Lack of Inhibition by Inhalational Anesthetics of Myocardial Contraction Dependent on Intracellular Sodium Activity

Right ventricular papillary muscles were isolated from rabbits anesthetized with intravenous injections of pentobarbital (about 50 mg/kg). The procedure was approved by Animal Care and Use Committee of the University of Wisconsin, Madison. Muscles were stimulated with a pair of field electrodes using a Grass S48 stimulator (Grass Instruments, Quincy, MA). Stimuli lasting 4 ms and x1.5 threshold voltage were used. Isometric force of contraction was measured at 30 degrees Celsius with a force transducer (Gould-Statham UC-2, Oxnard, CA) and recorded on a polygraph (Gilson Medical Electronics, Middleton, WI). The medium (a Krebs-Henseleit medium, pH 7.4) containing 115 mM NaCl, 5.9 mM KCl, 2.5 mM CaCl², 1.2 mM MgCl², 1.2 mM NaH²PO sub 4, 1.2 mM Na²SO⁴, 25 mM NaHCO³, 5.6 mM glucose, and 0.05 mM EDTA was equilibrated with a gas mixture of 95% oxygen and 5% carbon dioxide. Isoflurane (1.5% and 2.4%) was added to the gas mixture using a calibrated Drager vaporizer (North American Drager, Telford, PA). The concentration of isoflurane in the medium was checked by gas chromatography (Perkin Elmer 910, Perkin-Elmer, Norwalk, CT) and found to be 0.36 mM (1.5% in gas phase) and 0.51 mM (2.4% in gas phase). Miao and Lynch [8]reported that the concentration of isoflurane in the medium is 40% higher at 30 degrees Celsius compared with at 37 degrees Celsius. Isoflurane concentration in the gas phase at a given vaporizer setting varied by about 10% to 20% of the values obtained in the initial calibration. After stabilization (2 to 3 h), L^max(the minimum muscle length at which developed force is maximum) was determined. All experiments were carried out at L^max. The diameter of the muscles (n = 27) was 1.0 +/- 0.2 mm (SD), and the length (L^max) was 4.5 +/- 1.2 mm (SD). During stabilization and between experiments, muscles were stimulated at 0.1 Hz unless otherwise specified. The muscles were stable for about 3 h, and the experiments were carried out then.

The effect of isoflurane on the time course of the force increase that occurs when the muscles are stimulated after a rest was measured. The time course of peak force increase under the control condition was measured at 0.5 Hz after 10 min of rest. This was followed by the control measurements at 1 Hz and then at 2 Hz. Each measurement was preceded by 10 min of rest. These measurements were then repeated 20 min after 1.5% isoflurane was added to the gas mixture and then 20 min after 2.4% isoflurane was added. Of nine muscles, one muscle had essentially no slow phase under the control condition when the muscle was stimulated at 2 Hz. Another muscle had a long refractory period that precluded stimulation at 2 Hz under the control condition. These two muscles were excluded.

(Figure 1) shows the time course of the increase in peak force that occurred when the muscles were stimulated at 0.5 Hz after 10 min of rest. The initial fast phase of the force increase was often complex, with a lag at the beginning and an overshoot toward the end. For this reason, we did not analyze the time course of the fast phase. The slow phase, after the end of the fast phase, can be described by an equation containing an exponential term; F = a + b x (1 - exp [-k^slowx t]) ... 1, where F is the force (mN/mm²) at time t (s); and k^slow(s sup -1) is a first-order rate constant. The rate constant (k^slow) was obtained by curve fitting the data using equation one. The constants a and b obtained by curve fitting are the calculated magnitudes of the fast phase and that of the slow phase extrapolated to t = 0 when the force increase was assumed to continue to t = infinity. Because of this, the calculated values of the constants a and b were not identical to the observed magnitude of the fast phase (total force minus slow phase) and the slow phase. The magnitude of the slow phase was determined from the observed steady-state force and the observed force at the end of the fast phase. The end of the fast phase was easy to see with most traces (56 of 63) but was not with a few (7 of 63). In the latter situations, the point at which the logarithm of the peak force increase versus time plot deviates from the straight line obtained by curve fitting the slow phase was considered to be the end of the fast phase.

