Background

In human ventricular myocardium, contractile force increases at higher stimulation frequencies (positive force-frequency relation). In failing hearts, the force-frequency relation (FFR) is negative. Data on the effect of volatile anesthetics on FFR are very limited.

Methods

The authors obtained left ventricular tissue from 18 explanted hearts from patients undergoing cardiac transplantation and tissue of 8 organ donors. The negative inotropic effect of halothane, isoflurane, and sevoflurane on isometric force of contraction of isolated muscle preparations at a stimulation frequency of 1 and 3 Hz and the effect of each anesthetic on the FFR were studied. Ryanodine and verapamil were studied for comparison. In addition, the effect of the anesthetics on Ca(2+)-dependent (3)H-ryanodine binding was investigated.

Results

In nonfailing myocardium, halothane was the strongest negative inotropic compound, and the positive FFR was not affected by either drug. In failing myocardium, halothane also showed the strongest negative inotropic effect, but the positive shape of FFR was restored by halothane and ryanodine. In contrast, isoflurane, sevoflurane, and verapamil did not change FFR. Only halothane shifted the Ca(2+)-dependent (3)H-ryanodine binding curve toward lower Ca(2+) concentrations.

Conclusion

In nonfailing human myocardium, none of the anesthetics affect FFR, but halothane is the strongest negative inotropic compound. In failing myocardium, halothane, but not isoflurane or sevoflurane, restores the positive shape of FFR. Both the more pronounced negative inotropic effect of halothane and the restoration of the positive shape of FFR in failing myocardium in the presence of halothane can be explained by its interaction with the myocardial sarcoplasmic reticulum calcium-release channel.

THE cardiodepressant effect of volatile anesthetics can be explained by an inhibition of the Ca2+-inward current, 1Ca2+depletion of the sarcoplasmic reticulum (SR) by an interaction with the Ca2+-release channel of the SR, 2,3and by a reduction of the Ca2+sensitivity of the myofibrils. 4Beyond these changes of the excitation–contraction coupling, volatile anesthetics interact with other regulatory mechanisms controlling contractile force. For example, the anesthetics modulate the β-adrenergic signal transduction pathway. 5,6 

A further important mechanism that regulates myocardial contractility in the heart is the force–frequency relation (FFR). In healthy human isolated myocardium, an increase in stimulation frequency results in an increase in developed contractile force. The positive FFR could also be demonstrated in vivo  7and is crucial for the adaptation of the heart during exercise. Several investigators have demonstrated a flat or even negative FFR in human myocardium of patients suffering from heart failure 8or cardiac hypertrophy. 9The negative FFR has been suggested to contribute to the maladaptation of the heart at high rates. Accordingly, a reduction of heart rate may improve cardiac performance in patients with depressed cardiac function. 10Whether this improvement of myocardial contractility at low frequencies also occurs during anesthesia is currently not known.

The effect of halogenated anesthetics on mechanical restitution has been addressed by a number of studies showing that halothane inhibits the postrest potentiation of contractile force by impairing the function of the SR. 11However, data on the interaction of volatile anesthetics with the FFR in healthy or diseased human myocardium are very limited. In one study, halothane restored the positive shape of the FFR in failing human myocardium. 12However, it is not known whether the halogenated anesthetics differentially alter the FFR in human myocardium or whether this depends on changes present in failing myocardium. Moreover, the negative inotropic effect of sevoflurane has only been evaluated in human atrial myocardium. 13 

In the light of the considerable differences in cardiac physiology in different species, it appears necessary to evaluate the mechanisms of cardiac depression by volatile anesthetics and their interaction with the FFR in human cardiac tissue. Accordingly, the importance of studies on human tissue samples has recently been stressed by several investigators. 13,14 

In the current study, we compared the negative inotropic effects of halothane, isoflurane, and sevoflurane on isolated ventricular human myocardium from healthy organ donors and from patients with end-stage heart failure at different stimulation frequencies. In search for an explanation for the different extent of the negative inotropic effects, we studied the influence of the three anesthetics on the Ca2+sensitivity of the human Ca2+-release channel of the SR.

Methods

Patients

Left ventricular myocardium was obtained from 18 patients suffering from end-stage heart failure. The patients underwent cardiac transplantation as a result of dilated (n = 10) or ischemic (n = 8) cardiomyopathy. Nonfailing left ventricular myocardium was obtained from eight multiorgan donors whose hearts could not be transplanted for technical reasons. All organ donors showed a normal left ventricular pump function as shown by echocardiography (ejection fraction > 55%), and none of the donors had any pathologic cardiovascular history. Because brain death may lead to a pronounced depression of cardiac performance, all patients with brain death–induced cardiac dysfunction were excluded. 15The study was approved by the institutional review board on human research of the University Hospital, Aachen. All patients gave written informed consent. In case of the organ donors, consent was obtained from the relatives.

