Intravenous anesthetics etomidate, propofol, and midazolam produce negative inotropic effects of various degrees. The mechanism underlying these differences is largely unknown.
The effects of intravenous anesthetics on L-type Ca2+, transient outward and inward-rectifier K+ channel currents (ICa, IKto, and IK1) were compared in canine ventricular cells using the whole-cell voltage-clamp technique. ICa and IK were elicited by progressively depolarizing cells from -40 to +40 mV, and from -90 to +60 mV, respectively. The peak amplitude and time-dependent inactivation rate of ICa and IK were measured before, during, and after the administration of equimolar concentrations (5, 30, or 60 microM) of etomidate, propofol, or midazolam.
Exposure to etomidate, propofol, and midazolam produced a concentration-dependent inhibition of ICa. Midazolam was the most potent intravenous anesthetic; at 60 microM, etomidate, propofol, and midazolam decreased peak ICa by 16 +/- 4% (mean +/- SEM), 33 +/- 5%, and 47 +/- 5%, respectively. Etomidate, propofol, and midazolam given in a 60-microM concentration decreased IKto by 8 +/- 3%, 9 +/- 2%, and 23 +/- 3%, respectively. IK1 was decreased by 60 microM etomidate and midazolam by 20 +/- 6% and 14% +/- 5%, respectively. Propofol had no effect on IK1.
At equimolar concentrations, intravenous anesthetics decreased the peak ICa, IKto, and IK1 with various degrees of potency. Effects of anesthetics on ICa were significantly greater compared with their effects on K+ currents. These findings suggest that the negative inotropic actions of etomidate, propofol, and midazolam are related, at least in part, to decreased ICa. Some effects, such as IK inhibition, may partially antagonize effects of decreased ICa. Indeed, the final effect of these intravenous anesthetics on myocardium will be the sum of these and other sarcolemmal and intracellular effects.
Key words: Anesthetics, intravenous: etomidate; midazolam; propofol. Animal: dog. Current: calcium; potassium. Tissue: myocardium; ventricular.
Intravenous anesthetics etomidate, propofol, and midazolam are used to induce and maintain general anesthesia and to provide sedation during local and regional anesthesia. These agents can depress cardiovascular function in humans [1-3]and animals. [4,5]
The mechanisms underlying in vivo cardiovascular depression by intravenous anesthetics are not well understood but probably include a reduction in afterload [6,7]and preload [8,9]and direct myocardial depression. [10,11]Although the mechanisms of intravenous anesthetic-induced negative inotropic effects appear to be diverse, [6,11-14]increasing evidence suggests that these agents exert direct negative inotropic actions in vivo [1-5]and in vitro. [13,15,16],* Because changes in contractile force reflect an interaction between Calcium2+ influx and Potassium sup + efflux through the sarcolemma, Calcium2+ release and sequestration by the sarcoplasmic reticulum, activity of membrane Calcium2+ and Potassium sup + pumps, and the Calcium2+ sensitivity of the contractile proteins, it is possible that these agents may interfere with any one of these steps, thus decreasing contractility. Although all intravenous anesthetics have negative inotropic effects, researchers frequently contend that etomidate is the least potent. [4,11],** Preliminary studies implicate sarcolemmal ion channels as a potential site of intravenous anesthetic-induced negative inotropic action. ,*,***
Our goal was to gain greater insight into the mechanisms underlying the negative inotropic effects of intravenous anesthetics by evaluating and comparing the effects of etomidate, propofol, and midazolam on the high-threshold Calcium2+ current (ICa), low-threshold transient Potassium sup + current (IKto), and the inward-rectifier Potassium sup + current (IK1) in single canine ventricular cells using the whole-cell patch-clamp technique.
