Mechanism of the Negative Inotropic Effect of Thiopental in Isolated Ferret Ventricular Myocardium 

This study was approved by the Animal Care and Use Committee of the Mayo Foundation. We used papillary muscles from the right ventricle of adult male ferrets (weight 1,100–1,500 g, aged 16–19 weeks). The animals were anesthetized with sodium pentobarbital (100 mg/kg, intraperitoneally), and the heart was quickly removed through a left thoracotomy. Immediately after cardiectomy, the heart was placed in a warm (30 degrees Celsius) oxygenated physiologic solution for a few minutes, during which the solution was changed at least once. The hearts kept beating vigorously throughout this period as well as during the dissection of papillary muscles, which was carried out under generous superfusion of the opened right ventricle. Suitable papillary muscles were excised and mounted in a temperature—controlled (30 degrees Celsius) muscle chamber that contained a physiologic salt solution of the following composition (mmol *symbol* 1¹): Sodium sup + 135, Potassium sup + 5, Calcium²+ 2.25, Magnesium²+ 1, Chlorine 103.5, HCO³24, HPO⁴²1, SO⁴²1, acetate 20, and glucose 10. This solution was equilibrated with 95% Oxygen²and 5% CO²(pH = 7.4). Suitable preparations were selected on the basis of previously used criteria. [17] The muscles were held between the lever of a force-length transducer (Innovi, Belgium) and a miniature Lucite clip with a built-in stimulation electrode. Muscles were stimulated at a stimulus frequency of 0.25 Hz, with rectangular pulses of 5 ms duration and an intensity 10% above threshold. Muscles were made to contract in alternating series of four isometric and four isotonic twitches at preload only during a 2-h period of stabilization before the onset of the experiment. The solution was changed at least once during this stabilization period. The entirety of the dissection, superfusion, and bathing procedures and the time elapsed from anesthetizing the animal to the actual experiment most likely has allowed for any residual pentobarbital to wash out and its effect to disappear. All experiments were carried out with the initial muscle length set at L^max, i.e., the muscle length at which active force development is maximal. All ferret papillary muscles were pretreated with (plus/minus)-bupranolol HCl 10⁷M (Sanol, Germany)[18] before the onset of the experiment to abolish any beta-adrenergic effects. Kaumann et al. [18] demonstrated that the equilibrium dissociation constant (K^B) derived from the antagonism of the chronotropic and inotropic effects of isoproterenol in spontaneously beating right atria and electrically stimulated left atrial strips from kitten and guinea pigs was 8.8–9.2 for (plus/minus) bupranolol. Consequently, the concentrations of (plus/minus) bupranolol used in the studies (10⁷M) is 2 orders of magnitude larger than assessed from the K^Bvalues and should be adequate to block beta-adrenergic effects.

Four protocols were used to examine the mechanism of thiopental's inotropic effect. Each muscle served as its own control.

In group 1 muscles (n = 8), thiopental's effect on contractility and relaxation was defined. Papillary muscles were exposed to cumulative concentrations of 10⁵to 10³M thiopental in 0.5 log M increments. (Thiopental sodium, USP sterile powder, Abbott, North Chicago, IL, was dissolved in Krebs-Ringer's solution to constitute a stock solution of 0.1 M). Thiopental serum concentration 30 s after a 6 mg/kg intravenous bolus injection of thiopental in patients averaged 93 micro gram/ml (3 x 10⁴M), [19] and thiopental serum concentration declined to 6.9 micro gram/ml by 15 min after the intravenous bolus. [19] Total plasma thiopental levels of 39–42 micro gram/ml (1.56–1.68 x 10⁴M) and free plasma thiopental levels of 5.9–6.3 micro gram/ml (2.3–2.5 x 10⁵M) produce surgical anesthesia in humans. [20] Assuming the high degree of protein binding of thiopental (83–86%), [19,20] the average anesthetic and peak concentrations of nonprotein bound thiopental would be in the range of 3 x 10⁶M to 6 x 10⁵M in humans. The concentrations studied in our experiments span those of other investigators and encompass the range present in clinical situations in humans.