Contraction Experiments

Immediately after surgical resection, the tissue was placed into bathing solution (2.5 mm CaCl2, 119.8 mm NaCl, 1.04 mm MgCl2, 5.36 mm KCl, 22.6 mm NaHCO3, 0.42 mm NaH2PO4, 5.05 mm glucose, 0.28 mm ascorbic acid, 0.05 mm Na2EDTA, pH 7.4, gassed with 5% CO2–95% O2, room temperature) and transferred to the laboratory. In parallel to muscle fiber direction, thin myocardial strips were prepared under stereomicroscopic control. The diameters of the muscle preparations were less than 0.6 mm to avoid core hypoxia and were comparable to those used by other investigators in similar experiments. 16The muscle preparations were transferred into an organ bath filled with prewarmed (37°C) bathing solution and electrically stimulated at 1 Hz with rectangular impulses (5-ms duration, 5–10% above threshold voltage). After an equilibration period of 30 min, the muscles were stretched by increasing the resting tension from 1 mN stepwise by 0.5 mN until the muscle length providing maximal active force generation was reached. Table 1shows the main mechanical characteristics of the muscle preparations. In a first group of muscle preparations, the concentration-dependent negative inotropic effect of the volatile anesthetics was studied (10–12 muscle preparations from 5 donor hearts and 22–23 muscle preparations from 13 failing hearts). The anesthetics were added to the carbogen using vaporizers (Dräger [Lübeck, Germany] for halothane and sevoflurane or Ohmeda [Herts, United Kingdom] for isoflurane) and bubbled through the bathing solution. Gas chromatography measurements revealed that equilibration of the anesthetics with the bathing solution was complete within 5 min. The concentration of the anesthetics was increased in steps of 0.5 minimum alveolar concentration (MAC) up to 3 MAC for isoflurane and halothane. In the case of sevoflurane, 2.5 MAC was the maximal concentration that could be delivered by the vaporizer. If force of contraction after wash-out of the anesthetic was less than 5% lower than the predrug value, the next anesthetic was studied. Halothane, isoflurane, and sevoflurane were studied in a random order. In a second group of muscle preparations, the effect of the anesthetics on the FFR was studied (10–12 muscle preparations from 5 donor hearts and 19–24 muscle preparations from 13 failing hearts). The stimulation frequency was increased from 0.5 to 3 Hz at control conditions, at 1.5 MAC, and at 2.5 MAC of each anesthetic. In 12 muscle preparations from 5 donor hearts and 20 muscle preparations from 13 failing hearts, the effect of ryanodine (10 nm) and verapamil (100 nm) on the FFR was studied for comparison. The effect of low Ca2+concentrations in the bathing solution (1.5 and 0.5 mm) on the FFR was studied in 10 muscle preparations from 3 failing hearts.

Table 1. Dimensions and Baseline Forces (37°C, 1 Hz) of All Muscle Preparations

*P < 0.05 versus  nonfailing myocardium.

Table 1. Dimensions and Baseline Forces (37°C, 1 Hz) of All Muscle Preparations
Table 1. Dimensions and Baseline Forces (37°C, 1 Hz) of All Muscle Preparations

To exclude that hypoxia occurs in the preparations, at the end of the experiment the muscle strips were kept in a condition with a high oxygen demand (i.e. , a high stimulation frequency). After force of contraction had reached steady state, the gas bubbling (95% O2, 5% CO2) was changed to 80% O2plus 15% N2and 5% CO2for a period of 30 min. All muscle preparations showing a decline in force of contraction of more than 5% during this period were excluded from the study (< 5% of all muscle preparations). 17 

3H-ryanodine Binding

Immediately after explantation, the myocardium was cut into small pieces, immersed in liquid nitrogen, and stored at −80°C until use. 3H-ryanodine binding experiments were performed as previously described. 18Briefly, 70 mg myocardium of each heart were homogenized three times for 15 s with an Ultraturrax (IKA, Staufen, Germany) and afterward with 25 bursts with a glas potter at 1,100 rpm in 3 ml ice-cold buffer A: 1 m KCl, 20 mm HEPES, and 1 mm EGTA at pH 7.4. For saturation binding experiments, the free Ca2+concentration was adjusted to 105m according to the calculations of Fabiato. 19Radioligand binding was started by addition of 150 μl of the heart homogenates (100 μg protein) to 100 μl buffer A containing 0.3–40 nm 3H-ryanodine. The reaction was stopped by the addition of 5 ml ice-cold buffer A, immediately followed by rapid filtration through Whatman GF/C filters. The filters were washed twice with 5 ml buffer A and counted after the addition of 5 ml Hionic Fluor (Canberra-Packard, Dreieich, Germany). Nonspecific radioactivity was determined by the addition of 10 μm unlabeled ryanodine into the binding assays and was approximately 15% at 1 nm 3H-ryanodine. To investigate Ca2+-dependent 3H-ryanodine binding, 100 μg protein was incubated with 12 nm 3H-ryanodine in the presence of 15 different Ca2+concentrations ranging from 109to 101m. Ca2+ions were buffered with 1 mm EGTA and adjusted to the respective final free concentrations according to Fabiato. 19All other steps were performed as described previously. To study the effect of volatile anesthetics on Ca2+-dependent binding, the membrane preparation was gassed for 15 min with anesthetic in nitrogen or with pure nitrogen for control to avoid oxidative alterations of membrane lipids. The radioligand binding experiments were performed in anesthetic–nitrogen atmosphere or pure nitrogen, respectively. As determined by gas chromatography, equilibration of anesthetic concentration in the medium was achieved within 15 min. In these experiments, the assays were performed in the presence of 10 different Ca2+concentrations ranging from 108to 105m.