Materials and Methods
Preparation of Single Ventricular Cells
These experiments were approved by the Medical College of Wisconsin Animal Care Committee, and all experimental procedures strictly conformed to the standards of American Association for Accreditation of Laboratory Animal Care. Adult mongrel dogs weighing 15 to 25 kg were placed in a Plexiglas box and anesthetized with halothane. After surgical anesthesia was attained, the trachea was intubated and the lungs were ventilated with 1.5% halothane in oxygen. The chest was opened and the heart was excised rapidly and placed in cold Krebs' solution. Thin strips of the ventricular tissue were cut into 5-mm lengths and placed in cold cardioplegia solution composed of 10 mM NaCl, 60 mM KCl, 5 mM MgCl2, 20 mM glucose, 100 mM sucrose, 5 mM pyruvate, 20 mM taurine, 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 0.5 mM disodium salt of ethylenediamine tetraacetic acid (Sodium2-EDTA). The washed ventricular segments were placed in Calcium2+ -free Tyrode's solution composed of 140 mM NaCl, 5.4 mM KCl, 5 mM MgCl2, 5.5 mM glucose, 5 mM HEPES, 4 mg/ml collagenase (Type I, 161 units/mg; Worthington, Freehold, NJ), 2 mg/ml bovine albumin (Sigma Chemical Co., St. Louis, MO), 40 micro Meter CaCl2, 5 mM pyruvic acid, and 5.5 mM disodium salt of adenosine triphosphate (Sodium2-ATP); the pH was adjusted to 6.2 with NaOH. The solution containing ventricular tissue was incubated at 37 degrees Celsius for 1 to 2 h in a slow shaker. After this incubation, single ventricular cells were washed three times in cold potassium glutamate (K-glutamate) solution composed of 130 mM Potassium-glutamate, 5.7 mM MgCl2, 5 mM HEPES, 5.5 mM glucose, 5 mM Sodium2-ATP, and 0.12 mM Sodium2-EDTA; the pH was adjusted to 7.4 with KOH. Dispersed cells were stored in Potassium-glutamate solution at 4 degrees Celsius before use.
A drop of dispersed single canine ventricular cells was placed in a perfusion chamber (22 degrees Celsius) on the stage of an inverted microscope (Olympus IMT-2; Leeds Instruments, Minneapolis, MN) equipped with modulation contrast. At 500x magnification, a hydraulic micromanipulator (Narishige, Tokyo, Japan) was used to position heat-polished borosilicate patch pipettes with tip resistance of 4 to 6 M Ohm on the membrane of ventricular cells. High-resistance seals (3 to 30 G Ohm) were formed, after which the pipette patch was removed by negative pressure to obtain the electrical access to the whole cell as previously described. Whole-cell currents were elicited by 200-ms depolarizing pulses generated by a computerized system (pClamp software; Axon Instruments, Burlingame, CA) every 5 to 10 s. The currents were amplified by a List EPC-7 patch-clamp amplifier (Adams & List Associates, Great Neck, NY), and the amplifier output was low-pass filtered at 500 Hz. All data were digitized (sampling rate = 10,000/s) and stored on a hard disk to permit analysis at a later time. For the I sub Ca, the leak and capacitative currents were subtracted from each record by linearly summating scaled currents obtained during 10-mV hyperpolarizing pulses.
The external solution used to measure ICacontained: 10 mM BaCl2, 135 mM tetraethylammonium chloride, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH = 7.4). The pipette solution used to measure ICacontained: 130 mM CsCl, 5 mM adenosine triphosphate (magnesium salt), 5 mM ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), 10 mM HEPES, 1 mM MgCl2, and 10 mM glucose (pH = 7.2).
The external solution used to measure IKcontained: 135 mM NaCl, 5.5 mM dextrose, 0.5 mM MgCl2, 4 mM KCl, and 2 mM CaCl2(pH = 7.4). The pipette solution used to measure IKcontained: 125 mM potassium aspartate, 20 mM KCl, 10 mM EGTA, 5 mM adenosine triphosphate (magnesium salt), 1 mM MgCl2, and 5 mM HEPES (pH = 7.2).
Repetitive current-voltage curves were obtained in the control solution to monitor time-dependent changes in ICaand IK, followed by exposure to 5, 30, or 60 micro Meter etomidate, midazolam, or propofol. Effects of anesthetics on ICaand IKwere complete within 6 min and were reversible with washout.