After 15 min of equilibration at a particular concentration, variables of contraction and relaxation were determined from three types of twitches (Figure 1). The first contraction was an isotonic twitch at the preload of L^maxfrom which peak shortening (DL), peak velocity of shortening (+V), peak velocity of lengthening (-V), and time to peak shortening (TDL) were measured. The second contraction was a “zero-load-clamp” twitch, that is, an isotonic twitch at the preload of L^maxwhere load was rapidly (< 3 ms) reduced to zero just before the stimulus was applied, and this unloading step was critically damped. [21] Maximal unloaded velocity of shortening (MUVS) and time to MUVS (TMUVS) were measured from zero-load-clamped twitches. The third contraction was an isometric twitch, from which peak developed force (DF), maximal rate of rise (+dF/dt), and fall of force (-dF/dt), time to peak force (TPF), and time from peak developed force to half isometric relaxation (RTH) were measured. Each of these three contractions was separated by seven isotonic twitches at the preload of L^maxto eliminate effects of loading history of preceding contractions. [22,23] Load-sensitivity of relaxation was determined as in earlier studies, [24] from an isometric twitch and six afterloaded isotonic twitches. In brief, the ratio of time to initiation of isometric relaxation in afterloaded isotonic twitches relative to time for the isometric twitch to decline to corresponding force levels was plotted against the ratio of force of the afterloaded isotonic twitches relative to peak developed force of the isometric twitch. The slope of this time ratio versus force relationship is a quantitative measure of load-sensitivity of relaxation.

After the initial 2-h stabilization period, electrical stimulation was stopped and multiple superficial cells were microinjected with the Calcium²+-regulated photoprotein aequorin [16] to allow for subsequent detection of the intracellular Calcium sup 2+ transient. Aequorin (> 95% pure) was generously donated to us by J.R. Blinks, M.D. (Friday Harbor Laboratories, University of Washington, Friday Harbor, WA). In brief, 1 micro liter of filtered aequorin solution (2 mg/ml in 5 mM PIPES, 150 mM KCl, pH = 7.0) is loaded in a glass micropipette pulled from capillary tubing with an internal filament (TW 150F-4, World Precision, Sarasota, FL) to a resistance of 5–10 M Omega. The pipette is inserted into a holder that provides for an airtight seal at the butt of the pipette with a Teflon collar. A platinum wire inside the pipette makes electrical contact with the solution in the electrode tip. A superficial cell is impaled by the pipette mounted on a micromanipulator until a reasonable and stable membrane potential is obtained. Nitrogen pressure is then applied to the inside of the pipette using the apparatus described by Blinks et al. [16] Injection is discontinued at the first sign of appreciable changes in resting membrane potential; the microelectrode is then withdrawn and another cell is impaled for microinjection of aequorin. It was usually necessary to microinject 30–100 cells. After microinjection, muscles were not stimulated for 2 h to allow for resealing of the plasma membranes of the injected cells. The muscles were then carefully transferred to and mounted in a vertical muscle chamber that allows for simultaneous detection of variables of contractility and of aequorin luminescence. [25–27] The aequorin-injected muscle was positioned in a narrow glass extension at the base of the organ chamber at one focal point of a bifocal ellipsoidal reflector. The photocathode of a bialkali photomultiplier (EMI 9235QA, Fairfield, NJ) was located at the other focal point. Muscles were made to contract isometrically at L^maxthroughout experiments in which aequorin luminescence was measured. It was usually necessary to average luminescence and force signals of 16–256 twitches to obtain a satisfactory signal-to-noise ratio in aequorin luminescence signals. This was accomplished on a digital oscilloscope (Nicolet 4904C, Madison, WI). We quantified the following variables from the aequorin luminescence signals (Figure 2): diastolic aequorin luminescence (measured as the average value of aequorin luminescence during a time span of 200 ms immediately preceding the electrical stimulus), peak systolic aequorin luminescence, time to peak aequorin luminescence, and t¹.25, the time from the stimulus to the time when aequorin luminescence had decreased to 25% of its peak value (Figure 2). To assess possible effects of thiopental on Calcium²+ uptake by the sarcoplasmic reticulum, the time course of decline of the aequorin luminescence was analyzed as follows. The natural logarithm of aequorin luminescence from 50% peak to 10% peak and from 40% to 10% and from 35% to 5% of peak aequorin luminescence was subject to least-squares linear regression. The slopes of each regression fit k⁵⁰/10, k⁴⁰/10, and k³⁵/05 were compared between control and various thiopental concentrations and during Calcium²+ back titration experiments (see below;Figure 2, insert).