Protein content of the homogenates was measured by the method of Bradford 20using γ-globulin as standard.

Materials

3H-ryanodine (68.4–74.8 Ci/mmol) was obtained from NEN DuPont de Nemours (Bad Homburg, Germany), and ryanodine was obtained from Calbiochem-Novabiochem (Bad Soden, Germany). All chemicals were of analytical or best commercial grade available. Deionized water was used throughout.

Statistical Analysis

Saturation curves, Ca2+-dependent 3H-ryanodine binding curves, and Scatchard plots 21were fitted with computer-assisted least square regression analysis using software designed by Graphpad (San Diego, CA). Data are expressed as mean ± SD. For EC50and EC40values, 95% confidence intervals are given. Statistical significance was determined with the unpaired Student t  test (for comparison of two groups) or by one-way analysis of variance with Newman-Keuls test for comparison of multiple groups. A P  value < 0.05 was considered statistically significant.

Results

Negative Inotropic Effect of Halogenated Anesthetics

A total of 34 muscle preparations from 5 donor hearts (in 3 donors only frozen tissue was obtained) and 68 preparations from 16 failing hearts were studied. Length, diameter, and resting force at the maximal active force generation of the preparations did not differ between nonfailing and failing myocardium, whereas the active force at a stimulation frequency of 1 Hz and the ratio between active and resting force were significantly higher in nonfailing myocardium (table 1).

Figure 1(top) shows the concentration-dependent negative inotropic effect of all three anesthetics in left ventricular myocardium of five organ donors. The negative inotropic effect of halothane was more pronounced than that of isoflurane or sevoflurane. At a physiologic rate (1 Hz), 0.72 MAC halothane reduced contractile force by 40% (EC40), whereas approximately 1.4 MAC isoflurane or sevoflurane was needed to elicit the same effect (P < 0.05;table 2). This difference was even more pronounced at a stimulation frequency of 3 Hz. Interestingly, only the first part of the concentration–response curve of halothane, at concentrations up to 1.5 MAC, was steeper compared with the other compounds. At greater than 1.5 MAC, the contractile force decreased parallel to the decrease with isoflurane and sevoflurane. In failing human myocardium, at a stimulation frequency of 1 Hz, the negative inotropic effect of halothane was also most pronounced (fig 1, bottom). Approximately 0.5 MAC halothane was sufficient to reduce contractile force by 40% (table 2). Similar as in nonfailing myocardium, the negative inotropic potency of isoflurane and sevoflurane was lower (P < 0.05;table 1). Again, only the initial part of the concentration–response curve of halothane was steeper. At concentrations greater than 1.5 MAC, force of contraction decreased parallel in all three anesthetics. At 3 Hz, the negative inotropic effect of all three anesthetics was similar. The concentration–response curves were nearly superimposable. Forty percent reduction of contractile force was reached at equianesthetic concentrations of halothane, isoflurane, and sevoflurane (nonsignificant;table 2). Interestingly, the negative inotropic effect of halothane was much less pronounced at 3 Hz compared with 1 Hz.

Fig. 1. Negative inotropic effect of anesthetics on force of contraction of nonfailing (10–12 preparations, 5 organ donors, top ) and failing myocardium (22–23 preparations from 13 patients, bottom ) at 1 or 3 Hz stimulation frequency. *P < 0.05. Hal = halothane; Iso = isoflurane; Sevo = sevoflurane; MAC = minimum alveolar concentration.

Fig. 1. Negative inotropic effect of anesthetics on force of contraction of nonfailing (10–12 preparations, 5 organ donors, top ) and failing myocardium (22–23 preparations from 13 patients, bottom ) at 1 or 3 Hz stimulation frequency. *P < 0.05. Hal = halothane; Iso = isoflurane; Sevo = sevoflurane; MAC = minimum alveolar concentration.

Table 2. Negative Inotropic Potency of Volatile Anesthetics

Concentration of each anesthetic (times minimum alveolar concentration) eliciting a 40% reduction in contractile force (EC40value).

*P < 0.05 versus  halothane.

Table 2. Negative Inotropic Potency of Volatile Anesthetics
Table 2. Negative Inotropic Potency of Volatile Anesthetics

Effect of Halogenated Anesthetics on Force–Frequency Relation

Figure 2shows the influence of the anesthetics on the FFR. In nonfailing myocardium and in the absence of anesthetics (baseline), the FFR was positive. All three anesthetics reduced contractile force equally at all stimulation frequencies, with halothane being the strongest compound, but none of them changed the positive shape of the FFR. In contrast, in failing myocardium, the FFR was differentially affected by the anesthetics. In the absence of anesthetics, the FFR was negative in failing myocardium. As previously mentioned, the negative inotropic effect of halothane was relatively strong at 1 Hz, whereas it was much less pronounced at 3 Hz. As a result, the shape of the FFR became positive in the presence of halothane, although the baseline FFR was negative. The restoration of the positive shape of the FFR could be demonstrated at 1.5 and 2.5 MAC halothane. In contrast, isoflurane and sevoflurane did not alter the negative shape of the FFR.

