterns sampled from the video image (using a Nikon ×40 objective) with a UP1200 Mavigraph Color Video Printer (Sony, Japan).

Dye-loading procedures. Before the trabeculae were loaded with fura 2, autofluorescence (emission at 510 nm) was measured at a pacing rate of 0.5 Hz and 30°C. Based on a method already described (2), preparations were then loaded by iontophoretic injection of fura 2 dye in MKS with extracellular [Ca²⁺] ([Ca²⁺]_o) of 0.5 mM at 22°C with no stimulation. Micropipettes were pulled on a Flaming/Brown micropipette puller (model P87, Sutter Instrument), and tips were filled with 1 µl of 2 mM fura 2 (pentapotassium salt, Molecular Probes, Eugene, OR). The pipettes were then backfilled with a 150 mM potassium acetate solution (pH = 7.3) and connected via a micropipette holder to the headstage of a WPI electrometer module (model S-7071A). Tip resistances of 200-300 M were typical in micropipettes filled with the fura 2 solution before impalement of single myocytes, and stable membrane potentials of 40 to 60 mV were required for successful dye loading. After impalement, injection of the dye into the myocyte was achieved by passing a negative current of 3-5 nA. After loading was completed, the preparation was superfused again with the standard MKS, the temperature was increased to 30°C from room temperature, and the pacing was resumed at 0.5 Hz. About 40 min were allowed to let the dye diffuse, yielding a complete uniformity of fluorescence. Fura 2 was loaded to levels such that the added fluorescence was about three times the autofluorescence observed at 358 nm (the isosbestic point for fura 2) before loading.

Application of isoflurane. Using a Dräger vaporizer, we added isoflurane to the superfusion solution in the treatment bottle to produce four doses (in mM): 0.23, 0.42, 0.62, and 1.2. The isoflurane concentrations were determined using a Hewlett-Packard 5890 Series II gas chromatograph with an HP 624 column and a flame ionization detector. According to a recent report (14), 1 MAC for isoflurane in non-blood-containing aqueous media at below-normal body temperature in the rat should be ~0.31 mM, which is higher than that given for halothane, 0.27 mM. The first three doses of isoflurane listed above, therefore, in this work, were chosen to be approximately equipotent to the three doses of halothane used in our previous work (in mM): 0.18, 0.37, and 0.55 (21). The fourth dose of isoflurane given above (1.2 mM) or near 4 MAC was chosen independently of our previous work with halothane, and this dose was chosen to produce a more nearly maximal depression of the twitch force and still yield complete recovery on washing out the drug. The MAC concept is related to an intact whole body response to a noxious stimulus signaled by purposeful movement in 50% of the subjects. The use of the MAC concept would seem to set an upper limit on vapor agent dose levels that would fall within the clinically relevant range. This is an important consideration, since all of the vapor agents, if used at sufficiently high doses, can severely attenuate the [Ca²⁺]_i signal and twitch force.

Rapid-cooling procedure. Rapid cooling was used to assay SR Ca²⁺ content using both contracture force as well as the [Ca²⁺]_i signal reported by fura 2. A simple procedure was devised using a fixed-volume (2,000 µl) tip ejector pipetter to deliver ejectate into the muscle chamber (250-µl volume). The superfusate for both control and treatments in the pipetter tip was precooled to 0°C by placing the tip in a solution of ice and distilled water. The temperature near the preparation was monitored using a thermistor needle probe, with a response time of ~0.15 s. This showed that the technique could produce a temperature change in the chamber solution from 30°C to 3°C in ~1 s. The cold injectates could be reliably delivered at ~2 s after the last electrical stimulation. Just before the injection, flow of the perfusate as well as electrical stimulation was interrupted and then resumed after the cold response was recorded (both fura 2 signal response and contracture force). The peak contracture force and the peak [Ca²⁺]_i were used to estimate the amount of Ca²⁺ released from the SR in response to an rapid cooling as previously reported (6, 30).

Application of caffeine. Caffeine (Sigma Chemical, St. Louis, MO) was directly added into the standard MKS, equilibrated with 1% isoflurane (0.62 mM), used to elicit a cold response to yield a final concentration of 30 mM. The MKS containing both caffeine and isoflurane was precooled and used to assay SR Ca²⁺ content.