In group 2 muscles (n = 9), we determined possible changes in the intracellular Calcium²+ transient during exposure to thiopental. After beta-adrenoceptor blockade with (plus/minus)-bupranolol HCI 10 sup -7 M, a dose-response curve to thiopental was obtained in each of nine muscles (group 2). The following steps were used: control and thiopental 10 sup -5 M, 3.3 x 10 sup -5 M, 10 sup -4 M, 3.3. x 10 sup -4 M, and 10 sup -3 M. The muscles were exposed to each concentration of thiopental until a steady state of force and systolic aequorin luminescence was obtained.

In group 3 muscles (n = 6), we determined whether thiopental possibly alters myocardial relaxation or myofibrillar Calcium²+ sensitivity. Each of six muscles was pretreated with 10 sup -7 M (plus/minus)-bupranolol and subjected to “Calcium²+-back titration” experiments: after measurement of control variables of the isometric twitch, muscles were exposed to 10 sup -1 M thiopental. Extracellular [Calcium²+] was rapidly raised by adding small aliquots of a concentrated CaCl²solution (112.5 mM) to the bathing solution, until peak developed force was equal to that in the control twitch. This protocol allowed us to compare contraction, relaxation, and time variables and aequorin luminescence signals in control and in the presence of 10 sup -1 M thiopental at equal peak developed force.

In group 4 muscles (n = 5), we attempted to determine whether transsarcolemmal Calcium²+ exchange is affected by thiopental. Pretreatment with the plant alkaloid ryanodine (60% ryanodine, 40% 1,29-didehydroryanodine, in combination 90–95% pure; Polysciences, Warrington, PA) was used to exclude the contribution of the SR to Calcium²+ release. Each of four muscles was exposed to 1 micro Meter ryanodine, after which the effects of 10 sup -5 M, 3.3 x 10 sup -5 M, 10 sup -4 M, 3.3 x 10 sup -1 M, and 10 sup -3 M thiopental, respectively, on contractile variables and aequorin luminescence were assessed. Thiopental's effects on contractility and aequorin luminescence in the presence of functional SR (group 2) were compared to its effects in muscles lacking a functional SR (group 4).

A separate group of experiments (group 5) was performed to assess the stability of the ryanodine-treated preparation with time (n = 5). At time 0, papillary muscles pre-injected with aequorin were exposed to 1 micro Meter ryanodine, and developed force and aequorin luminescence were repeatedly recorded over the next 6 h. Data of these control experiments have been reported [28] and are used as a basis of comparison against thiopental's effect in ryanodine-treated muscles (group 4).

In group 6 muscles (n = 6), we assessed the effects of thiopental on frog ventricular myocardium, a species that is primarily dependent on transsarcolemmal Calcium²+ exchange for activation. [29] Ventricular strips were cut from the ventricle of pithed frogs (Rana pipiens) and were mounted vertically in the muscle chamber for measurements of contractility variables during thiopental dose-response experiments. The physiologic salt solution was diluted to 80% of its original composition with distilled water to approximate the composition of extracellular fluid in frogs. [30] Frog ventricular strip experiments were carried out at 25 degrees Celsius. In each group 7 muscle (n = 4), we measured the effects of thiopental on the action potential of frog ventricular myocardium.

All waveforms of aequorin luminescence, force, length, and velocity were displayed as a function of time on a four-channel digital oscilloscope (Nicolet 4094C, Madison, WI), stored permanently on 5.25-inch floppy disks and recorded at slow speed on a four-channel pen recorder (Honeywell 1400, Minneapolis, MN). All waveforms of interest recorded on the digital oscilloscope were transferred to a computer (Reason Technology 486/33 MHz, Minneapolis, MN) by software programs written in Waveform Basic (Blue Feather Software, New Glarus, WI). Variables of contraction and relaxation, aequorin luminescence, and corresponding time values were determined automatically by software written in Waveform Basic language. We have written numerous programs over the years for automatic transfer, storage, analysis, and data-compiling in Waveform Basic. Each of these programs has been extensively tested for correctness, accuracy, and reproducibility, and data measured by each of these programs were compared against measurements carried out without the use of these programs.