Fig. 2. Effect of 1.5 and 2.5 minimum alveolar concentration (MAC) of each anesthetic on the force–frequency relation in nonfailing (10–12 preparations from 5 donor hearts) and failing myocardium (19–24 muscle preparations, 13 patients). *P < 0.05. Hal = halothane; Iso = isoflurane; Sevo = sevoflurane.

Fig. 2. Effect of 1.5 and 2.5 minimum alveolar concentration (MAC) of each anesthetic on the force–frequency relation in nonfailing (10–12 preparations from 5 donor hearts) and failing myocardium (19–24 muscle preparations, 13 patients). *P < 0.05. Hal = halothane; Iso = isoflurane; Sevo = sevoflurane.

Effects of Verapamil, Ryanodine, and Low Ca2+Concentrations on Force–Frequency Relation

In nonfailing myocardium, neither verapamil nor ryanodine changed the positive shape of the FFR, although both drugs showed a strong negative inotropic effect (fig. 3). In failing myocardium, verapamil also did not alter the (negative) shape of the FFR, but blockade of the SR by ryanodine reduced contractile force preferentially at low stimulation frequencies. Similar to halothane, ryanodine restored the positive shape of the FFR. In failing myocardium, lowering the availability of Ca2+for the contraction by low Ca2+concentrations in the bathing solutions (1.5 and 0.5 mm) did not change the shape of the FFR despite the pronounced negative inotropic effect (table 3).

Fig. 3. Effect of verapamil (Vera; 100 nm) and ryanodine (Rya; 10 nm) on the force–frequency relation in nonfailing (12 muscle preparations, 5 organ donors) and failing (20 muscle preparations, 13 patients) myocardium. Forces in nonfailing myocardium at 0.5 Hz were 12.4 ± 4.9 mN/mm2(baseline), 3.0 ± 1.8 mN/mm2in the presence of ryanodine (P < 0.05 vs.  baseline), and 5.8 ± 2.9 mN/mm2in the verapamil group (P < 0.05 vs.  baseline). Forces in failing myocardium at 0.5 Hz were 11.3 ± 4.3 mN/mm2(baseline), 1.8 ± 1.0 mN/mm2in the presence of ryanodine (P < 0.05 vs.  baseline), and 7.0 ± 2.7 mN/mm2in the verapamil group (P < 0.05 vs.  baseline and ryanodine). *P < 0.05.

Fig. 3. Effect of verapamil (Vera; 100 nm) and ryanodine (Rya; 10 nm) on the force–frequency relation in nonfailing (12 muscle preparations, 5 organ donors) and failing (20 muscle preparations, 13 patients) myocardium. Forces in nonfailing myocardium at 0.5 Hz were 12.4 ± 4.9 mN/mm2(baseline), 3.0 ± 1.8 mN/mm2in the presence of ryanodine (P < 0.05 vs.  baseline), and 5.8 ± 2.9 mN/mm2in the verapamil group (P < 0.05 vs.  baseline). Forces in failing myocardium at 0.5 Hz were 11.3 ± 4.3 mN/mm2(baseline), 1.8 ± 1.0 mN/mm2in the presence of ryanodine (P < 0.05 vs.  baseline), and 7.0 ± 2.7 mN/mm2in the verapamil group (P < 0.05 vs.  baseline and ryanodine). *P < 0.05.

Table 3. Effect of Low Ca2+Concentrations on the FFR in Failing Human Myocardium

*P < 0.05 versus  2.5 mm Ca2+.

P < 0.05 versus  0.5 Hz.

FFR = force–frequency relation.

Table 3. Effect of Low Ca2+Concentrations on the FFR in Failing Human Myocardium
Table 3. Effect of Low Ca2+Concentrations on the FFR in Failing Human Myocardium

3H-ryanodine Binding Studies

Binding of the radioligand 3H-ryanodine to left ventricular homogenates showed saturation characteristics (fig. 4A), and the Scatchard plot (fig. 4B) was linear, indicating that 3H-ryanodine bound to one distinct receptor binding site. Similar to our previous observation, 18the 3H-ryanodine binding site density was the same in nonfailing (86 ± 11 fmol/mg protein; n = 8) and failing myocardium (91 ± 30 fmol/mg protein; n = 11; nonsignificant).

Fig. 4. (A ) Representative saturation binding of 3H-ryanodine to a homogenate of left ventricular tissue of a patient with dilated cardiomyopathy as a function of increasing 3H-ryanodine concentrations. Binding was performed at 7 concentrations of 3H-ryanodine ranging from 0.5 to 38 nm in the absence (total binding) and in the presence (nonspecific binding) of 10 μm unlabeled ryanodine to determine specific binding. Free Ca2+concentration was 105m. (B ) Scatchard analysis of specific 3H-ryanodine binding of the experiment shown in (A ). The ratio of specifically bound 3H-ryanodine to free 3H-ryanodine is plotted as a function of specifically bound 3H-ryanodine. (C ) Bell-shaped pattern of Ca2+-dependent 3H-ryanodine binding to a homogenate of the same specimen. Binding was performed at a saturating concentration of 3H-ryanodine (12 nm).