Fluorescence system. The PTI Deltascan 4000 system was used in these experiments, and a 75-W xenon lamp was the source for excitation light. The light passed through a scanning monochromator that was programmed to select wavelengths of 345 and 380 nm for excitation of fura 2. Light exiting the monochromator was carried by a quartz fiberoptic bundle to a filter cassette coupled to the microscope objective. On entering the cassette, the light was first short-pass filtered (<470 nm) to remove the residual 510-nm light before reflecting off a dichroic mirror (400 nm long pass) en route to the objective, an Olympus ×10 (Dapo UV/340; numerical aperature 0.4). Fluorescence from the preparation passed back through the objective and dichroic mirror to the sideport of the microscope. Just before reaching the photomultiplier tubes, light signals were passed through emission filters, which transmitted wavelengths of 510 ± 20 nm.

Calibration of fura 2. A complete description of our method for calibrating fura 2 in the heart has been published (20). Particularly for peak values of [Ca²⁺]_i, our values for [Ca²⁺]_i are considerably higher than those usually reported in the past. However, the value for [Ca²⁺]_i, obtained by a transformation of the fura 2 R signal, depends on the method used for calibration, and calibration of fura 2 has been done in various ways. For example, some of the [Ca²⁺]_i values reported previously were calculated using maximal and minimal fluorescence ratio (R_max and R_min, respectively) values obtained from both in vitro and in vivo calibrations that were similar, thus leading to peak values for the transient in [Ca²⁺]_i <1 µM (2). Recently, we have done a careful study to compare the values particularly for R_max and dissociation constant (K_d) for fura 2 obtained from both in vitro and in vivo conditions (20). For the in vivo calibration, the intra- and extracellular [Ca²⁺] were measured at equilibrium (4). Under these conditions, the [Ca²⁺]_i at the peak of the transient calculated using our in vivo calibration parameters is about three times higher than that obtained using our in vitro calibration parameters, owing mainly to a decrease in R_max and an increase in K_d for the in vivo condition. We therefore believe the peak value for the transient in [Ca²⁺]_i is ~2-3 µM (20-22), which is higher than previously reported. Similar results have also been obtained using indo 1 to report [Ca²⁺]_i in intact rabbit myocytes (4).

Data collection and analysis. Fluorescence signals were collected in either photon-counting mode (entrance and exit slit widths set to 2 nm) or analog mode (entrance and exit slit widths set to 10 nm). The fluorescence system in the analog mode was controlled by PTI software "Oscar" running on an IBM-compatible 486 computer (American Megatrends) used to control shutter openings, monochromator settings, and other automated data acquisition programs. The fluorescence and twitch force signals were recorded using the oscilloscope, and all traces presented here are averages of nine sweeps. The autofluorescence at excitation wavelengths 345 nm (F_345a) and 380 nm (F_380a) were interleaved in time so that a sweep at 345 nm was followed by one at 380 nm and so on until a total of 18 sweeps were recorded. These were then averaged in two groups of nine. In this way, a noise reduction by a factor of 3 was achieved. The fluorescence values after dye loading were collected in the same way as for the autofluorescence yielding total fluorescence at the two excitation wavelengths (F_345t and F_380t). The F_345a and F_380a were then subtracted from the F_345t and F_380t, respectively, yielding the dye-related fluorescence F₃₄₅ and F₃₈₀. The R signal was formed by dividing F₃₄₅ by F₃₈₀. Fluorescence and twitch force were analyzed using Vu-Point 3 data analysis software (Maxwell Laboratories).

When a photon-counting mode was used, the excitation wavelengths 345 and 380 nm were alternated by a chopper at a rate of 200/s, and the "Felix" program of PTI was used to record the fluorescence intensities of F₃₄₅ and F₃₈₀ from photomultiplier A. Dye loading was monitored also using photomultiplier A. The autofluorescence values of F₃₄₅ and F₃₈₀ were obtained before dye loading, and these were subtracted from the raw fluorescence signals after dye loading, thus yielding corrected fluorescence signals in real time. The R signal and the corresponding force trace were also recorded and displayed using the same program.