To determine whether thiopental influences the Calcium²+-sensitivity of aequorin, we used an aequorin in vitro assay apparatus [16] to measure aequorin luminescence in the presence and absence of 10 sup -5, 10 sup -1, and 10 sup -3 M thiopental in a solution containing 150 mM KCl, 20 mM PIPES (piperazine-N,N bis-2-ethanesulfonic acid), 2 mM EGTA [ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid], and 2 mM Ca EGTA, pH 7.00. This solution approximates the ionic composition of the intracellular milieu and produces, at 22 degrees Celsius, a pCa of 6.4, which is in the range of myoplasmic-free Calcium²+ concentrations encountered during a twitch. We used 20 mM PIPES instead of the traditionally used 2 mM PIPES concentration to enhance buffering against increases in pH induced by thiopental. With a control pH = 7.00, 10 sup -4 M thiopental increased the PIPES-KCl solution's pH to 7.01, 3.3 x 10 sup -4 M thiopental raised pH to 7.03, while pH was 7.09 in 10 sup -3 M thiopental. This small change in pH indicates adequate buffering capacity by 20 mM PIPES and does not appreciably by itself alter aequorin bioluminescence's Calcium²+ sensitivity. The effects of 10 sup -5, 10 sup -4, 10 sup -3 M thiopental on the Calcium²+ sensitivity of aequorin luminescence was assessed in a Calcium²+-free solution containing only KCl and PIPES in the same concentration as listed above to determine whether thiopental alters Calcium²+-independent aequorin luminescence.

Thiopental caused a small alkalinization of the Krebs-Ringer's solution used for the muscle experiments as well. While being bubbled with 95% Oxygen²-5% CO²at 30 degrees Celsius, the Krebs-Ringer's solution underwent a 0.04 pH unit increase in 10 sup -4 M thiopental, a further 0.03 pH unit increase in 3.3 x 10 sup -4 M thiopental, and a further 0.03 pH unit increase in 10 sup -3 M thiopental.

The effects of thiopental (groups 1, 2, 4, 6) on contractility variables and on aequorin luminescence variables were assessed with repeated measures analysis of variance. When appropriate, Dunnett's test was used to compare effects of individual drug concentrations with control. All values are expressed as mean plus/minus SD. For group 3 muscles, aequorin luminescence and contractility variables of control twitches and after Calcium²+ back-titration were compared by repeated measures analysis of variance followed by Dunnett's test where applicable. To compare the dose-response curves to thiopental alone (n = 9), to thiopental after ryanodine (n = 5), and to ryanodine, time controls ([28], n = 5), the following procedure was used. First, the inotropic responses in each muscle were expressed as percent of control. For each muscle in each of the three groups, the slope of the least squares linear regression between aequorin luminescence and contractile response (in percent of control) versus thiopental concentration was calculated. Slopes of dose-response curves were compared between groups with one-way ANOVA followed by Student-Newman-Keuls test when appropriate. All tests were two-tailed and P < 0.05 was taken as the level for statistical significance of differences.

Among the five ferret muscle groups, there were no statistically significant differences in muscle length at L^max, mean cross-sectional area (CSA), peak developed force (DF), and ratio of resting to total peak isometric force at L^max(R/T)(one-way ANOVA). When all ferret muscle characteristics are pooled, L^maxwas 6.4 plus/minus 1.3 mm, CSA was 0.60 plus/minus 0.27 mm², DF was 31.1 plus/minus 16.3 mN *symbol* mm sup -2, and R/T was 0.24 plus/minus 0.07 (all data are mean plus/minus SD; n = 33). Frog ventricular strip characteristics were: L^max, 5.8 plus/minus 0.8 mm; CSA, 0.89 plus/minus 0.23 mm²DF, 19.8 plus/minus 6.5 mN/mm²; R/T, 0.23 plus/minus 0.08 (mean plus/minus SD, n = 6). Thiopental (10 sup -5 M-10 sup -3 M) did not alter the Calcium²+-independent aequorin luminescence, nor the Calcium²+-sensitivity of aequorin at pCa 6.4 (22 degrees Celsius, pH 7.0) in in vitro assays.