The effect of isoflurane (1.5%) on the positive inotropic effect of ouabain (Sigma Chemical Co., St. Louis, MO) was measured with seven muscles as follows. After control force measurements at different stimulation frequencies, isoflurane was added and the force measurements were repeated after 20 min. Ouabain (1 micro Meter) was then added and the muscles were stimulated at 0.5 Hz until a steady state was reached. The time course of the force increase was monitored by recording the force of contraction at 0.5 Hz every 10 min. After the steady state was reached, the force in the presence of isoflurane and ouabain was measured at different frequencies. Finally, the force was measured 30 min after isoflurane was removed. The last measurements represented the force in the presence of ouabain alone. The time course of force increase in the absence of isoflurane was determined with a separate group of 13 muscles.

Data were expressed as means +/- SEM. Statistical significance of the effects of isoflurane measured with the same group of muscles was analyzed by repeated-measures analysis of variance and Dunnett's t test. Logarithmic transformation was used when comparisons involved data with small means and small variance and large means with large variance (data of Figure 5). Data obtained with two different groups of muscles were tested using unpaired t tests. Differences were considered significant at P < 0.05. Curve fitting was done with MacCurveFit 1.0.3 or 1.1 (Kevin Raner Software, Mt. Waverley, Victoria, Australia).

Isoflurane decreased the total force at all frequencies but had a relatively small effect on the magnitude of the slow phase of force increase when the muscle was stimulated at 2 Hz (Figure 2). The results obtained with seven muscles indicated that isoflurane (1.5 and 2.4%) had no statistically significant effect on the magnitude of the slow phase of force increase when the muscle was stimulated at 2 Hz (Figure 3). Thus, with the muscle stimulated at 2 Hz, the negative inotropic effect of isoflurane was largely due to the decrease in the magnitude of the fast phase (difference between the total force and the slow phase of Figure 3). At lower frequencies, the magnitude of the slow phase was also decreased by isoflurane.

Although isoflurane had little effect on the magnitude of the slow phase of force increase when the muscle was stimulated at 2 Hz, the anesthetic prolonged the time required for the force of contraction to reach the steady state. As shown in Table 1, isoflurane decreased the rate constant (k^slow) for the slow phase of the force increase. This effect was statistically significant when the muscle was stimulated at 0.5 Hz or 2 Hz but not at 1 Hz. Table 1also shows that isoflurane decreased constants a (calculated magnitude of the fast phase) and b (calculated magnitude of the slow phase) of equation one (See Materials and Methods) except that the anesthetic had no statistically significant effect on constant b at 2 Hz.

(Figure 4) shows that the development of the positive inotropic effect of ouabain (1 micro Meter) was slowed in the presence of isoflurane (1.5%). The rate constant for the ouabain-induced force increase after the initial lag was significantly (P < 0.05) decreased in the presence of isoflurane (1.5%) from the control value of 0.041 +/- 0.004 min sup -1 (n = 13) to 0.020 +/- 0.003 min sup -1 (n = 7).

(Figure 5) shows the effect of isoflurane (1.5%) on the force of contraction (total force) in the absence and the presence of ouabain (1 micro Meter). The effect of isoflurane on the positive inotropic effect of ouabain was assessed by comparing the effect in the absence of isoflurane (0 - C; see Figure 5) and the effect in the presence of isoflurane ([O + I] - I; see Figure 5). At high frequencies, where the positive inotropic effect of ouabain was relatively small under the control condition, the positive inotropic effect of ouabain was larger in the presence of isoflurane than in its absence.

The effects of 2.4% isoflurane on the rate constants for ouabain-induced force increase (data not shown) and on the positive inotropic effect of ouabain [9]were similar to those of 1.5% isoflurane reported in this study. Compared with the muscles used in this study, however, the group of muscles used to measure the effects of 2.4% isoflurane had a much stronger average force of contraction under the control condition. Accordingly, the data obtained with this group of muscles were not included in this report.