	RESULTS

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A typical time-dependent response of an isolated trabecula to isoflurane (0.62 mM, 2 MAC) is shown in Fig. 1. Isoflurane caused a gradual decrease in the peak [Ca²⁺]_i transient (Fig. 1A) and, to a greater extent, the peak twitch force (Fig. 1B). Then both the peak [Ca²⁺]_i transient and the peak twitch force reached steady-state levels ~80 s from the beginning of treatment. For this preparation, in the steady-state, isoflurane at a dose near 2 MAC depressed the peak twitch force over 50%, whereas the peak [Ca²⁺]_i transient was not depressed in proportion. There was no detectable effect on either diastolic force and diastolic [Ca²⁺]_i. Washing out isoflurane led to a complete recovery of both twitch force and [Ca²⁺]_i transient (data not shown). This result shows that treatment with a relatively high dose of isoflurane does cause a substantial time-dependent reduction in peak twitch force, although this is not accompanied by a proportional decrease in the peak [Ca²⁺]_i transient. This experiment was repeated in seven trabeculae with similar results.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   Time-dependent effects of isoflurane on intracellular [Ca2+] ([Ca2+]i) transients and twitch force. Treatment with isoflurane (0.62 mM or 2 minimum alveolar concentration) was as indicated by the solid bar shown in middle. A: [Ca2+]i transients. B: corresponding twitch force. Trabecula was paced at a rate of 0.5 Hz at 30°C, extracellular [Ca2+] ([Ca2+]o) 2 mM. Data for R signal were collected in photon-counting mode at a rate of 200/s. [Ca2+]i was obtained by transformation of R signal using minimum R (Rmin), maximum R (Rmax), and dissociation constant (Kd) ×  obtained from an in vivo calibration (20). Note that isoflurane in steady state substantially reduced peak twitch force, whereas peak [Ca2+]i transients were not proportionally depressed.

The effects of isoflurane in the steady state at different doses are shown in the montage presented in Fig. 2 for [Ca²⁺]_i transients (Fig. 2A) and twitch forces (Fig. 2B). Isoflurane (0.23, 0.42, and 0.62 mM) was applied in equipotent doses to those we used in our previous work with halothane (21), that is, equipotent in the sense that the halothane doses were incremented by ~15% to account for the higher 1 MAC dose level of isoflurane in the rat (14). The high 1.2 mM dose was chosen arbitrarily (near twice the 0.62 mM dose or near 4 MAC) to produce more profound depressions in peak [Ca²⁺]_i and peak twitch force. Remarkably, the 1.2 mM dose did not eliminate either the [Ca²⁺]_i response or the twitch force response. Washing out isoflurane resulted in nearly complete recovery of both peak twitch force and peak [Ca²⁺]_i transient as shown in the postcontrol, which included treatment with the 1.2 mM dose. The reductions in peak twitch force are larger than the reductions in the corresponding peak [Ca²⁺]_i transient, as was the case for our findings with halothane (21). At the highest dose of isoflurane (1.2 mM) there was still no detectable elevation of diastolic [Ca²⁺]_i

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Steady-state effects of varying isoflurane concentrations on [Ca2+]i transients (A) and the corresponding twitch force (B). Preparation was paced at 0.5 Hz at 30°C, [Ca2+]o 2 mM. Data were collected in analog mode at a rate of 2,000/s. For this preparation, isoflurane was introduced in the order 0.23, 0.42, 0.62, and 1.2 mM after precontrol twitch was obtained. For each dose of isoflurane, data were obtained in steady state of maximal depression of peak twitch force. Postcontrol twitch was obtained after washing out isoflurane and allowing the twitches to recover to steady-state values. Note that in the steady state of isoflurane action, reduction of peak twitch force is more prominent than is the depression of the corresponding peak [Ca2+]i transient. [Ca2+]i was obtained by transformation of R signal using Rmin, Rmax, and Kd ×  obtained from an in vivo calibration (20).