(Table 1) shows the absolute values of variables of contractility in control conditions at the onset of the experiment for group 1. Figure 3and Figure 4show measurements of contractility in the dose-response experiments to thiopental (group 1; n = 8). Thiopental, greater or equal to 10 sup -4 M, decreased DF, DL, and MUVS. Thiopental caused a dose-dependent decrease in the maximal rate of force development (+dF/dt) and decline (-dF/dt;Figure 4) of isometric twitches. In isotonic twitches, thiopental decreased +V and -V in a dose-dependent fashion. Thiopental shortened time to peak force (TPF) and time to half-isometric relaxation (RTH) of isometric twitches, increased time to maximal unloaded velocity of shortening (TMUVS) of zero-load clamped twitches, and had no effect on time to peak shortening (TDL). Relaxation of ferret papillary muscle is sensitive to load during control conditions. Thiopental, > 10 sup -4 M, decreased load sensitivity of relaxation (Figure 5).

(Figure 6) illustrates a representative example of a dose-response experiment to incremental concentrations of thiopental on aequorin luminescence and developed force. Figure 7and Table 2summarize the results of nine experiments and demonstrate that:(1) diastolic aequorin luminescence was not altered by thiopental;(2) thiopental, greater or equal to 3.3 x 10 sup -5 M, decreases peak aequorin luminescence;(3) thiopental, greater or equal to 10 sup -4 M, decreases peak developed force;(4) thiopental has no significant effect on the time to peak aequorin luminescence (except at 10 sup -3 M where it is slightly increased);(5) thiopental does not significantly alter the time course of decline of aequorin luminescence [lack of significant changes in t¹.25 and lack of significant changes of the slopes of the ln (aequorin luminescence during decline from 50% to 10%, from 40% to 10% and from 35% to 5% of peak)];(5) thiopental does not significantly alter the time course of the isometric twitches (as measured by TPF and RTH).

To determine whether thiopental alters myofibrillar Calcium sup 2+ responsiveness, aequorin luminescence was measured in each of six muscles (group 3) and compared at equal peak developed force in control conditions (Figure 8, left) and after exposure to 10⁴M thiopental in elevated [Calcium²⁴]⁰(Figure 8, right). Table 3lists the values of aequorin luminscence and of contractile variables in control, in 10⁴M thiopental and in 10⁴M thiopental in higher [Calcium²⁴]⁰. The [Calcium²⁴]⁰achieved during Calcium²⁴back-titration to equal control peak force was 3.56 plus/minus 0.76 mM (mean plus/minus SD; n = 6). As observed for group 1 and 2 muscles, thiopental decreased peak developed force (DF), time to peak force (TPF), time to half isometric relaxation (RTH), and aequorin luminescence. When thiopental's negative inotropic effect on DF was corrected by raising [Calcium²⁴]⁰, peak aequorin luminescence was significantly less than control. Peak developed force, diastolic aequorin luminescence, time to peak aequorin luminescence, and TPF were unchanged from control (Table 3).

To assess the effects of thiopental on contractility independent of the sacroplasmic reticulum Calcium²⁴release, aequorin luminscence and contractility variables were measured under isometric conditions in ferret papillary muscle after exposure to ryanodine 1 micro Meter (group 4). Consistent with its effects on the sarcoplasmic reticulum, ryanodine decreased peak developed force and peak aequorin luminescence from control conditions (Figure 9, far left, and Table 4). In muscles pretreated with ryanodine 10 sup -6 M, force and aequorin luminescence were further decreased by thiopental (Figure 9, three right panels, and Table 4).

The role of the sarcoplasmic reticulum in thiopental's negative inotropic effect was evaluated further by comparing the relative effects of thiopental on DF and acquorin luminescence in group 1 muscles with intact SR (Figure 10, top) with those in group 4 muscles with ryanodine-inactivated SR (Figure 10, bottom). The relative effects of thiopental on DF and aequorin luminescence with or without functional SR did not differ (P > 0.05 for DF and AL; ANOVA and Student-Newman-Keuls tests).