Fig. 4. (A ) Representative saturation binding of 3H-ryanodine to a homogenate of left ventricular tissue of a patient with dilated cardiomyopathy as a function of increasing 3H-ryanodine concentrations. Binding was performed at 7 concentrations of 3H-ryanodine ranging from 0.5 to 38 nm in the absence (total binding) and in the presence (nonspecific binding) of 10 μm unlabeled ryanodine to determine specific binding. Free Ca2+concentration was 105m. (B ) Scatchard analysis of specific 3H-ryanodine binding of the experiment shown in (A ). The ratio of specifically bound 3H-ryanodine to free 3H-ryanodine is plotted as a function of specifically bound 3H-ryanodine. (C ) Bell-shaped pattern of Ca2+-dependent 3H-ryanodine binding to a homogenate of the same specimen. Binding was performed at a saturating concentration of 3H-ryanodine (12 nm).

A representative example of Ca2+-dependent 3H-ryanodine binding to the human Ca2+-release channel is shown in figure 4C. 3H-ryanodine binding showed a bell-shaped pattern with an increase in specific binding at submicromolar Ca2+concentrations and a decrease at Ca2+concentrations higher than 0.5 mm.

Because it has been shown that increasing specific 3H-ryanodine binding at submicromolar Ca2+concentrations reflects increasing open probability of the channel and the role of decreasing 3H-ryanodine binding at Ca2+concentrations in the millimolar range is still unknown, in the following experiments we concentrated on Ca2+concentrations ranging from 108to 105m. Ca2+-dependent 3H-ryanodine binding to the SR Ca2+-release channel from nonfailing and failing human myocardium showed a similar pattern, and EC50values of the increase in specific 3H-ryanodine binding were the same in nonfailing and failing myocardium (table 4). Figure 5shows the influence of the halogenated anesthetics (1.5 MAC) on the Ca2+-dependent 3H-ryanodine binding. Halothane shifted the curve toward lower Ca2+concentrations. The extent of this effect was similar in failing and nonfailing myocardium. In contrast, isoflurane and sevoflurane (1.5 and 2.5 MAC) did not alter Ca2+-dependent 3H-ryanodine binding. In addition, the maximal binding of ryanodine was not changed by either compound. Table 4shows the statistical analysis of the binding data. Halothane at 2.5 MAC did not provoke a further shift of the binding curve to the left. Both in nonfailing and failing myocardium, the maximal effect of halothane on the SR Ca2+-release channel was already reached at 1.5 MAC.

Table 4. Effect of Volatile Anesthetics on Ca2+-dependent 3H-ryanodine Binding

Maximal specific binding (Bmax) and EC50values of Ca2+-dependent 3H-ryanodine binding. Bmaxare given as mean ± SD, and EC50values are given with 95% confidence intervals.

*P < 0.05 versus  baseline.

MAC = minimum alveolar concentration.

Table 4. Effect of Volatile Anesthetics on Ca2+-dependent 3H-ryanodine Binding
Table 4. Effect of Volatile Anesthetics on Ca2+-dependent 3H-ryanodine Binding

Fig. 5. Ca2+-dependent 3H-ryanodine binding to homogenates from nonfailing (n = 8) and failing (n = 11) myocardium at Ca2+concentrations ranging from 108to 105m. Hal = halothane; Iso = isoflurane; Sevo = sevoflurane.

Fig. 5. Ca2+-dependent 3H-ryanodine binding to homogenates from nonfailing (n = 8) and failing (n = 11) myocardium at Ca2+concentrations ranging from 108to 105m. Hal = halothane; Iso = isoflurane; Sevo = sevoflurane.

Discussion

The current study provides a direct comparison of the negative inotropic effects of halothane, isoflurane, and sevoflurane in nonfailing and failing ventricular myocardium at different stimulation frequencies. We demonstrated that in both failing and nonfailing myocardium, halothane exerts the most pronounced negative inotropic effect. In failing myocardium, halothane and ryanodine restored the positive shape of the FFR, whereas isoflurane and sevoflurane did not change FFR. Only halothane activated the myocardial Ca2+-release channel of the SR, which offers an explanation for the differential effect of volatile anesthetics on the FFR and for the more pronounced negative inotropic effect of halothane.

Interaction of Volatile Anesthetics with the Sarcoplasmic Reticulum Ca2+-Release Channel

As recently reported, 18Ca2+-dependent specific 3H-ryanodine binding can be used as a probe for the activation of the human cardiac SR Ca2+-release channel. The plant alkaloid ryanodine only binds to the open state of the channel. 22Accordingly, a good correlation between channel activation by Ca2+(i.e. , opening of the channel) and increased specific 3H-ryanodine binding at submicromolar Ca2+concentrations has been reported during various conditions. 22–24Although 3H-ryanodine binding studies are an indirect method to assess channel activation, there is increasing evidence that Ca2+-dependent 3H-ryanodine binding reflects Ca2+-induced opening of the channel. 22 

As expected, specific 3H-ryanodine binding was absent at nanomolar Ca2+concentrations, indicating that the channel is closed during these conditions. At submicromolar Ca2+concentrations, 3H-ryanodine binding increased reflecting enhanced open probability of the channel. The Ca2+concentration eliciting the half-maximal 3H-ryanodine binding can be used as a measure of the Ca2+sensitivity of the channel. 21The increase of Ca2+-dependent 3H-ryanodine binding could be observed at lower Ca2+concentrations when 1.5 MAC halothane was present, indicating that halothane increased the Ca2+sensitivity. Halothane at 3.0 MAC did not provoke a further leftward shift of the Ca2+-dependant 3H-ryanodine binding curve, indicating that relatively low halothane concentrations already elicit the maximal effect. In contrast, isoflurane and sevoflurane did not change the Ca2+sensitivity of the SR Ca2+-release channel.