To establish the factors that contribute to its depressant influence, the results obtained with isoflurane are shown in Fig. 3, which is a plot of peak twitch force against the corresponding peak [Ca²⁺]_i transient. The isoflurane results are shown by the thick solid line with error bars. To better delineate the effects of isoflurane, it was compared in Fig. 3 with the effects produced by two other agents with well-defined mechanisms of negative inotropic action in the heart. One intervention was by lowering [Ca²⁺]_o and thus lowering the peak [Ca²⁺]_i transient, which should reduce contractility by decreasing the [Ca²⁺]_i transient for force generation without significant alteration of the Ca²⁺ responsiveness of the contractile system. The other intervention was by using 2,3-butanedione monoxime (BDM), which is an agent that markedly reduces muscle force generation by a peripheral mechanism interfering with cross-bridge formation with little effect on the peak [Ca²⁺]_i transient in the doses used here (1). These treatment effects are shown by the thin lines in Fig. 3, which summarize results obtained previously (21). Previous results obtained with halothane (21) are also included in Fig. 3 as indicated by the thin line labeled as halothane. From Fig. 3, it is apparent that isoflurane in increasing doses causes a near-linear decrease in the peak twitch force versus the corresponding peak [Ca²⁺]_i transient. Note that the extrapolation of the curve to zero force would yield a peak [Ca²⁺]_i transient decreased by only 56%. Importantly, the isoflurane dose-response curve falls approximately halfway between those obtained by lowering [Ca²⁺]_o or by applying BDM. It is also apparent in Fig. 3 that the isoflurane response is different from that produced by halothane, even though the isoflurane doses were adjusted to be equipotent, except for the 1.2 mM dose.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Plot of peak twitch force as a function of corresponding peak [Ca2+]i transients in absence and presence of various doses of isoflurane. Data shown for isoflurane (thick solid line labeled isoflurane) are presented as means ± SE from 5 experiments. Thin lines labeled [Ca2+]o, 2,3-butanedione monoxime (BDM), and halothane indicate results from an earlier study (21). For isoflurane, linear regression equation was Y = 75.8 + 1.73X. Note that this would indicate zero twitch force for a peak [Ca2+]i transient reduction of 56.2%. * Peak value for transient in [Ca2+]i of a twitch obtained with isoflurane was significantly lower than that obtained with halothane at a similar level of peak twitch force reduction.

In accordance with our previous work with halothane (21), the question also arises here as to what extent isoflurane's negative inotropic effect in the heart could be due to diminished Ca²⁺ content in the SR. To pursue the answer to this inquiry, we show in Fig. 4 a typical response of a trabecula used in this series to rapid cooling, which has been often used in both skeletal and cardiac muscle to assay "on-line" the Ca²⁺ content of the SR (5). Note that the trabecula in Fig. 4 was not exposed to isoflurane. In Fig. 4A the [Ca²⁺]_i response to the cold MKS is shown. It is apparent that the sudden drop of temperature causes a rapid rise in [Ca²⁺]_i to levels near those associated with the responses to electrical stimulation. This indicates that the SR contained a store of Ca²⁺ similar to that present under normal pacing conditions. In Fig. 4B, the corresponding force responses are shown. Again, the sudden drop of temperature is associated with a rapid rise of force to reach a level near that of the twitch responses. The results shown in Fig. 4 are entirely those to be expected from application of rapid cooling during normal pacing conditions, and they verify that rapid cooling can be used to assay SR Ca²⁺ content.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Typical rapid-cooling responses for control conditions with no isoflurane or caffeine. The trabecula was paced at a rate of 0.5 Hz, at 30°C, with [Ca2+]o of 2 mM. Rapid cooling was initiated after the stimulator was turned off and 2 s from the last stimulus. Solid smooth line in each panel shows temperature response in chamber near trabecula from 30°C to near 0°C. Data for R signals (not shown) were collected in photon-counting mode at a rate of 200/s. [Ca2+]i was obtained by transformation of R signal using the Rmin, Rmax, and Kd ×  obtained from an in vivo calibration (20). A: [Ca2+]i responses are shown, including three twitches followed by rapid cooling. Note that upstroke of rapid-cooling response is nearly as rapid as that for the 3 preceding twitches with a similar peak amplitude value. B: corresponding relative force responses are shown. Note rapid initial upstroke of force produced by rapid cooling.