The recommended intravenous doses to induce anesthesia are approximately 0.2 to 0.4 mg/kg for midazolam, 0.3 mg/kg for etomidate, and 2.5 mg/kg for propofol. It was reported that plasma protein-bound fraction of midazolam and propofol is 97%, whereas for etomidate it is 76%. Peak plasma concentrations during induction with these doses vary widely but are reported to be approximately 0.5 to 3 micro Meter for midazolam, [1,18-20]3 micro Meter for etomidate, and 50 micro Meter for propofol. [22,23],**** For our study, intravenous anesthetics were obtained commercially in their vehicles (2 mg/ml etomidate, 5 mg/ml midazolam, and 10 mg/ml propofol). Each anesthetic was diluted in a given extracellular solution to make 1 mM stock solution that was divided into aliquots, frozen at -15 degrees Celsius, and thawed for daily use. Measured volumes of the 1-mM concentration of each drug were diluted into a known volume of perfusate to obtain perfusate drug concentrations of 5, 30, and 60 micro Meter. Cells were randomly exposed to one of the anesthetic agents, at one of three concentrations. 4-aminopyridine (4-AP; Sigma Chemical Co.) and barium chloride (Barium2+; Fisher Scientific, Itasca, IL) were dissolved directly in the recording solution. Nifedipine (Sigma Chemical Co.) was dissolved in 70% ethanol to make 1 mM stock solution, yielding a final solvent concentration in the recording solution of 0.07%, which by itself did not affect the current. Cobalt chloride (Cobalt2+; Sigma Chemical Co.) was dissolved directly in the recording solution. Tetrodotoxin (TTX; Sigma Chemical Co.) was dissolved in the recording solution to make 1 mM stock solution.
Vehicle preparations of etomidate (350 mg/mL propylene glycol in H2O), propofol (Intralipid; 100 mg/ml soybean oil, 22.5 mg/ml glycerol, and 12 mg/ml egg lecithin in water), and midazolam (0.01% disodium edetate and 1% benzyl alcohol in water, adjusted to pH of 3 with 0.1 NHCl3) were also prepared and administered in separate groups of cells (n = 2 to 4) at the concentrations that would correspond to the highest drug concentration given in this study (60 micro Meter) over the same time period (6 to 10 min). None of the drug vehicles had any effect (change < 5%) on either ICa, IKto, or IK1.
All currents are expressed as means +/- SEM and were analyzed by two-way analysis of variance. If the F-test showed significance, Fisher's test for least significant differences was performed, with the level of significance designated at P less or equal to 0.05.
Calcium current (ICa) was generated by depolarizing pulses (10 mV; 200 ms) from a holding potential of -40 mV to a command potential as high as +40 mV. All experiments were performed with Barium sup 2+ (10 mM) as the charge carrier in place of external Calcium2+ (2 mM); this substitution increased the peak of ICaand decreased the rate of inactivation. In 64 cells (each group containing 5 to 9 cells), the threshold activation occurred at approximately -30 mV and the maximal activation was reached between -10 and 0 mV. This ICaresembled high-threshold, long-lasting (L-type) Calcium2+ channel current previously described by researchers in our laboratory and by others. [25-27] Figure 1(upper panel) illustrates the effects of 60 micro Meter etomidate (A), propofol (B), and midazolam (C) on the peak ICaelicited by the test pulse from a holding potential of -40 mV to 0 mV in three canine ventricular cells. Peak ICa(Figure 1(A-C), lower panel) was plotted as a function of membrane potential to analyze the effects of etomidate (A), propofol (B), and midazolam (C) on the current-voltage relations for ICaactivation. Figure 1(D) summarizes decreases of the peak ICaamplitude by all three anesthetics. Etomidate at concentrations of 5 micro Meter, 30 micro Meter, and 60 micro Meter decreased the peak ICaamplitude by 3 +/- 3% (n = 7, NS), 10 +/- 6% (n = 7, NS) and 16 +/- 4% (n = 6; P less or equal to 0.05), respectively. Propofol at concentrations of 5 micro Meter, 30 micro Meter, and 60 micro Meter also decreased the peak ICaamplitude by 10 +/- 3% (n = 6, NS), 21 +/- 4% (n = 9; P less or equal to 0.05), and 33 +/- 6% (n = 9; P less or equal to 0.05), respectively. In a dose-dependent manner, 5 micro Meter, 30 micro Meter, and 60 micro Meter midazolam decreased the peak ICaamplitude by 7 +/- 7% (n = 7, NS), 30 +/- 4% (n = 5; P less or equal to 0.05), and 47 +/- 5% (n = 8; P less or equal to 0.05), respectively.