As previously reported, [28] the contractile performance of ryanodine-treated muscles were stable over at least 6 h. The effects of thiopental on DF and AL in control and in ryanodine-pretreated muscle were significantly different from time-control muscles (P < 0.05 for DF and AL; ANOVA and Studen-Newman-Keuls test).

In frog ventricular muscle (group 6; n = 6), thiopental had no significant effect on either peak force of isometric twitches (DF) or on peak shortening of isotonic twitches (DL) at concentrations up to 3.3 x 10⁵M (Figure 11, left, and Figure 12). Thiopental, at concentrations of 10⁴M and higher, caused a marked dose-dependent negative inotropic effect (Figure 11, left, and Figure 12). DF was decreased from a control value of 19.8 plus/minus 6.6 mN *symbol* mm²to 15.1 plus/minus 4.8 mN *symbol* mm²in 10²M thiopental and to 5.5 plus/minus 2.0 mN *symbol* mm²in 3.3 x 10⁴M thiopental. DL was decreased from a control value of 0.16 plus/minus 0.04 L *symbol* L^max1to 0.12 plus/minus 0.04 L *symbol* L^max1in 10⁴M thiopental and to 0.03 plus/minus 0.02 L *symbol* L^max1in 3.3 x 10⁴M thiopental. The amplitude of the twitch was unmeasurably small in 10³M, and thiopental's effects were fully reversible after washout, Figure 11(right) illustrates the pronounced prolongation of the action potential by thiopental in frog ventricular myocardium. Thiopental also decreased the overshoot of the action potential.

Thiopental is sued extensively in clinical practice as intravenous induction agent. Thiopental is well known to cause significant depressant cardiovascular effects in humans, [11] animals, [10–13] and isolated cardiac tissue. [2–9] Thiopental caused a greater decreased in contractility at higher pacing rates and effectively reversed the positive staircase in isolated ventricular muscle. [3,5] Komai and Rusy [5] found that thiopental decreases the force of potentiated contractions more than control and increased the length of the period required for maximizing potentiated state contractions. They postulated that thiopental inhibits Calcium²⁴entry via the sarcolemma and decreases the rate of movement of Calcium sup 24 within the sarcoplasmic reticulum from sites of uptake to sites of release, with little or no effect on the total amount of Calcium²⁴available with the SR.

An effect of thiopental to inhibit transsarcolemmal Calcium sup 24 entry finds support in the observation of thiopental's effect to reduce the amplitude and maximal rate of rise of the slow action potential in isolated dog papillary muscles.²Probably the most comprehensive studies on thiopental's mechanical and electrophysiologic effects have been carried out by Lynch et al. [8,15] Thiopental (10–100 micro Meter) increased action potential duration and caused contractile depression both in normal Tyrode solution and in partially depolarized beta-adrenergically stimulated myocardium. They found that 30–100 micro Meter thiopental increased “slow” action potential duration (consistent with decreased potassium conductance), and 100 micro Meter thiopental decreased dV/dt^max(consistent with decreased calcium channel ionic influx), [8] A thiopental-induced decrease in potassium conductance was suggested by Komai et al., [31] and Pancrazio et al. [15] demonstrated in whole-cell patch clamped frog atrial and guinea pig myocytes that thiopental caused a reduction of the inward rectifying Potassium sup + current (I^kl). They demonstrated in a computer simulation that a blockade of I^kl, as produced by thiopental, would slightly prolong the action potential duration and decrease the time-to-threshold for action potential generation. [32] An increase in action potential duration is certainly consistent with the prolongation of the isometric twitch in frog ventricle after thiopental. Thiopental did not alter the rate of In (aequorin luminescence) in its terminal phase (from 50% to 10%, 40% to 10%, 35% to 5% of peak, respectively measured as k⁵⁰/10, k⁴⁰/10, k³⁵/5). Because the natural logarithm of aequorin luminescence is a linear representation of [Calcium²+]o it would be reasonable to conclude that the rate of decline of [Calcium²⁴]ⁱin its terminal phase is not affected by thiopental. Because the terminal phase of aequorin luminescence decline is determined mainly by SR Calcium²⁴uptake, one would conclude that thiopental does not alter SR Calcium²⁴uptake. This would be consistent with the lack of significant effect of thiopental on SR Calcium²⁴uptake [33] and with Komai and Rusy's [5] observations and points toward other sites of action.