However, the results shown in Fig. 5 indicate that isoflurane has the unique property of blocking the usual response to rapid cooling. In this trabecula, 0.62 mM isoflurane was applied, and the steady-state [Ca²⁺]_i and twitch responses were obtained before application of rapid cooling (note the reduced peak twitch forces compared with Fig. 4). Remarkably, as clearly shown in Fig. 5A, there is no sign of an increase in [Ca²⁺]_i following rapid cooling, and this was the response observed in six of nine trabecula with the other three showing only small increases (see Fig. 7A). The corresponding force responses are shown in Fig. 5B where it can be seen that essentially no force was generated either in response to the rapid cooling.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effect of isoflurane (0.62 mM) on rapid-cooling response. Data were obtained from same preparation as for Fig. 4 at a pacing rate of 0.5 Hz, at 30°C, with [Ca2+]o of 2 mM. Data for R signals and transformation to [Ca2+]i are as in Fig. 4, as are solid smooth lines in each panel together with the ordinate scales. Rapid cooling was initiated in presence of 0.62 mM isoflurane. A: response to rapid cooling is shown together with four preceding twitch responses. Most common response observed (for n = 9) was complete elimination of any sign of Ca2+ release from sarcoplasmic reticulum as indicated by the flat [Ca2+]i trace after onset of rapid cooling. B: corresponding force responses are shown. Note four twitches in response to electrical stimulation followed by no sign of force generation at the application of rapid cooling, which is to be expected from results shown in A. Note that the small glitch in the force record in response to rapid cooling has no counterpart in [Ca2+]i trace shown in A. This is because the [Ca2+] signal was obtained using fura 2 in the ratioing mode so that excellent cancellation of motion artifact was obtained.

Because isoflurane blocks the rapid-cooling response, it is impossible to use rapid cooling alone to estimate SR Ca²⁺ content. It could be argued that the reason why no increase in [Ca²⁺]_i was observed in Fig. 5 was because isoflurane had actually depleted the SR of releasable Ca²⁺. This is very unlikely. It is known that the transient increases in [Ca²⁺]_i observed following normal pacing are largely due to release of Ca²⁺ from the SR (7). It is obvious in Fig. 5 that the transient increases in [Ca²⁺]_i preceding the rapid cooling show kinetic responses quite similar to those in Fig. 4 in the absence of isoflurane, with some decrease in amplitude. It would seem more likely, in Fig. 5, that the SR Ca²⁺ content was near the control level and that isoflurane acted specifically and reversibly to block the rapid-cooling response of the SR. Nevertheless, it is important to demonstrate that isoflurane does not produce negative inotropy by substantially emptying the SR of Ca²⁺. In work with permeabilized rat cardiac preparations, caffeine was added to the cold solution, thus overriding the blocking action of isoflurane (17). We adopted this approach by adding caffeine into the cold MKS as it was done in the permeabilized preparation (17). A typical result is shown in Fig. 6. In Fig. 6A, the [Ca²⁺]_i responses are shown where the rapid-cooling solution containing 30 mM caffeine was applied while the trabecula was treated in the steady state with 0.62 mM isoflurane. It can be seen that the result is a slow rise of [Ca²⁺]_i, much slower than shown in Fig. 4, to approach a peak value below those associated with the preceding pacing responses. This indicates that substantial Ca²⁺ remained in the SR even with isoflurane treatment. In Fig. 6B, the associated force response is shown, which indicates a slower, more persistent effect compared with that shown in Fig. 4, following rapid cooling. It is necessary to apply caffeine in the cold solution for reasons that are probably similar to those with ordinary use of rapid cooling, i.e., to inhibit the SR Ca²⁺ uptake pump and the Na/Ca exchanger thus allowing significant increases in [Ca²⁺]_i (5).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of caffeine (30 mM) added to rapid-cooling solution in presence of 0.62 mM isoflurane on response to rapid cooling. Data were collected from same preparation used in Figs. 4 and 5, with pacing at a rate of 0.5 Hz, at 30°C, with [Ca2+]o of 2 mM. Data for R signals and transformation to [Ca2+]i are as in the legend for Fig. 4, as are solid smooth lines in each panel together with ordinate scales. A: [Ca2+]i transients to three preceding twitches followed by rapid-cooling response are shown. Note, compared with first three twitch responses, that rise of [Ca2+]i signal in response to rapid cooling is slower to reach a lower maximal value, which is in contrast to results shown in Fig. 4. B: corresponding force responses are shown. As with [Ca2+]i signal, rise of force is slower compared with control response shown in Fig. 4.