At equimolar concentrations, midazolam was most potent among the intravenous anesthetics in inhibiting ICa. The effects of these agents on ICawere voltage independent (i.e., there was no difference in percentage decrease among different voltages).
Further analysis of the current traces produced by the depolarization step from a holding potential of -40 mV to 0 mV revealed that all three anesthetics not only decreased the peak ICabut also hastened the inactivation of ICaand thus further contributed to a decrease of total Calcium2+ influx. The inactivation phase of ICatrace was best fitted with a single exponential curve with an average control inactivation constant (tau) of 0.185 +/- 0.013 s. Although all three anesthetics decreased the inactivation constant at 60 micro Meter, etomidate given at 5 and 30 micro Meter did not significantly alter the inactivation constant (Table 1). Although propofol significantly decreased tau at two higher concentrations, it produced a significantly smaller (P less or equal to 0.05) decrease compared with the same concentration of midazolam. The effects of intravenous anesthetics on I sub Ca were readily reversible with washout.
During the 200-ms depolarizing pulses from -90 mV to consecutively more positive voltages as high as +60 mV, cells showed transient outward Potassium sup + current (IKto) and an inward-rectifier Potassium sup + current (IK1). To measure IKtoand IK1, all experiments were done in the presence of 20 micro Meter tetrodotoxin (TTX) and 2 mM cobalt chloride (Cobalt2+) in the external solution to block Sodium sup + and Calcium2+ currents, respectively. Addition of nifedipine (1 micro Meter) in the external solution produced no significant changes in IKamplitude, indicating that this current is not influenced by the Calcium2+ influx via the L-type Calcium2+ channel. Voltage-dependent IK1was activated approximately at voltages negative to -20 mV and was completely abolished by 1 mM Barium2+ in the external solution, as described previously. [28-30]Voltage-dependent IKtowas identified as IKto1subtype because it was blocked by 2 mM 4-aminopyridine (4-AP) in the external solution. [31-35]
(Figure 2(A)) shows actual recordings of the peak IK1and IKtoelicited by depolarizing pulses from a holding potential of -90 to -50 mV, and from -90 to +60 mV, respectively, in control solution and during exposure to 60 micro Meter etomidate, propofol, and midazolam in three ventricular cells. Peak IKtoand IK1were plotted as a function of membrane potential to analyze the mean anesthetic effect on the current amplitude (Figure 2(B)). At equimolar concentrations, etomidate, propofol, and midazolam caused reversible decreases in the peak IKtoamplitude (at +60 mV) by 8 +/- 3% (n = 8; P less or equal to 0.05), 9 +/- 2% (n = 6; P less or equal to 0.05), and 23 +/- 3% (n = 15; P less or equal to 0.05), respectively. At the same concentration, etomidate and midazolam produced reversible decreases in the peak IK1amplitude (at -50 mV) by 20 +/- 6% (n = 8; P less or equal to 0.05), and 14 +/- 5% (n = 15; P less or equal to 0.05). Propofol had no effect on IK1(n = 6). The effects of intravenous anesthetics on IKtoand IK1were not voltage dependent. The decrease of IKtoand IK1amplitude by intravenous anesthetics was completely reversed with washout.
At equimolar concentrations, all three anesthetics significantly decreased IKto; however, midazolam was more potent than etomidate and propofol (Figure 2(C)). At the same concentration, etomidate and midazolam produced similar and statistically significant decreases in IK1, whereas propofol had no effect (Figure 2(D)).
Similar to ICa, IKtocurrent that was obtained by a depolarization step from a holding potential of -90 mV to +60 mV was also fitted with a single exponential curve to study its inactivation kinetics. The control inactivation constant (tau) was 0.034 +/- 0.0014 s. Neither etomidate nor propofol changed the inactivation constant but midazolam decreased the inactivation constant by nearly 40% (Table 1).