The increased Ca2+sensitivity of the channel in the presence of halothane will increase the average open probability of the channel 25as a response to physiologic variations in intracellular Ca2+concentrations. It has been suggested that this effect leads to a decrease in the ability of the SR to store and accumulate Ca2+and thus to a Ca2+depletion of the SR. 3 

Comparison of the Negative Inotropic Effect of Volatile Anesthetics

Numerous studies in various mammalian species demonstrated a pronounced, concentration-dependent, and reversible cardiodepressant effect of all halogenated anesthetics. 5,26,27Because important physiologic differences exist between animal myocardium and human cardiac tissue, 28especially with regard to the FFR, 29there is a need for studies on the inotropic effect of the anesthetics in human myocardium. Only very recently was an extensive work on the differential negative inotropic effect of the most frequently used volatile anesthetics on isolated atrial human myocardium published. 13It showed that the negative inotropic effect of halothane was more pronounced than that of isoflurane, sevoflurane, and desflurane. Some properties of electrical and mechanical function in ventricular myocardium, however, might differ from the behavior of atrial myocardium. Action potential duration 30and contraction times 31have been shown to be shorter in atrial myocardium, and isometric force development is lower than in ventricular myocardium. 31Moreover, altered Ca2+homeostasis in failing human myocardium 32might have an influence on the negative inotropic mechanism of volatile anesthetics. Our data demonstrate that, in failing and nonfailing human ventricular myocardium, the negative inotropic effect of halothane is more pronounced than that of isoflurane and sevoflurane, which depress myocardial contractility to a similar extent. Interestingly, the slope of the concentration–response curve of halothane was steepest at low concentrations up to 1.5 MAC, resulting in a higher negative inotropic potency (lower EC40value). At concentrations greater than this, contractile force declined parallel with the negative inotropic effect of isoflurane and sevoflurane. Because 1.5 MAC halothane exerts the maximal effect on the Ca2+sensitivity of the SR Ca2+-release channel, these data suggest that the initial steep decline of contractile force is mainly caused by activation of the SR Ca2+-release channel and subsequent Ca2+depletion of the SR. At concentrations greater than 1.5 MAC, no further activation of the SR Ca2+-release channel occurs, and all anesthetics reduce force of contraction with a similar slope of the concentration–response curve, probably by inhibition of the Ca2+inward current via  the L-type Ca2+channel. 33Isoflurane and sevoflurane do not interact with the SR Ca2+-release channel and therefore do not show the steep decrease in contractile force at low concentrations of the anesthetics.

Effect of Volatile Anesthetics on Force–Frequency Relation

Schmidt et al.  12reported that halothane restores the positive FFR in human ventricular myocardium and proposed that prevention of diastolic Ca2+overload by the antagonistic effects of halothane on L-type Ca2+channels or activation of the SR Ca2+-release channel might have caused the restoration of the FFR. Moreover, a decrease of the Ca2+sensitivity of myofilaments might contribute to the phenomenon, but the exact molecular mechanism remained unclear. In concordance with the findings of Schmidt et al. , 12we also found that the positive shape of the FFR is restored by halothane in failing human myocardium. The comparison with other volatile anesthetics and with the effect of verapamil and ryanodine allow further conclusions to be drawn on the intracellular mechanism. With respect to inhibition of the Ca2+inward current via  the L-type Ca2+channel, no major differences have been reported between halothane, isoflurane, and sevoflurane. 33Because only halothane restored the positive shape of the FFR in failing human myocardium, the interaction of the anesthetics with L-type Ca2+channels cannot account for this effect of halothane. Accordingly, the Ca2+antagonist verapamil did not influence the shape of the FFR in failing myocardium. Similarly, the low availability of Ca2+itself cannot account for the positive shape of the FFR in the presence of halothane. At low Ca2+concentrations in the bathing solution (1.5 or 0.5 mm), the FFR was still negative in failing myocardium.

A decrease in Ca2+sensitivity of the myofilaments also cannot explain why halothane, but not isoflurane, alters the FFR in failing myocardium. Halothane and isoflurane depressed the Ca2+sensitivity of human ventricular myofilaments to a similar extent. 4 