Figure 7A shows that isoflurane (0.62 mM) reduces the SR cold response by 96% in terms of the peak value of the [Ca²⁺]_i compared with the precontrol value. Thus, overall, only a relatively very small increase in [Ca²⁺]_i was recorded following application of rapid cooling with isoflurane present. In Fig. 7B, we summarize our findings with respect to the alternate assay of SR Ca²⁺ content in the presence of isoflurane using 30 mM caffeine in the cold solution. Here the peak responses of the [Ca²⁺]_i signal obtained with 30 mM caffeine added to the rapid-cooling MKS, at steady-state in the presence of 0.62 mM isoflurane, are presented as a percentage of the precontrol value. As is evident in Fig. 7B, the SR Ca²⁺ content assayed in this way in the presence of isoflurane reveals that on average the SR was filled to at least 71% of the control value in the absence of isoflurane. However, it is not necessarily true that using the peak values of [Ca²⁺]_i as indicators is optimal particularly in the case of the caffeine response in the presence of isoflurane, which is much slower than the cold response without isoflurane. Therefore, in Fig. 7C, the responses observed as in Fig. 4 (cold without isoflurane) and Fig. 6 (cold with caffeine and isoflurane) are shown on the same time base to emphasize the differences in speeds of response. The time to peak of the [Ca²⁺]_i rise in response to rapid cooling alone is more than 10 times shorter than that of rapid cooling plus caffeine and isoflurane. Because the time to peak is prolonged with rapid cooling plus caffeine and isoflurane, the peak value will be more sensitive to Ca²⁺ release and uptake processes. Thus the true SR content of Ca²⁺ may be >71% compared with precontrol in the presence of 0.62 mM isoflurane.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Effect of isoflurane on rapid-cooling response. A: peak values of [Ca2+]i elicited by rapid cooling in presence of 0.62 mM isoflurane and for postcontrol (97%) are given as a percentage of precontrol value (100%) for n = 9. B: peak values of [Ca2+]i elicited by rapid cooling plus 30 mM caffeine in presence of 0.62 isoflurane (71%) and for postcontrol (93%) are given as a percentage of precontrol value (100%) for n = 5. C: [Ca2+]i responses from same trabecula induced by rapid cooling for control (from Fig. 4A) and by rapid cooling plus 30 mM in presence of 0.62 mM isoflurane (from Fig. 6A) are shown on an expanded time base to emphasize dissimilarity of speeds of response. Here, zero marks beginning of rapid-cooling response, and 10 s is time required for [Ca2+]i increase induced by rapid cooling plus caffeine in presence of isoflurane to reach a peak.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The study of the depressant effect of isoflurane on cardiac muscle contraction is not new (19, 25-27). Work using intact papillary muscles and the photoprotein aequorin to report [Ca²⁺]_i concluded that isoflurane has weaker negative inotropic effects than does halothane (9). Similar results were obtained using isolated myocytes loaded with the membrane-permeant AM form of fura 2 (35). However, it is generally believed that isoflurane, compared with halothane, exerts a greater depression on the Ca²⁺ responsiveness of the myofibrillar apparatus (8, 18). Also, it has been reported that isoflurane inhibits contracture force development to rapid cooling (24). In our previous work, we found that halothane had a strong effect to depress the Ca²⁺ responsiveness of the myofibrillar apparatus at relatively low doses (21). We also developed a technique to measure [Ca²⁺]_i rather than contracture force (20). This study was designed, therefore, to fill gaps in our knowledge concerning the effects in the heart of isoflurane, relative to halothane. We did this by 1) using the technique of iontophoretically injecting fura 2 salt to report [Ca²⁺]_i in intact myocardium; 2) reexamining the question of whether isoflurane, compared with halothane, exerts more or less depression on the Ca²⁺ responsiveness of the myofibrillar apparatus; 3) determining whether use of a contracture response to reveal the effects of rapid cooling on SR Ca²⁺ content is valid using isoflurane; and 4) evaluating the effects of isoflurane, compared with halothane, on the mechanism of interaction with the SR Ca²⁺ release channel during the rapid-cooling response. Our results suggest that vapor agents cause negative inotropic actions in the heart by different mechanisms; thus these agents can be selective in their actions on specific targets rather than exerting largely nonspecific effects, just as they may do in neurons (15).