Intravenous anesthetics cause various degrees of cardiovascular depression in vivo [1-5]and in vitro. [13,15,16],** The differences in cardiovascular depression could result from their differential effects on systemic vascular resistance, [6,7]venous capacitance, [8,9]the autonomic nervous system, and the heart. [10,11]
Etomidate produces only minimal effects on cardiovascular dynamics [2,11]and therefore is widely recommended for patients with compromised cardiac function and hypotension. On the other hand, propofol produces cardiovascular depression to a larger extent than does etomidate, [4,11],** and thus propofol should be used cautiously in patients with hypovolemia. It was reported that induction of anesthesia with midazolam, even in patients with limited coronary flow, was accompanied by no change in cardiac output or central venous pressure and only a modest reduction in peripheral vascular resistance. At equimolar concentrations as great as 50 micro Meter in isolated guinea pig heart preparation, propofol has been shown to be more potent than etomidate and less potent than midazolam in depressing myocardial contractility. However, the authors suggested that at equivalent induction concentrations (concentration that produces the same depth of anesthesia), propofol would be the most potent, midazolam less potent, and etomidate the least potent negative inotrope. Despite the extensive literature documenting differences in negative inotropic effects among intravenous anesthetics, very little is known about the mechanisms underlying these differences.
Our purpose in this study was to identify and compare the actions of etomidate, propofol, and midazolam on macroscopic Calcium2+ and Potassium sup + currents in isolated canine ventricular myocytes to identify possible mechanisms for the observed differences in negative inotropic effects of these anesthetics.
Inward Calcium2+ current (ICa), transient outward Potassium sup + current (IKto), and inward-rectifier Potassium sup + current (IK1) contribute to the electrical activity in human, rabbit and canine ventricular myocytes and are the major determinants of the action potential duration in these cells. [34,39]The efflux of Potassium sup + through Potassium sup + channels is functioning as an important modulator of the action potential in canine ventricular myocytes, [35,40,41]offsetting Calcium2+ current and thus preventing early slow-response action potentials while maintaining a high resting membrane potential in latent pacemaker cells. 
Our results show that etomidate, propofol, and midazolam reduce the amplitude of ICa, IKto, and IK1in these cells. Furthermore, these three channel types were not equally sensitive to block by these anesthetics, and ICawas blocked more effectively than IKtoor IK1. Midazolam was the most potent in decreasing peak ICa, whereas etomidate was the least potent. A recent study using the whole-cell and single-channel recordings in guinea pig ventricular myocytes found that 4.4 and 27.4 micro Meter etomidate decreased the whole-cell L-type ICa. Similar concentrations of etomidate did not produce attenuation of ICain this study, suggesting the possibility of differences between species (guinea pig vs. dog). Another possibility is that the large decrease in ICaobserved by Takahashi and Terrar is partially due to a significant ICarundown in guinea pig myocytes as reported by the same authors. The relative potency of intravenous anesthetics in depressing ICacorrelates well with in vitro and in vivo depression of contractility, supporting the hypothesis that the block of ICais one of the most important factors in determining the negative inotropic effect of intravenous anesthetics. Effects of all three anesthetics on ICawere dose dependent and readily reversible.
In addition to decreasing the peak current, intravenous anesthetics could decrease a total ICaby increasing the rate of inactivation of open Calcium2+ channels. Indeed, analysis (single exponential fit) of the inactivation portion of the ICarevealed that midazolam dramatically increased the rate of ICainactivation. Propofol was less potent, whereas only 60 micro Meter etomidate significantly increased the rate of ICainactivation. Absence of the effect of 5 micro Meter etomidate on ICainactivation rate that we found correlates with the findings of Takahashi and Terrar, who reported no effect of a similar dose of etomidate on the single Calcium2+ channel kinetics. At 27.4 micro Meter, etomidate reduced the mean open time and increased the mean closed time, favoring the closed state without any effect on conductance. In our study, 30 micro Meter etomidate did not alter the ICainactivation rate, which could be consistent with the relatively greater effect seen in their study. The same group reported that a high concentration of propofol (100 micro Meter) also decreased the mean open time and increased the mean closed time without changing ICachannel conductance. Decreased peak current and increased rate of inactivation by propofol seen in this study are consistent with their observations at the single-channel level. The increased rate of ICainactivation suggests that midazolam, and to a lesser degree propofol and etomidate, bind to the open Calcium2+ channel and through an interaction with the gating mechanism increase the rate of transition of the channel from an open to inactivated state. Further studies investigating frequency and voltage dependence, rate of recovery from inactivation, and the single-channel recordings are needed to determine the exact nature of this interaction.
