Ketamine has a species-dependent inotropic effect on myocardium. The authors' aim was to investigate the direct inotropic effect and the corresponding intracellular Ca2+ transients of ketamine and its isomers on human myocardium.
Right auricular myocardial strips obtained during open heart surgery were exposed to increasing concentrations (73 microM, 360 microM, and 730 microM) of racemic ketamine (n = 12), S(+)-ketamine (n = 12), or R(-)-ketamine (n = 11). Isometric force, isotonic shortening, contractility, relaxation, and time to maximal isotonic and isometric force were assessed. Ten muscle strips in each group were loaded with the calcium-sensitive fluorescent dye FURA-2/AM for simultaneous measurements of calcium transients.
Compared with the initial control maximal isometric developed force, maximal isotonic shortening amplitude, contractility, and relaxation increased by 12.5-22.4% after perfusion with S(+)-ketamine at the concentration of 73 microM (P < 0.05). In contrast, no changes were seen after addition of 73 microM R(-)-ketamine. The effect of racemic ketamine (73 microM) was between that of the two isomers. At the highest concentration (730 microM) ketamine and its isomers decreased maximal isometric developed force, maximal shortening amplitude, contractility, and relaxation by 26.8-57.4% (P < 0.05), accompanied by a significant decrease of the intracellular calcium transient (by 21.0-32.2%, P < 0.05).
In contrast to R(-)-ketamine, S(+)-ketamine increased isometric force, isotonic shortening, contractility, and relaxation at low concentrations (73 microM) compared with the initial control. At higher concentrations (730 microM) a direct negative inotropic action was observed after perfusion with ketamine and its isomers, which was accompanied by a decreased intracellular Ca2+ transient.
This article is featured in "This Month in Anesthesiology." Please see this issue of Anesthesiology, page 7A.
BECAUSE of the sympathomimetic effects of ketamine, its direct actions on the heart are of particular interest. This question is of clinical importance because a potentially direct negative inotropic effect of ketamine on myocardium can be masked by the central stimulation. Ketamine directly depresses myocardial contractility in vitro after inactivation of sympathetic nerve endings in most animals. [1–4] One exception is the rat, in which ketamine has positive inotropic effects on muscle strips despite [Greek small letter beta]-adrenergic receptor ([Greek small letter beta]-AR) blockade, [5,6] but a negative inotropic action on ventricular myocytes in concentrations above clinical relevance.  The general negative inotropic effect is caused by a decrease in intracellular Ca2+and net transsarcolemmal Ca2+influx. [2–4,8,9] In the rat, ketamine's nondepressant action may be explained by the observation that contraction of the ventricle is largely dependent on sarcoplasmic reticulum Ca2+stores. 
Pharmacodynamically, S(+)-ketamine is more effective than R(-)-ketamine or the racemic mixture because it produces similar analgesia and hypnosis at lower concentrations, and unwanted side effects such as agitated behavior and psychomimetic emergence reactions occur less often.  In smooth muscle it has been shown that the extraneuronal catecholamine uptake block is solely caused by the action of the S(+)-isomer.  In the untreated isolated perfused guinea pig heart, catecholamine uptake is blocked in a stereoselective way by ketamine isomers.  In contrast, the action of ketamine isomers on pretreated catecholamine-depleted or [Greek small letter beta]-AR-blocked isolated perfused guinea pig hearts is nonstereoselective. [13,14] There is clinical evidence that ketamine can have negative inotropic effects in humans, as severely ill patients undergoing anesthesia with ketamine show a deterioration of cardiac performance.  However, little is known about the direct effect of ketamine and its isomers on human muscle.
Our aim was to investigate in detail the direct effects of ketamine and its isomers on inotropic variables of human myocardium. We also measured simultaneously the effect of ketamine and its isomers on calcium transients.
Muscle Specimens and Mechanical Measurements
Informed consent was obtained from the patients, and the study was approved by the ethics committee of the medical faculty of the University of Heidelberg. Right auricular myocardium was obtained during cardiac operations for bypass surgery or valve replacement. A list of the operations and patients' characteristics is summarized in Table 1. Muscle strips were exposed to increasing concentrations (73 [micro sign]M, 360 [micro sign]M, and 730 [micro sign]M) of racemic ketamine (n = 12), S(+)ketamine (n = 12), or R(-)-ketamine (n = 11). The lengths were 3.50 +/- 1.31, 3.83 +/- 1.59, and 3.46 +/- 1.51 mm, and diameters were 0.66 +/- 0.08, 0.65 +/- 0.17, and 0.63 +/- 0.16 mm in the racemic, S(+)-, and R(-)-ketamine group, respectively (mean +/- SD).