Some of the major new findings in this study can be appreciated by a careful examination of the results shown in Fig. 3. The steady-state peak twitch force versus peak [Ca²⁺]_i transient shown by isoflurane falls intermediate between those relationships produced by lowering [Ca²⁺]_o or by treatment with BDM. Treatment with BDM (up to 10 mM) produces a nearly vertical relationship, indicating a nearly pure downstream action at the cross-bridge level with very little effect on peak [Ca²⁺]_i. This is in agreement with a previous report (1). In contrast, lowering [Ca²⁺]_o also produces a nearly linear relationship, but in this case peak force and peak [Ca²⁺]_i transient both tend to zero as the [Ca²⁺]_o drops to near zero. That is, in the case of lowering [Ca²⁺]_o, the effect on peak twitch force is mainly upstream on the Ca²⁺ transient with little effect at the cross-bridge level. The boundaries of the two relationships set by lowering [Ca²⁺]_o and treatment with BDM should serve to define the mechanism of action of any vapor agent having negative inotropic effects unless quite different mechanisms are involved. In fact, we have found that the dose-response relationships of both halothane and isoflurane fall within the boundary lines defined by [Ca²⁺]_o and BDM, as shown in Fig. 3. The simplest interpretation of these results is that both halothane and isoflurane act by combinations of depressant upstream and downstream actions.

The specific combinations cannot, however, be the same as evidenced by the different relationships for peak twitch force versus peak [Ca²⁺]_i transient for halothane and isoflurane shown in Fig. 3. This is so even though the isoflurane doses were adjusted to be equipotent to those of halothane, except for the highest dose, 1.2 mM. For both agents, extrapolation of the relationships would indicate that active force generation would be abolished considerably before the [Ca²⁺]_i transient disappeared. This should focus interest on the downstream effects of these agents, which does not seem in the past to have been sufficiently emphasized (8). Even though the relationships in Fig. 3 for peak twitch force versus peak [Ca²⁺]_i transient for halothane and isoflurane fall in between those for lowering [Ca²⁺]_o and treatment with BDM, the slope of the relationship for isoflurane is closer to that for lowering [Ca²⁺]_o, whereas the slope for halothane is closer to that for BDM. This indicates that at equipotent doses, isoflurane depresses contractility through a reduction of the responsiveness of the contractile system to [Ca²⁺]_i less than does halothane.

One pertinent question is whether the reduction of the peak [Ca²⁺]_i transient caused by isoflurane is due to a diminished SR Ca²⁺ content. A common approach to estimate SR Ca²⁺ content is by using rapid cooling (5). The difficulty using this approach when isoflurane is applied is that isoflurane depresses the contracture induced by rapid cooling (24). Because isoflurane also reduces the responsiveness of the contractile system to [Ca²⁺]_i, it is difficult to estimate how much the reduction in contracture induced by rapid cooling is caused by reduction of SR Ca²⁺ content. Here, we measured both [Ca²⁺]_i and the contracture in response to rapid cooling. The new and striking findings in this work are shown in Figs. 4-7, because previous results indicating some kind of blocking action of isoflurane (or sevoflurane) on the rapid-cooling response were obtained using contracture force as the index of Ca²⁺ release (24, 31). Here, we present strong evidence indicating that isoflurane has a specific and unique effect to block the release of Ca²⁺ from the SR induced by rapid cooling. This effect of isoflurane is in complete contrast to that of halothane, since we have shown that halothane does not block the rise in [Ca²⁺]_i signaling, a release of Ca²⁺ from the SR initiated by rapid cooling (21). Our result indicates that isoflurane eliminates the contracture induced by rapid cooling by blocking the Ca²⁺ release from the SR rather than through reduction of the responsiveness of the contactile system to [Ca²⁺]_i, and that isoflurane is very different from halothane with respect to the rapid-cooling response regardless of the particular mechanisms of release involved.