Midazolam was the most potent and propofol the least potent in depressing IKtoin ventricular cells. The decrease in amplitude of IKtoby these anesthetics suggests that these agents may influence the phase 1 repolarization and early plateau phase of the action potential. These alterations of plateau voltage may result in changes in other voltage-dependent currents during the plateau phase, including augmented ICa, and therefore cytosolic Calcium2+ concentration, contractility, and duration of the action potential in the ventricular myocytes. [31,38]However, the magnitude of IKdecrease may not be able to overcome a large decrease in ICa, as suggested by shortened action potential duration in the presence of propofol. It is of great interest that midazolam increased the rate of IKtoinactivation, whereas propofol and etomidate did not change channel inactivation. The mechanism(s) underlying similar effects of midazolam on ICaand IKtoinactivation are not clear.
Etomidate exerted the greatest depressant effect on IK1, whereas propofol had no effect. Baum has reported the lack of propofol's effect on IK1in guinea pig ventricular myocytes. Blockade of IK1can affect the duration of the action potential and the rate of repolarization because this current is activated during the repolarization of the cardiac action potential. This current plays a critical role in determining the amplitude and shape of the subthreshold response, [29,48]thus influencing the excitability of ventricular myocytes. Therefore, a depressant effect of intravenous anesthetics on the outward IKwould alter resting membrane potential and affect action potential duration and cellular excitability.
Despite the inhibition of both ICaand IK, the greater decrease of ICamay be responsible for the negative inotropic effect observed with these agents. Although midazolam produced the largest decrease of ICa, the final effect on myocardial contractility would be dramatically attenuated due to a decrease of I sub K and its significantly smaller induction dose. Because the induction concentration of midazolam is less than 5 micro Meter, midazolam would probably be free of effects on ICawhen anesthesia is induced.
Etomidate, propofol, and midazolam produced a dose-dependent decrease of Calcium2+ influx and a type-dependent decrease of Potassium sup + efflux. These anesthetics, particularly midazolam, also increased the rate of ICaand IK(only midazolam) inactivation, thus further decreasing the total current. Our data suggest that cardiac depression caused by intravenous anesthetics is due, at least in part, to a decreased amplitude of ICa, as directly measured by the whole-cell voltage-clamp method. The relative magnitude of a decrease in ICacorrelates well with clinically and experimentally observed negative inotropic effects of these anesthetics. These membrane alterations probably interact with other actions of these intravenous anesthetics, including the changes in Potassium sup + currents leading to an overall effect on myocardial contractility and excitability.
*Rusy BF, Thomas-King PY, King GP, Komai H: Effects of propofol on the contractile state of isolated rabbit papillary muscles under various stimulation conditions (Abstract). Anesthesiology 1990; 73:A560.
**Williams JP, McArthur DJ, Walker EW, Rietsema K, Teunissen E, Bonnenkamp H, Goderie P, Stanley HT: A comparison of the hemodynamics of Diprivan (propofol), thiopental, and etomidate for induction of anesthesia in patients with coronary artery disease. Semin Anesth 1988; 7:112-5.
***Wegrzynowicz ES, Matsuda J, Volk K, Shibata E, Wachtel R: Propofol decreases voltage activated calcium currents in rabbit heart myocytes (Abstract). Anesthesiology 1990; 73:A592.
****White PF: Propofol: Pharmacokinetics and pharmacodynamics. Semin Anesth 1988; 7:4-20.