Muscle strips were transported and prepared in an oxygenated Krebs-Henseleit solution (KHL; composition in mM: NaCl, 118; NaHCO3, 25; KCl, 4.1; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 1.8; glucose, 12) with 30 mM 2,3-butanedione monoxime at 4 [degree sign]C. We added 2,3-butanedione monoxime to protect human myocardium from cutting injury by reversibly desensitizing the contractile proteins to calcium ions, inhibiting cross-bridge interaction, reducing oxygen demand during ischemic periods, and resulting in higher peak twitch tensions. [16,17] Subsequently 10 muscle strips of each group (racemic, S(+)-, and R(-)-ketamine) were incubated for 4 h in oxygenated KHL without 2,3-butanedione monoxime containing 14 [micro sign]M of the calcium-sensitive fluorescence dye FURA-2/AM and the noncytotoxic detergent Cremophor EL (0.5%; BASF, Ludwigshafen, Germany) at room temperature (22–23 [degree sign]C) in darkness. Thereafter, muscle strips were mounted between force transducer and a fixed hook and stretched to slack length (for details see Vahl et al. ). For at least 10 min muscle strips were equilibrated in oxygenated KHL (37 [degree sign]C) and electrically stimulated (pulse duration, 5 ms; amplitude, 10% above threshold; range, 3–5 V; frequency, 1 Hz, square wave). Then they were stretched slowly until force augmentation reached a plateau. After steady states of force development and shortening amplitudes were recorded for at least 20 min, measurements were carried out. After baseline measurements with KHL, the myocardial strips were superfused with either the racemic mixture of ketamine (Sigma, Heidelberg, Germany), R(-)-ketamine, or S(+)-ketamine, with increasing concentrations: 73 [micro sign]M, 360 [micro sign]M, and 730 [micro sign]M for at least 15 min in each solution. After 10 min of steady state, measurements were performed. Ketamine isomers were kindly provided by Parke-Davis (Freiburg, Germany). The chemical purities were 100% for R(-)-ketamine and 99.9% for S(+)-ketamine. For measurements with [Greek small letter beta]-AR antagonists, esmolol HCl (Gensia, Offenbach am Main, Germany) 3 [micro sign]M was used in four muscle strips. Dobutamine (Lilly, Bad Homburg, Germany) was used at a concentration of 0.296 [micro sign]M (0.1 [micro sign]g/ml; n = 4).
Contractile variables included isometric measurements-maximal developed isometric force (mN/mm), contractility (maximal rate of force development, +dF/dt; mN [middle dot] s-1[middle dot] mm-2), relaxation (maximal rate of relaxation, -dF/dt; mN [middle dot] s-1[middle dot] mm-2) and time to maximal isometric force (ms) and isotonic measurements-isotonic shortening (maximal shortening amplitude, % change of muscle length) and time to maximal isotonic force (ms). Measurements were performed with the Muscle Research System by Guth Scientific Instruments (Heidelberg, Germany).
Measurement of Intracellular Calcium Transients
Before incubation in FURA-2/AM, muscle strips were tested for contractility under isometric conditions at 37 [degree sign]C in each group. Fibers were used for measurements only when isometric force remained 90% after the incubation. A xenon lamp provided the excitation light. Alternating excitation wave lengths of either 340 nm or 380 nm were obtained by a rotating filter wheel (rotation frequency: 125 Hz). A photomultiplier collected the emitted fluorescence signals. The ratio of both FURA-2 fluorescence signals, which is proportional to the intracellular calcium concentration, was continuously measured after background fluorescence subtraction. The previously described [18,19] in vivo calibration of calcium measurements was not carried out because it was not possible to "clamp" the intracellular calcium transient of intact human myocardium at maximal and minimal levels under physiologic conditions. Therefore the intracellular calcium transients are presented as percentages of the initial control transients. Quenching of the FURA-2 signal was excluded by adding increasing concentrations of ketamine (73–730 [micro sign]M) to the KHL solution, as no obvious changes of the 340/380 ratio were observed. With each measurement a rundown of the fluorescence signal occurs. This was fitted to a time-dependent linear rundown of 8.55% every 15 min; with y (rundown)= 100 - 0.570 x time (min). Peak Calcium transients were normalized according to the rundown of the FURA-2 signal.
