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CARDIOVASCULAR ANESTHESIOLOGY

Etomidate Has No Effect on Hypoxia Reoxygenation and Hypoxic Preconditioning in Isolated Human Right Atrial Myocardium

Jean-Luc Hanouz, MD, PhD^*, Sandrine Lemoine, PhD, Lan Zhu, MD, Olivier Lepage, MD, Gerard Babatasi, MD, PhD, Massimo Massetti, MD, PhD, André Khayat, MD, Benoit Plaud, MD, PhD^*, and Jean-Louis Gérard, MD, PhD^*

From the *Department of Anesthesiology, Laboratory of Experimental Anesthesiology and Cellular Physiology, Department of Cardiac and Thoracic Surgery, CHU Caen, Caen Cedex, France.

Address correspondence and reprint requests to Dr. Jean-Luc Hanouz, Département d'Anesthésie-Réanimation, CHU de Caen, Avenue Côte de Nacre, 14033 Caen Cedex, France. Address e-mail to hanouz-jl{at}chu-caen.fr.

Abstract

BACKGROUND: We examined the effects of etomidate on recovery of contractile function after hypoxia reoxygenation and hypoxic preconditioning in vitro using isolated human myocardium.

METHODS: Human right atrial myocardium were obtained at the time of cardiac surgery from 38 adults patients. We recorded isometric force of contraction (FoC) of atrial trabeculae suspended in an oxygenated Tyrode's solution (34°C, stimulation frequency 1 Hz). In all groups, a 30-min hypoxic period was followed by 60 min of reoxygenation (HR). In separate groups, muscles were exposed to etomidate (10^–7, 10^–6, 10^–5 M) 10 min before and throughout the HR periods. Hypoxic preconditioning was induced by 4-min hypoxia followed by 7-min reoxygenation applied before HR periods. Etomidate 10^–5 M was administered before, throughout, and after the hypoxic preconditioning stimulus. Recovery of FoC (expressed as % of baseline value) at the end of HR was compared among groups.

RESULTS: Compared with the control group (FoC: 52% ± 10%), etomidate 10^–7 M (FoC: 57% ± 9%; P = 0.24), 10^–6 M (FoC: 61% ± 11%; P = 0.10), and 10^–5 M (FoC: 54% ± 9%; P = 0.29) did not modify the recovery of FoC after HR. Hypoxic preconditioning-induced increase in the recovery of FoC (87% ± 5%; P < 0.001 vs control group) was not modified in the presence of etomidate 10^–5 M (FoC: 86% ± 7%; P = 0.74 vs hypoxic preconditioning group).

CONCLUSIONS: Etomidate did not modify the in vitro FoC of human myocardium exposed to HR. Furthermore, etomidate did not modify the protective effect of hypoxic preconditioning.

Etomidate is an imadazole derivative that is often used for induction of anesthesia in patients with cardiac disease and patients who are hemodynamically compromised since it has minimal cardiovascular depressive effects.1–3 Such high-risk patients can suffer perioperative episodes of myocardial ischemia and reperfusion, which have been associated with increased morbidity and mortality.4 Despite extensive literature documenting the direct myocardial actions of etomidate,5–7 its effects on myocardial ischemia and reperfusion are reported in only one experimental study that showed that etomidate did not modify regional myocardial function in normal and acutely ischemic heart segments in dogs.8 Consequently, we tested the hypothesis that etomidate does not modify contractile force of isolated human myocardium exposed to hypoxia and reoxygenation. Ischemic preconditioning has been shown to be a powerful protective mechanism against subsequent myocardial ischemia and reperfusion injury.9 Little is known about the effects of etomidate on myocardial ischemic preconditioning. Zaugg et al. have shown that etomidate did not modify the diazoxide-induced opening of mitochondrial adenosine triphosphate-sensitive potassium channels, and important mechanisms of ischemic preconditioning.10 Thus, we further hypothesized that etomidate would not modify the protective effect of hypoxic preconditioning (PC) in isolated human myocardium.

METHODS

After the approval of local medical ethics committee and written informed consent, right atrial appendages were obtained from 38 patients scheduled for routine coronary artery bypass surgery or aortic valve replacement. The specimens were the normally discarded right atrial appendage removed during venous cannulation for cardiopulmonary bypass as previously described.11 In 32 patients, 1 trabeculae was dissected, but in 6 patients, 2 trabeculae were dissected from the appendage. All patients received total IV anesthesia with propofol and sufentanil. Patients with chronic atrial dysrhythmia, diabetes mellitus, and those taking oral hypoglycemic medications were excluded from the study.