A more promising explanation for the restoration of the positive FFR by halothane is its interaction with the SR Ca2+-release channel of the SR. As demonstrated by our 3H-ryanodine radioligand studies, only halothane, but not isoflurane or sevoflurane, increased the Ca2+sensitivity of the Ca2+-release channel, leading to an increased open probability of the channel and Ca2+depletion of the SR. The Ca2+depletion of the SR lowers contractile force, particularly at low stimulation frequencies at which time for Ca2+reuptake into the SR is not limited, and thus the contribution of intracellular Ca2+cycling via  the SR to contractile force is high. Because the activity of the SR Ca2+–adenosine triphosphatase is reduced in failing human myocardium, 34the contribution of SR Ca2+cycling to contractile force becomes less important at higher stimulation frequencies with decreased time for reuptake of Ca2+into the SR. 35This could explain why, in failing human myocardium, halothane decreased contractile force preferentially at low stimulation frequencies, resulting in the restoration of the FFR. The hypothesis is strongly supported by our observation that ryanodine also restored the positive shape of the FFR in failing myocardium. Ryanodine keeps the SR Ca2+-release channel in a 40% subconductance state and thereby blocks the repetitive reuptake and release of Ca2+by the SR, resulting in Ca2+depletion of the intracellular Ca2+stores. 22The similarity of halothane and ryanodine with respect to their interaction with the SR and their effect on the FFR in failing myocardium strongly suggests that halothane restores the FFR by increasing the open probability of the SR Ca2+-release channel, thereby depleting the sarcoplasmic reticular Ca2+stores, leading to a cardiodepressant effect particularly at low stimulation frequencies. Because reduction of the heart rate has been suggested to improve myocardial contractility in patients with heart failure, 10knowledge about the influence of halogenated anesthetics on the FFR might help to improve the clinical outcome of anesthetized patients with depressed myocardial function.

Although the negative inotropic effect of halothane was also more pronounced than that of isoflurane and sevoflurane in nonfailing human myocardium, the positive shape of the FFR was not altered by any anesthetic. Because the activity of the SR Ca2+–adenosine triphosphatase was not depressed, intracellular Ca2+cycling via  the SR strongly contributed to the active force development at all stimulation frequencies. Thus, inhibitors of the sarcoplasmic reticular function are expected to reduce contractile force at all rates. Indeed, our data demonstrate that both ryanodine and halothane depress contractile force to a similar extent at all stimulation frequencies. Accordingly, in nonfailing myocardium, the positive shape of the FFR was maintained in the presence of all drugs.

Limitations

Extrapolation of our data to the situation in vivo  should be performed with caution. Although changes in the shape of the FFR in vitro  go hand-in-hand with respective alterations of the FFR in  vivo, 7the effect of volatile anesthetics on frequency-dependent force development might be influenced by a number of additional factors beyond changes of intrinsic myocardial contractility. Changes of cardiac performance during the administration of anesthetics may also depend on modifications of the physiologic variations in sympathetic and parasympathetic tone, venous return, and vascular tone.

Moreover, the number of specimens from nonfailing hearts was small because of the limited access to appropriate tissue samples. However, in the absence of volatile anesthetics, baseline force of contraction and the shape of the FFR was very similar to results previously reported in similar tissue samples, 8and the variability within our population of muscle preparations was not extraordinarily high. Thus, we suggest that the data obtained from the limited number of specimens investigated here represent the typical contractile behavior of nonfailing myocardium studied during similar conditions.