Because thiopental's inotropic effects most likely involve alterations in intracellular Calcium²⁴homeostasis in cardiac myocytes on a beat-to-beat basis, this study was undertaken to determine to what extent thiopental's negative inotropic effect is accompanied by a decrease in intracellular Calcium²+ availability and to what extent, if any, changes in myofibrillar Calcium²+ sensitivity plays a role.

Ferret ventricular myocardium is a good model to study drug actions because it shares certain physiologic characteristics with human ventricular myocardium. The density of sympathetic innervation of the ferret right ventricle parallels that of human right ventricle. [34] The regulation of activator Calcium²+ by sarcolemma and sarcoplasmic reticulum is similar in the two species. This is expressed in the mechanical characteristics of the two tissues exhibiting similar force-frequency relationships and mechanical restitution curves. [35,36] Furthermore, information from experiments carried out in different species can help identify the predominant site of drug action because of different contributions to excitation-contraction coupling by SR Calcium²+ release and transsarcolemmal Calcium²+ entry in rat, ferret, rabbit, and frog ventricular myocardium.

This in vitro study demonstrates the negative inotropic effect of thiopental in ferret ventricular myocardium at concentrations of 10⁴M and greater. This concentration is in the range of the free plasma thiopental concentration typically found after an intravenous bolus injection of thiopental. [19,20] The negative inotropic effect of thiopental observed in our study is accompanied by a decrease in peak acquorin luminescence. The concomitant decrease in the amplitude of contraction and the amplitude of the intracellular Calcium sup 2+ transient is most likely due to a reduced availability of intracellular Calcium²+. This decreased intracellular Calcium [21] concentration can result from decreased Calcium²+ release by the SR and/or decreased net transsarcolemmal Calcium²+ entry.

To differentiate between these potential sites of action, we examined thiopental's effects in myocardial tissue pretreated with the plant alkaloid ryanodine. Ryanodine abolishes cardiac SR Calcium²+ release [37,38] but has no effect on the SR Calcium²+ uptake pump or the Sodium sup +-Calcium²+ exchanger [39–42] and, therefore, can be used as a tool to make contractility dependent on transsarcolemmal Calcium²+ movement. [42] The significant reduction in all contractile variables from control measurements after 10⁶M ryanodine is similar in magnitude to that reported previously in similar [42] and identical [44] experimental conditions and reflects the substantial contribution of the SR to the activator Calcium sup 2+ pool in ferret ventricular myocardium. Changes in contractility and in the intracellular Calcium²+ transient caused by a drug that occur over and above those produced by ryanodine reflect the action of that drug on sarcolemmal function. [42,43] The relative loss of contractility and of aequorin luminescence produced by thiopental after ryanodine pretreatment (no SR Calcium²+ release) did not differ from that in muscles not pretreated with ryanodine (SR Calcium²+ release unimpaired). This strongly suggests that thiopental decreases intracellular Calcium²+ availability by interfering with transsarcolemmal Calcium²+ influx. Frog ventricular myocardium has a poorly developed SR and is almost entirely dependent on transsarcolemmal Calcium²+ influx to activate the myofibrillar apparatus. The thiopental-induced decrease in contractility in frog ventricle is, therefore, consistent with an effect of thiopental to decrease net transsarcolemmal Calcium²+ influx. In addition, the thiopental-induced prolongation of the period of tension development may result from the prolongation of the action potential caused by a decrease in [Calcium²+], and an inhibitory effect of thiopental on I^lambdaI. [15,32].