When cardiac muscles are stimulated after a rest, the peak force increases with a biphasic time course. [5]Bers [10]showed that the Calcium²+ content of the sarcoplasmic reticulum, as determined by rapid cooling contractures, increases faster than the twitch force. One of the possibilities that Bers [10]considered to account for the slow increase in the twitch force is accumulation of Calcium²+ dependent on an increase in intracellular Sodium sup + activity. Seibel [5]showed that the slow phase of force increase is inhibited by tetrodotoxin and enhanced by dihydroouabain or veratridine. Tetrodotoxin inhibits Sodium sup + influx, and dihydroouabain increases intracellular Sodium sup + activity by inhibiting Sodium/Potassium-adenosine triphosphatase. Veratridine is a plant alkaloid that increases Sodium sup + influx. Thus the slow phase of the force increase appears to involve Calcium²+ accumulation by Sodium-Calcium exchange after an increase in intracellular Sodium sup + activity. In this regard, Harrison and colleagues [6]showed that intracellular Sodium sup + activity of ventricular myocytes increases after stimulation after a rest or when strophanthidin is added. Recently Lotan and associates [11]also reported an increase in intracellular Sodium sup + concentration in rat hearts that showed an increase in systolic pressure accompanying an increase in pacing rate.

The results indicate that isoflurane had no statistically significant effect on the magnitude of the slow phase of force increase at 2 Hz. Isoflurane had no statistically significant effect on constant b of equation one (the magnitude of the slow phase obtained by curve fitting) at 2 Hz. Thus, provided the muscle is stimulated at a sufficiently high frequency, isoflurane seems to have little effect on Calcium²+ accumulation dependent on an increase in intracellular Sodium sup + activity. The requirement for high-frequency stimulation suggests that a short diastolic interval that minimizes the Calcium²+ efflux is essential. Lynch [4]reported that isoflurane has a smaller negative inotropic effect in low Sodium sup + medium than in normal medium. This is consistent with the notion that isoflurane has little effect on Calcium²+ accumulation dependent on an increase in intracellular Sodium sup + activity, because low extracellular Sodium sup + and high intracellular Sodium sup + similarly affect the transsarcolemmal Sodium sup + gradient.

Because isoflurane had little effect on the magnitude of the slow phase of force increase at 2 Hz, the negative inotropic effect at this frequency may be attributed largely to the decrease in the magnitude of the fast phase. The force of contraction at the end of the fast phase is probably activated by the influx of the extracellular Calcium²+ and Calcium²+ released from the sarcoplasmic reticulum, with no contribution of Calcium²+ accumulated after an increase in intracellular Sodium sup + activity. Decreases in Calcium sup 2+ influx [4,12,13]and Calcium²+ sensitivity of the myofibrils [14,15]probably contribute to the effect of isoflurane on the force of contraction that is independent of intracellular Sodium sup + activity. The effect of isoflurane is completely compensated by high-frequency stimulation in the presence of isoproterenol. [16]Thus the effect of isoflurane on the fast phase of force increase must be completely compensated by isoproterenol as our results showed that isoflurane had little effect on the magnitude of the slow phase of force increase. In contrast, high-frequency stimulation in the presence of isoproterenol failed to reverse the negative inotropic effect of halothane. [16]Note that halothane but not isoflurane enhances sarcoplasmic reticulum Calcium²+ release [17]and decreases sarcoplasmic reticulum Calcium²+ content. [18-20]Thus it seems likely that catecholamines can compensate for the negative inotropic effect of inhalational anesthetics due to the inhibition of the transsarcolemmal Calcium²+ influx and decrease in Calcium²+ sensitivity of the myofibril but not for the effect due to the halothane depletion of sarcoplasmic reticulum Calcium²+.

Although isoflurane differs from halothane with respect to its effect on the fast phase of force increase, these anesthetics are similar with respect to their lack of significant inhibitory effect on the magnitude of the slow phase of force increase. Thus we reported earlier that the magnitude of the slow phase was increased by halothane. [21]In that study, we did not exclude the muscles that did not have a slow phase under the control condition. Instead we included them as those that have the magnitude of the slow phase = 0. This obviously decreased the mean control value, partly accounting for the reported increase in the magnitude of the slow phase in the presence of halothane. Because isoflurane and halothane are representative volatile anesthetics with different chemical structure (halogenated ether and halogenated hydrocarbon), we speculate that the lack of inhibition of the component of contraction dependent on an increase in intracellular Sodium sup + activity may be a general property of inhalational anesthetics.