References

References
1.
Bosnjak ZJ, Supan FD, Rusch NJ: The effects of halothane, enflurane, and isoflurane on calcium current in isolated canine ventricular cells. A nesthesiology 1991; 74: 340–5
2.
Katsuoka M, Ohnishi ST: Inhalation anaesthetics decrease calcium content of cardiac sarcoplasmic reticulum. Br J Anaesth 1989; 62: 669–73
3.
Lynch C, Frazer MJ: Anesthetic alteration of ryanodine binding by cardiac calcium release channels. Biochim Biophys Acta 1994; 1194: 109–17
4.
Tavernier BM, Adnet PJ, Imbenotte M, Etchrivi TS, Reyford H, Haudecoeur G, Scherpereel P, Krivosic HR: Halothane and isoflurane decrease calcium sensitivity and maximal force in human skinned cardiac fibers. A nesthesiology 1994; 80: 625–33
5.
Schotten U, Schumacher C, Sigmund M, Karlein C, Rose H, Kammermeier H, Sivarajan M, Hanrath P: Halothane, but not isoflurane, impairs the beta-adrenergic responsiveness in rat myocardium. A nesthesiology 1998; 88: 1330–9
6.
Schmidt U, Schwinger RH, Böhm M: Interaction of halothane with inhibitory G-proteins in the human myocardium. A nesthesiology 1995; 83: 353–60
7.
Hasenfuss G, Holubarsch C, Hermann HP, Astheimer K, Pieske B, Just H: Influence of the force-frequency relationship on haemodynamics and left ventricular function in patients with non-failing hearts and in patients with dilated cardiomyopathy. Eur Heart J 1994; 15: 164–70
8.
Mulieri LA, Hasenfuss G, Leavitt B, Allen PD, Alpert NR: Altered myocardial force-frequency relation in human heart failure. Circulation 1992; 85: 1743–50
9.
Schotten U, Voss S, Wiederin TB, Voss M, Schoendube F, Hanrath P, Schumacher C: Altered force-frequency relation in hypertrophic obstructive cardiomyopathy. Basic Res Cardiol 1999; 94: 120–7
10.
Böhm M, La Rosee K, Schmidt U, Schulz R, Schwinger RH, Erdmann E: Force-frequency relation and inotropic stimulation in the nonfailing and failing human myocardium: Implications for the medical treatment of heart failure. J Clin Invest 1992; 70: 471–5
11.
Gueugniaud PY, Hanouz JL, Vivien B, Lecarpentier Y, Coriat P, Riou B: Effects of desflurane in rat myocardium: Comparison with isoflurane and halothane. A nesthesiology 1997; 87: 599–609
12.
Schmidt U, Schwinger RH, Böhm M: Halothane restores the altered force-frequency relationship in failing human myocardium. A nesthesiology 1995; 82: 1456–62
13.
Hanouz JL, Massetti M, Guesne G, Chanel S, Babatasi G, Rouet R, Ducouret P, Khayat A, Galateau F, Bricard H, Gérard JL: In-vitro effects of desflurane, sevoflurane, isoflurane, and halothane in isolated human right atria. A nesthesiology 2000; 92: 116–24
14.
Riou B: Halogenated anesthetics and human myocardium. A nesthesiology 2000; 92: 1–2
15.
Riou B, Dreux S, Roche S, Arthaud M, Goarin JP, Leger P, Saada M, Viars P: Circulating cardiac troponin T in potential heart transplant donors. Circulation 1995; 92: 409–14
16.
Pieske B, Kretschmann B, Meyer M, Holubarsch C, Weirich J, Posival H, Minami K, Just H, Hasenfuss G: Alterations in intracellular calcium handling associated with the inverse force-frequency relation in human dilated cardiomyopathy. Circulation 1995; 92: 1169–78
17.
Paradise NF, Schmitter JL, Surmitis JM: Criteria for adequate oxygenation of isometric kitten papillary muscle. Am J Physiol 1981; 241: H348–53
18.
Schotten U, Schumacher C, Conrads V, Braun V, Schöndube F, Voss M, Hanrath P: Calcium-sensitivity of the SR calcium release channel in failing and nonfailing human myocardium. Basic Res Cardiol 1999; 94: 145–51
19.
Fabiato A: Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 1988; 157: 378–417
20.
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–54
21.
Scatchard G: The attraction of proteins for small molecules and ions. Ann NY Acad Sci 1949; 51: 660–72
22.
Coronado R, Morrissette J, Sukhareva M, Vaughan DM: Structure and function of ryanodine receptors. Am J Physiol 1994; 266: C1485–504
23.
Chu A, Díaz-Muñoz M, Hawkes MJ, Brush K, Hamilton SL: Ryanodine as a probe for the functional state of the skeletal muscle sarcoplasmic reticulum calcium release channel. Mol Pharmacol 1990; 37: 735–41
24.
Holmberg SR, Williams AJ: The cardiac sarcoplasmic reticulum calcium-release channel: Modulation of ryanodine binding and single-channel activity. Biochim Biophys Acta 1990; 1022: 187–93
25.
Connelly TJ, Coronado R: Activation of the Ca2+release channel of cardiac sarcoplasmic reticulum by volatile anesthetics. A nesthesiology 1994; 81: 459–69
26.
Komai H, Rusy BF: Negative inotropic effects of isoflurane and halothane in rabbit papillary muscles. Anesth Analg 1987; 66: 29–33
27.
Luk HN, Lin CI, Chang CL, Lee AR: Differential inotropic effects of halothane and isoflurane in dog ventricular tissues. Eur J Pharmacol 1987; 136: 409–13
28.
Schouten VJ, Schipperheyn JJ, Rijk Zwikker GL, Swier GP: Calcium metabolism and depressed contractility in isolated human and porcine heart muscle. Basic Res Cardiol 1990; 85: 563–74
29.
Buckley NM, Penefsky ZJ, Litwak RS: Comparative force-frequency relationships in human and other mammalian ventricular myocardium. Pflugers Arch 1972; 332: 259–70
30.
Janvier NC, Boyett MR: The role of Na-Ca exchange current in the cardiac action potential. Cardiovasc Res 1996; 32: 69–84
31.
Minajeva A, Kaasik A, Paju K, Seppet E, Lompré AM, Veksler V, Ventura CR: Sarcoplasmic reticulum function in determining atrioventricular contractile differences in rat heart. Am J Physiol 1997; 273: H2498–507
32.
Beuckelmann DJ, Näbauer M, Erdmann E: Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 1992; 85: 1046–55
33.
Park WK, Pancrazio JJ, Suh CK, Lynch C: Myocardial depressant effects of sevoflurane: Mechanical and electrophysiologic actions in vitro. A nesthesiology 1996; 84: 1166–76
34.
Schwinger RH, Bohm M, Schmidt U, Karczewski P, Bavendiek U, Flesch M, Krause EG, Erdmann E: Unchanged protein levels of SERCA II and phospholamban but reduced Ca2+uptake and Ca2+-ATPase activity of cardiac sarcoplasmic reticulum from dilated cardiomyopathy patients compared with patients with nonfailing hearts. Circulation 1995; 92: 3220–8
35.
Hasenfuss G, Reinecke H, Studer R, Meyer M, Pieske B, Holtz J, Holubarsch C, Posival H, Just H, Drexler H: Relation between myocardial function and expression of sarcoplasmic reticulum Ca2+-ATPase in failing and nonfailing human myocardium. Circ Res 1994; 75: 434–42