All values are presented as mean +/- SD. Each group was tested for normal distribution by application of the Kolmogorov-Smirnov test. For comparison among groups (comparison of the S(+)-isomer, the R(-)-isomer, and racemic ketamine all at equimolar concentrations vs. each other in the same concentration group) the one-way analysis of variance was applied. One-way repeated-measures analysis of variance was used for within-group comparison (comparison of increasing concentrations and the controls of either the isomers or racemic ketamine vs. each other). When the differences between the groups were greater than would be expected by chance, the multiple comparison Bonferroni t test was applied. A significant difference was defined by a P value < 0.05.
In Figure 1, single recordings of the effect of racemic ketamine on the peak calcium transient from a patient undergoing mitral valve replacement (Figure 1A) and single recordings of the peak calcium transient (Figure 1B), isometric force (Figure 1C), and isotonic shortening (Figure 1D) from a patient receiving coronary artery bypass graft surgery are shown. These recordings demonstrate an increased isometric force at 73 [micro sign]M racemic ketamine. At the highest concentration of 730 [micro sign]M the force transient decreases, which is accompanied by a marked decrease of the calcium transient (Figure 1B, Figure 1C). The initial augmentation of force at 73 [micro sign]M and 360 [micro sign]M is completely inhibited by [Greek small letter beta]-AR blockade (Figure 1E).
Measured variables in the racemic ketamine group and the isomer groups revealed a normal distribution. Baseline contractile variables are shown in Table 2. Comparison of increasing concentrations of either the isomers or racemic ketamine and controls revealed differences between the groups when one-way repeated measures analysis of variance was applied. For a closer analysis, the multiple-comparison Bonferroni t test was applied.
Maximal isometric developed force was increased after perfusion with 73 [micro sign]M racemic ketamine (5.5%, P < 0.05) and S(+)-ketamine (14.5%, P < 0.05); R(-)-ketamine caused no changes compared with the initial control (Figure 2). Racemic ketamine and its isomers at 360 [micro sign]M caused no change from the initial control value, whereas the highest concentration (730 [micro sign]M) caused a reversible decrease of 53–58% in the maximal isometric developed force (P < 0.05). The addition of the [Greek small letter beta]-AR antagonist esmolol (3 [micro sign]M) in four muscle strips of the racemic ketamine group caused a decrease of maximal isometric developed force from 14.5 +/- 1.43 mN/mm2to 2.37 +/- 1.28 mN/mm2. After addition of 73, 360, and 730 [micro sign]M racemic ketamine, concentration-dependent effects on maximal isometric developed force of 37.7%, 64.7%, and 84.1%, respectively (P < 0.05;Figure 2), were observed.
The lowest concentration of S(+)-ketamine (73 [micro sign]M) increased the maximal shortening amplitude by 12.5% compared with the initial control (P < 0.05;Figure 2). At the highest concentration (730 [micro sign]M) ketamine and both isomers caused a reversible decrease of maximal isotonic shortening by 52–65% compared with the initial control (P < 0.05, Figure 2).
Compared with the initial control, the maximal rate of force development increased after perfusion with 73 [micro sign]M of racemic ketamine (10.4%, P < 0.05) and S(+)-ketamine (22.1%, P < 0.05), whereas at the highest concentration the maximal rate of force development decreased after addition of racemic ketamine by 52.9%, S(+)-ketamine by 57.4%, and R(-)-ketamine by 48.1%(P < 0.05;Figure 3). Compared with the initial control, perfusion with 73 [micro sign]M S(+)-ketamine and 360 [micro sign]M of R(-)-ketamine caused significant increases of the maximal rates of relaxation (with 22.4%, P < 0.05, and 29.3%, P < 0.05, respectively;Figure 3). At the highest concentration, there were significant decreases after perfusion with racemic ketamine (48.5%, P < 0.05), S(+)-ketamine (42.9%, P < 0.05), and R(-)-ketamine (26.8%, P < 0.05) compared with the initial control (Figure 3).
The negative inotropic effect of 730 [micro sign]M racemic ketamine was completely reversed by adding dobutamine (Figure 4).
Comparing each concentration group with the initial control, time to maximal isometric force decreased after perfusion with 360 and 730 [micro sign]M S(+)-ketamine (P < 0.05;Figure 5). Time to maximal isotonic force decreased after perfusion with 360 [micro sign]M and 730 [micro sign]M ketamine and its isomers compared with the initial control (P < 0.05, Figure 5).