The path through which Ca²⁺ is released from the SR, which is affected by both rapid cooling and caffeine, is most likely the Ca²⁺-release channel or the ryanodine receptor (10, 34). It is also known that the SR contains an ATP-dependent Ca²⁺ uptake pump, which, if run in the "reverse" mode, can cause ATP synthesis and Ca²⁺ release from the SR (3, 16). In isolated single Ca²⁺-release channels incorporated into lipid bilayers, temperature reduction causes an increase in the Ca²⁺ current through this channel, and this is interpreted as the basis for the rapid-cooling response observed in cardiac muscle preparations (33). However, other experiments using isolated preparations of cardiac SR indicate that rapid cooling-induced liberation of Ca²⁺ could still be observed, even though the SR channels were blocked with ryanodine or ruthenium red, and it was suggested that net Ca²⁺ release might take place through the SR Ca²⁺ pump (12). Perhaps the lipid bilayer experiments suffer from the presence of an artificial environment, whereas the isolated SR experiments are hindered by the time course of the temperature change, which is on a scale of minutes rather than within seconds. As can be seen in our Fig. 4, our rapid cooling took place in <1 s, and the rapid upstroke of the subsequent increase in [Ca²⁺]_i was also nearly complete in ~1 s, i.e., the changes in [Ca²⁺]_i were of speed and magnitude comparable to those seen in the previous electrically induced twitches.

The important observation here is that isoflurane nearly completely blocks Ca²⁺ release from the SR induced by rapid cooling, but it affects to much less extent the electrically induced Ca²⁺ release, as indicated by the peak [Ca²⁺]_i transient associated with a twitch. This implies that normal SR Ca²⁺ release channel function is well preserved during treatment with isoflurane. Therefore, it was possible that caffeine-induced Ca²⁺ release could still be used to estimate SR Ca²⁺ content in the presence of isoflurane. We found that caffeine (30 mM) added to the standard rapid-cooling solution caused SR Ca²⁺ release even in the presence of 0.62 mM isoflurane (Fig. 6). However, the rate of the [Ca²⁺]_i upstroke induced by rapid cooling plus caffeine in the presence of isoflurane is much slower than that by rapid cooling alone without isoflurane. Most likely, rapid cooling plus caffeine causes abrupt Ca²⁺ release by simply slowing the removal of Ca²⁺ from the myoplasm. We conclude that caffeine can be used to assay SR Ca²⁺ content in the presence of isoflurane.

With the use of rapid cooling plus caffeine, the effect of isoflurane on SR Ca²⁺ content was estimated. In the presence of 0.62 mM isoflurane, the peak value of [Ca²⁺]_i is 71% of that induced by rapid cooling alone in the absence of isoflurane compared with precontrol (Fig. 7B), although it is not statistically different from postcontrol. Because the Ca²⁺ release rate induced by rapid cooling plus caffeine in the presence of isoflurane is much slower than that of rapid cooling alone for the control, it will take longer to reach a peak as shown in Fig. 7C. Therefore, using the peak value alone may underestimate the SR Ca²⁺ content in the presence of isoflurane, because the [Ca²⁺]_i transient depends on the net difference between the rates of Ca²⁺ release into and removal from the myoplasm. Thus the slower Ca²⁺ release rate may allow more Ca²⁺ removal from the myoplasm with a consequent decrease in the peak height. In addition, even at 1.2 mM isoflurane, which is much higher than that used clinically, isoflurane did not cause a detectable elevation of the diastolic [Ca²⁺]_i. This indicates that isoflurane at clinically relevant doses does not cause an apparent Ca²⁺ leak from the SR, and that reduction of the SR Ca²⁺ content is not the major mechanism by which isoflurane exerts its negative inotropic effect.

For our purposes here, it does not matter by which precise mechanism rapid cooling induces Ca²⁺ release from the SR, although this is an important unresolved question. Our main point here is that isoflurane inhibits the rapid-cooling response in intact trabeculae, whereas halothane does not, using the intracellular fura 2 signal rather than contracture alone as an indicator of a rise in [Ca²⁺]_i. This strongly suggests a specific differential action by two vapor agents on a key intracellular organelle, the SR. This finding is in agreement with other recent ones from the central nervous system, indicating that vapor anesthetic agents can be selective in their actions possibly by binding directly to critical proteins to exert their effects (13, 28). This view is in contrast to the long-held one that vapor agents act in a nonspecific physicochemical way to perturb membrane lipids and thus exert their effects in both the brain and the heart (15). Further study is required to elucidate the exact mechanism by which isoflurane can specifically block the rapid-cooling response in intact trabeculae.