Experimental Preparation
Atrial trabeculae were suspended vertically between an isometric force transducer (MLT0202, ADInstruments, Sydney, Australia) and a stationary stainless-steel clip in a 200-mL jacketed reservoir filled with daily prepared Tyrode's modified solution containing: 120 mM NaCl, 3.5 mM KCl, 1.1 mM MgCl₂, 1.8 mM NaH₂PO₄, 25.7 mM NaHCO₃, 2.0 mM CaCl₂, and 5.5 mM glucose. The jacketed reservoir was maintained at 34°C using a thermostatic water circulator (Polystat Micropros, Bioblock, Illkirch, France). The bathing solution was insufflated with carbogen (95% O₂–5% CO₂), resulting in a pH of 7.40 and a partial pressure of oxygen of 600 mm Hg. Isolated muscles were field-stimulated at 1 Hz by two platinum electrodes with rectangular wave pulses of 5 ms duration 20% above threshold (CMS 95107, Bionic Instrument, Paris, France).

The atrial trabeculae were equilibrated for 60–90 min to allow stabilization of their optimal mechanical performance at the apex of the length-active isometric tension curve (L_max). The force developed was measured continuously, digitized at a sampling frequency of 400 Hz, and stored on a Writable Compact Disk for analysis (Chart v 5.0.1 and PowerLab 4SP, ADInstruments).

At the end of each experiment, the length and the weight of the muscle were measured. The muscle cross-sectional area (CSA) was calculated from its weight and length assuming a cylindrical shape and a density of 1. To avoid core hypoxia, trabeculae included had to have a CSA <1.0 mm², a force of contraction normalized per CSA (FoC) >5.0 mN/mm², and a ratio of resting force/total force (RF/TF) <0.45. We have previously shown that mechanical variables of isolated human trabeculae remain stable at least 2 h.11

Experimental Protocol
In all experimental groups, hypoxia was achieved by replacing 95% O₂–5% CO₂ with 95% N₂–5% CO₂ in the buffer for 30 min, followed by a 60-min oxygenated recovery period. Atrial trabeculae were randomly assigned to one of the six experimental groups through sealed envelopes containing two numbered envelopes. The second envelope, indicating a different experimental group, was opened if a second atrial trabeculae was obtained from the same appendage.

In the control group (control; n = 10), muscles were exposed to a 30-min hypoxic period, followed by a 60-min oxygenation recovery period. In separate groups, trabeculae were exposed to etomidate 10^–7 M (n = 6), 10^–6 M (n = 6), and 10^–5 M (n = 6) administered for 10 min before hypoxia and throughout the experiments. These concentrations were chosen because they encompass the peak plasma concentrations of etomidate measured after rapid injection of 0.2 and 0.3 mg/kg corresponding to 3.6 x 10^–6 and 5.0 x 10^–6 M, respectively.12 Because etomidate undergoes plasma protein binding to the extent of 75%, the free plasma concentration should range from 0.5 x 10^–6 M to 0.3 x 10^–5 M.

Hypoxic PC (n = 10) was induced by a 4-min hypoxic period followed by a 7-min reoxygenation period. Hypoxic PC was applied before the 30 min hypoxia and 60 min reoxygenation periods as described above and elsewhere.11 The effects of etomidate on hypoxic PC were studied in the presence of etomidate 10^–5 M (PC + Etomidate 10^–5 M; n = 6). Etomidate was administered 10 min before, and for 5 min after the 4-min hypoxic PC stimulus. A 2-min wash out period was then performed just before the 30-min hypoxia and 60-min reoxygenation periods. Etomidate was purchased from Sigma Aldrich (St Quentin Fallavier; France) and dissolved daily in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the organ bath was 0.01%.

Statistical Analysis
Data are expressed as mean ± sd. The distribution of preoperative medications among groups was compared with a ² test. Age, left ventricular ejection fraction, baseline values of the main mechanical variables, and values of FoC at 60 min of reperfusion (FoC₆₀; major end-point of the study) were compared among groups by a one-way analysis of variance with Newman-Keuls post hoc analysis. Within-group data were analyzed over time using a two-way analysis of variance for repeated-measures and Newman-Keuls post hoc analysis. All P values were two-tailed, and a P value of <0.05 was required to reject the null hypothesis. Statistical analysis was performed using Statview 5 software (Deltasoft, Meylan, France).