Although force-development parameters were restored to normal on washout of ketamine, the recovery of the peak calcium transient was incomplete. This change represents a decrease in signal strength, which may be caused by bleaching, leaking, and sequestration.  For compensation, values in Figure 6were normalized according to the calcium rundown (as described in the Methods section). Peak calcium transients showed a decrease of 21.0–32.2% compared with the initial control at the highest concentration of ketamine and its isomers (Figure 6). Time to peak calcium transients showed no change at increasing ketamine concentrations.
All variables investigated (maximal isometric developed force, maximal shortening amplitude, maximal rate of force development, maximal rate of relaxation, time to maximal isometric force, time to maximal isotonic force, and peak calcium transients) revealed no differences when the S(+)-isomer, the R(-)-isomer, and racemic ketamine were compared with each other at a given equimolar concentration.
In this study we have demonstrated a positive inotropic action of S(+)-ketamine on human myocardium at a concentration of 73 [micro sign]M and a negative inotropic action of ketamine and its isomers at higher concentrations. The direct positive inotropic action of 73 [micro sign]M racemic ketamine can be blocked by a [Greek small letter beta]-AR antagonist. This suggests that the positive inotropic effect is caused by a prolonged presence of catecholamines on the neuromuscular junction. The reason may be the inhibition of the catecholamine uptake by ketamine. These results support previous observations in animal models on isolated cardiac muscle strips in which ketamine inhibited extraneuronal uptake of norepinephrine (e.g., ). We observed that simultaneous assessments of intracellular calcium transients revealed a significant decrease at the highest concentration (730 [micro sign]M) of ketamine and its isomers, accompanying the negative inotropic action. This supports recent findings by Kanaya et al. (1998), who described dose-dependent decreases in peak [Ca2+]iand shortening in isolated rat ventricular myocytes.  However, in their experiments negative inotropic effects already begin in the concentration range of 300 [micro sign]M ketamine.
In clinical use serum concentrations reach 60 [micro sign]M 5 min after the injection of 2 mg/kg ketamine,  and they may reach up to 100–150 [micro sign]M within 1–3 min after injection. Interestingly, Gelissen et al.  observed a negative inotropic effect of racemic ketamine on human myocardium already within clinically relevant concentrations (around 100 [micro sign]M). These differing results may be explained by their use of ketamine in its clinical formulation, which includes the preservative benzethonium chloride (0.1 mg/ml), and also by different experimental conditions (30 [degree sign]C with a stimulus frequency of 0.5 Hz, in contrast to 37 [degree sign]C and 1 Hz in our study). A synergistic effect of benzethonium chloride with ketamine has been described on m1 muscarinic signaling in the Xenopus model.  In a recent study, performed with human atrial myocardial tissue, ketamine also exhibited a negative inotropic action at concentrations above clinical relevance.  At lower clinically relevant concentrations, however, a negative inotropic action starting at 88 [micro sign]M already was observed.  These differing results may be caused by different transport solutions. Their atrial tissue was transported in cardioplegic solution, whereas ours was transported in KHL plus 2,3-butanedione monoxime for protection from cutting injury. In addition most of our patients received a preoperative multiple drug regimen including [Greek small letter beta]-AR antagonists and angiotensin-converting enzyme inhibitors and intraoperatively no volatile agents, whereas only a few patients in the study by Sprung et al.  received a multiple drug regimen preoperatively and almost all their patients received volatile agents intraoperatively. Our finding that the negative inotropic effect of racemic ketamine can be completely reversed by addition of dobutamine, supports the results of the study by Sprung et al.  that the effect of high nonclinical concentrations of ketamine on atrial myocardium from patients undergoing coronary artery bypass grafting can be completely reversed by [Greek small letter beta]-adrenergic agonists.
The reduction of intracellular calcium transients, accompanying the decreased force and contractility measurements, can be caused by either reduced calcium influx via the L-type Ca2+channel, reduced calcium release from the sarcoplasmic reticulum, or both. However, recent findings by Connelly et al.  and by Kanaya et al.  suggest that the calcium-release channel of the sarcoplasmic reticulum is not involved in the cardiodepressive effect of ketamine.