The finding that the positive inotropic effect of ouabain was undiminished in the presence of isoflurane also suggests that isoflurane does not inhibit the force increase due to Calcium²+ accumulation after an increase in intracellular Sodium sup + activity. This is because the effect of supratherapeutic concentrations of cardiac glycosides involves accumulation of Calcium²+ secondary to a decrease in Calcium²+ efflux secondary to Sodium/Potassium-adenosine triphosphatase inhibition and consequent increase in intracellular Sodium sup + activity. [7]The effect of ouabain was most pronounced at low frequencies, where the force of contraction is weak under the control condition. At high frequencies, where the force under the control condition is strong, additional increase due to ouabain was small. This suggests that the force in the presence of ouabain was approaching the practical limit of the twitch force for the given muscle, even though cardiac myofibrils can develop as much as 145 mN/mm²of force. [22]In the presence of isoflurane, which decreased the force before ouabain was added, there would be a larger inotropic reserve. Schappert and associates [23]reported the effects of isoflurane and halothane on the force of contraction of guinea pig papillary muscles in the presence of 0.1 micro Meter digitoxin. Because different groups of muscles with different control forces were used in the absence and in the presence of digitoxin, it is difficult to evaluate the effect of the anesthetics on the positive inotropic effect of the cardiac glycoside. Nevertheless, digitoxin's positive inotropic effect appears to be preserved in the presence of isoflurane. Interestingly, these authors [23]showed that the negative inotropic effect of isoflurane was smaller than that of halothane at a given minimum alveolar concentration in the presence of digitoxin but not in its absence.

To account for the observed decrease in k^slowand the rate constant for the ouabain-induced force increase in the presence of isoflurane, we have considered the possibility that the decrease may be related to some effects of isoflurane on Calcium²+ fluxes across the sarcolemma. We assumed, as Adler and colleagues [24]did, that the rate of increase of intracellular Calcium²+ content is determined by the difference in the rate of Calcium²+ influx (A) minus the product of a first-order rate constant for efflux (B) and the Calcium²+ content (Calcium). Thus dCalcium/dt = A - B x Calcium. This equation is analogous to the rate equation for a first-order reaction, [25]and Calcium = (A/B) x (1 - exp [-B x t]). If the force-Calcium relation can be considered to be constant, the force of contraction will increase to a steady-state value (constant x A/B) with a first-order rate constant B. Based on this model, our results of this study are consistent with the possibility that isoflurane decreases both influx (A) and efflux (B) of Calcium²+ across the sarcolemma with a minimum effect on the steady-state Calcium (A/B). Isoflurane inhibits Sodium-Calcium exchange, [26]which is probably involved in both influx and efflux of Calcium²- that activates the force of contraction dependent on an increase in intracellular Sodium sup + activity.

We performed this study using isolated rabbit papillary muscles at 30 degrees Celsius, and caution is required to extrapolate the present results to clinical situations. Although the slow phase of force increase is known to involve a change in intracellular Sodium sup + activity, [5,6]Harrison and coworkers [27]reported that the process is also influenced by Calcium²+ influx through the Calcium sup 2+ channels. These authors stated that their results "may not contradict, but complement the more favoured theory that the increase in the strength of contraction during the staircase is the result of a rise of intracellular Sodium sup +." More recently, Harrison and Boyett [28]showed that the increase in contraction accompanying an increase in stimulation frequency from 0.5 Hz to 3 Hz involves an increase in intracellular Sodium sup + activity and outward shift in membrane current at the end of voltage clamp. The latter was considered to reflect Calcium²+ influx by Sodium-Calcium exchange. These authors also found that the rate-dependent increase in both contraction and intracellular Sodium sup + activity was abolished when the holding potential was -40 mV (and not -80 mV), suggesting that the rate-dependent increase in contraction is primarily the result of the increase in intracellular Sodium sup + activity and not due to Calcium sup 2+ accumulation by Calcium²+ influx through the Calcium²+ channel.

Our results of this study and previous studies from this laboratory [21]suggest that inhalational anesthetics have little effect on myocardial contraction dependent on intracellular Sodium sup + activity. This effect, together with the ability of a catecholamine to reverse the negative inotropic effect of isoflurane at high stimulation frequencies, [16]probably account for the minimal cardiac depressant effect of this anesthetic in clinical situations.

The authors thank Dr. Richard J. Chappell of the Department of Statistics for helpful suggestions and Richard E. Kunert of the Department of Anesthesiology for suggesting that we use MacCurveFit.