Although thiopental profoundly affects intracellular Calcium sup 2+ availability, we examined its effects on myofibrillar Calcium²+ sensitivity. We measured aequorin luminescence in Calcium²+ back-titration experiments in which aequorin luminescence was measured in the absence and presence of 10⁴M thiopental, at equal peak force obtained by adjusting the extracellular [Calcium²+] concentration upward in the presence of thiopental. The assumption implicit to this type of analysis is that the Calcium²+ occupancy of troponin Carbon at peak force is the same in either condition, so that myofibrillar Calcium²+ sensitivity can be assessed from the relationship between [Calcium²+], and Calcium²+ occupancy of troponin Carbon. If thiopental alters reaction mechanisms “downstream” from the binding of Calcium²+ to troponin Carbon and modifies the relationship between Calcium²+ occupancy of troponin Carbon and force, our approach would be invalid. Yet, so far there is no evidence that this occurs. Moreover, it is difficult to determine, in twitch contractions of intact living muscle fibers, whether a particular intervention changes myofibrillar Calcium²+ responsiveness by comparison of force and Calcium²+ transients alone, because the relationship between force and [Calcium²+]ⁱin twitch contractions does not each steady state. When the kinetics of the [Calcium²+]ⁱtransient are altered by the intervention, the changes may be impossible to interpret in terms of changes of myofibrillar responsiveness. [25] In our experiments, because time to peak aequorin luminescence was not changed in the Calcium²+ back-titration experiments, the conclusions based on our experimental data should be valid.

The observations that peak aequorin luminescence was slightly smaller at equal peak force in 10⁴M thiopental than in its absence point toward a small increase in myofibrillar Calcium²+ sensitivity in the presence of thiopental. Thiopental's slight alkalinization of the extracellular fluid (0.04 pH units by 10⁴M thiopental) is insufficient to account for any significant alterations in the sensitivity of aequorin to Calcium²+. [16] If anything, alkalinization of the intracellular environment from pH 6.6 to pH 7.4 would cause the opposite effect, that is, a leftward shift of the aequorin luminescence-Calcium²+ curve [16] caused by a slightly increased sensitivity to Calcium²+. By the same token, intracellular alkalization would increase myofibrillar sensitivity to calcium, and this effect could have become apparent in our experiments.

The negative inotropic effect of thiopental most likely can be attributed to an interference with cellular mechanisms that regulate intracellular Calcium²+ availability. The data from Calcium²+ back titration experiments suggest that thiopental may slightly increase myofibrillar Calcium²+ sensitivity. If this action is significant, it would counteract the negative inotropic effect. The mechanism of this effect is not clear at this time and may need to be addressed in future studies using skinned fibers.

Thiopental decreased load-sensitivity of relaxation in a dose-dependent fashion. This measure of relaxation is determined by the time course of isometric relaxation, the time course of isotonic relaxation, and the amplitude of force development. The time course of isometric relaxation, typically measured by RTH, reflects mainly the myofibrillar Calcium²+ sensitivity, whereas isotonic relaxation is strongly determined by sarcoplasmic reticulum function. Thiopental decreases both the rates of isometric and isotonic relaxation, most likely because it decreases the amplitude of the twitch in the first place. Decreases in [Calcium²+]⁰achieve the same effect, [44,45] and it is therefore likely that a decrease in load-sensitivity of relaxation reflects primarily a decrease in intracellular Calcium²+ availability. Therefore, in contrast to propofol, thiopental's effects on contraction and relaxation are not dissociated.

The results of this study must be interpreted in the context of the experimental conditions in which they were obtained. Results obtained here at 30 degrees Celsius and a stimulus interval of 4 s may differ from those that one could obtain at the more physiologic conditions of the animal, 37–38 degrees Celsius and 200 beats/min. Yet, the concentration of thiopental studied here (10⁵M-10³M) encompasses the range of free plasma thiopental concentrations found in humans. [20].

In conclusion, the evidence from the current study indicates that thiopental exerts a marked negative inotropic effect by decreasing transsarcolemmal Calcium²+ influx. The depressant effect may be caused by an inhibition of the sarcolemmal slow inward L-type Calcium sup 2+ current. Possible effects of thiopental on other membranous Calcium²+ exchange mechanisms (e.g., Sodium²+/Calcium²+ exchange, Calcium²+ ATPase export pump) cannot be excluded. The negative inotropic effect of thiopental is also consistent with a slowing of exchange of Calcium²+ from sites of uptake to sites of release within the sarcoplasmic reticulum. [5].

The authors thank Sharon Guy and Laurel Wanek, for technical assistance, and Janet Beckman, for secretarial support.