Our results revealed that S(+)-ketamine decreases the time to peak isometric force and that both isomers decrease the time to maximal isotonic shortening (Figure 6). These changes could suggest an effect on the kinetics of transsarcolemmal Ca2+influx, Ca2+release from the sarcoplasmic reticulum, or Ca2+sensitivity of the contractile proteins. However, our FURA signal did not reveal changes in time to peak Ca2+transients, and it was demonstrated previously that ketamine does not show an effect on the Ca2+release channel. [7,9] Therefore, the most likely reason for faster contractile kinetics could be that the sensitivity of the contractile proteins is increased with a simultaneous reduction of peak force. In contrast to our results on the timing variables Kanaya et al.  found no effect of ketamine on time to peak shortening, which may be explained by the different experimental set-up (investigation of single rat ventricular myocytes). However, it was not our primary objective to investigate specific effects of ketamine on the contractile apparatus; more sensitive assays, for example on skinned fibers, are necessary to further investigate these questions.
From animal models it is known that there is stereoselective inhibition of catecholamine uptake by ketamine isomers, however, differing in quantity between smooth muscle and myocardium. [12,13] Our results show that maximal isometric developed force and maximal rate of force development were significantly increased after the perfusion with 73 [micro sign]M S(+)-ketamine compared with the initial control, whereas R(-)-ketamine caused no significant changes (Figure 2 and Figure 3). However, when isometric force and contractility were compared directly between racemic ketamine and both isomers at the concentration of 73 [micro sign]M, there was no difference between ketamine and its isomers. The power of this comparison was revealed as 0.085 (with P = 0.317) and 0.40 (with P = 0.055) for isometric force and contractility, respectively, which is lower than the desired power of 0.80. A possible difference may become significant only after investigation of more patients. The number of patients, however, is not unlimited because of ethical reasons. Therefore, it remains open whether there is a stereoselective effect of ketamine isomers at the concentration of 73 [micro sign]M in human myocardium, but our data strongly suggest this effect.
Although the ideal of investigating left-ventricular myocardium from normal hearts of healthy patients is unrealistic, the drawbacks of working with potentially diseased myocardium from the right-atrial auricle of patients treated with different drugs, such as [Greek small letter beta]-AR antagonists or calcium antagonists, on the morning of the operation should not be ignored. Nevertheless, because of the reported species differences in myocardium, we think that the investigation of human myocardium in our model is necessary for understanding more about the pharmacodynamics of ketamine and its isomers. The finding of a massive decrease of force when a [Greek small letter beta]-AR antagonist is added without ketamine can be explained only by the previously mentioned characteristics of the tissues investigated. In animal models with normal hearts the suppression of left-ventricular performance was only around 30%. [13,26] However, further experiments about the effect of [Greek small letter beta]-AR antagonists on human myocardium-especially on normal left-ventricular myocardium-would be necessary to investigate this interesting effect.
Limitations of FURA-2/AM measurements for intracellular Ca2+concentrations include the FURA-2/AM loading procedure and high intracellular FURA-2 concentrations (reviewed in Roe et al. ). To minimize the possibility that FURA-2 acts as a calcium buffer, we included only fibers of which isometric force decreased by not more than 10% from the control value before the FURA-2/AM loading procedure. A rundown of the FURA-2 signal may be caused by leaking, bleaching, and sequestration. Leaking is corrected for by the ratio imaging and slowed down by low temperatures.  The latter could not be applied in our experiments because we aimed for physiologically relevant experimental conditions. Photobleaching was minimized by darkening the room and the experimental chamber. Although it can be lowered by decreasing the oxygenation, [20,27] this is not appropriate when working with myocardium. However, quantitative comparisons of intracellular calcium transients were not the aim of our investigation, and all muscle strips were analyzed under the same controlled experimental conditions. Therefore it was reasonable, despite these limitations, to compare the relative changes of the peak calcium transients with each concentration of ketamine and its isomers.
We conclude from our data that the effect of ketamine on human myocardium results in a small increased contractile force at low concentrations (73 [micro sign]M) of racemic ketamine and S(+)-ketamine, and in decreased force at higher concentrations (730 [micro sign]M) of ketamine and its isomers. The positive inotropic effect of racemic ketamine can be blocked by addition of esmolol. The negative inotropic effect of high doses of racemic ketamine and its isomers is associated with a significant decrease in the intracellular calcium transient and it can be reversed by dobutamine.
The authors thank R. H. A. Fink and P. Young for their critical reading of the manuscript. The authors also thank K. Meyer and H. Bauer for statistical assistance.