RESULTS

Patient demographic data, preoperative drug treatment, main medical conditions, and preoperative left ventricular ejection fraction are reported in Table 1. There were no differences in patients' characteristics among groups. Forty-four human right atrial trabeculae were studied. Morphological characteristics and baseline values for the mechanical variables of atrial trabeculae are reported in Table 2. There were no differences in control values for L_max, CSA, RF/TF, and the main mechanical variables among groups.

Direct Myocardial Effects of Etomidate
Ten minutes of exposure to etomidate 10^–7 (FoC: 100% ± 2% of baseline) and 10^–6 (FoC: 99% ± 5% of baseline) did not modify FoC. In contrast, etomidate 10^–5 M (FoC: 91% ± 6% of baseline; P = 0.008) resulted in a decrease in FoC compared with the control group and the etomidate 10^–7 and etomidate 10^–6 M groups.

Effects of Etomidate on Hypoxia Reoxygenation
The time course of FoC among experimental groups is shown in Figure 1. After 30 min of hypoxia, the reduction in FoC was not different among groups (control: 10% ± 5% of baseline; etomidate 10^–7 M: 14% ± 7% of baseline; etomidate 10^–6 M: 12% ± 9% of baseline; etomidate 10^–5 M: 19% ± 10% of baseline; P = 0.51). Compared with the control group (FoC, 52% ± 10% of baseline), the recovery of FoC after 60 min of reoxygenation was not different in the etomidate 10^–7 M (FoC: 57% ± 9%; P = 0.24 vs control), 10^–6 M (FoC: 61% ± 11%; P = 0.10 vs control), or 10^–5 M (FoC: 54% ± 9%; P = 0.29 vs control) groups.

View larger version (20K): [in this window] [in a new window]   Figure 1. Time course of the force of contraction of human atrial trabeculae expressed as percent of baseline value in control group (n = 10), etomidate 10–7 M (n = 6), 10–6 M (n = 6), and 10–5 M (n = 6). NS (not significant) indicates the result of between groups comparison. *P < 0.05 vs baseline values and values measured during the hypoxia period. #P < 0.05 vs baseline values and values measured during the reoxygenation period.

Effects of Etomidate on Hypoxic PC
The time course of FoC for the hypoxic PC and PC + etomidate 10^–5 M groups compared with the control group is shown in Figure 2. Four minutes of hypoxic pretreatment induced a decrease in FoC (42% ± 12% of baseline), followed by complete recovery after 7 min of reoxygenation (106% ± 6% of baseline). The FoC in the PC + etomidate 10^–5 M group (20% ± 10% of baseline) was not different after 30 min of hypoxia compared with the control (15% ± 12% of baseline; P = 0.32) and hypoxic PC groups (19% ± 10% of baseline; P = 0.86).

View larger version (17K): [in this window] [in a new window]   Figure 2. Time course of the force of contraction of human atrial trabeculae expressed as percent of baseline value in control group (n = 10), hypoxic preconditioning (hypoxic PC, n = 10) and etomidate 10–5 M bracketing hypoxic PC (PC + etomidate 10–5 M, n = 6). Hypoxic preconditioning was induced by 4-min hypoxia followed by 7-min reoxygenation. NS (not significant) and P < 0.05 indicate the result of between groups comparison. *P < 0.05 vs baseline values and values measured during the hypoxia period. #P < 0.05 vs baseline values and values measured during the reoxygenation period.

After 60 min of reoxygenation, FoC in the PC + etomidate 10^–5 M group was higher than that observed at this time point in the control group (86% ± 7% vs 52% ± 10% of baseline; P < 0.001). There was no difference in the FoC after 60 min of reoxygenation in the hypoxic PC and PC + etomidate 10^–5 M groups (86% ± 7% vs 87% ± 5% of baseline; P = 0.74).

DISCUSSION

This study showed that etomidate in clinically relevant concentrations did not alter the effects of simulated myocardial ischemia (hypoxia-reoxygenation) on FoC of isolated human atrial myocardium. We found that FoC after 30 min of hypoxia and 60 min of reoxygenation was not different in the etomidate 10^–7, 10^–6, and 10^–5M groups compared with the control group. We further found that hypoxia PC resulted in an increased recovery of FoC after 30 min of subsequent hypoxia and 60 min of reoxygenation compared with the control group. This enhanced recovery induced by hypoxia PC was unchanged by etomidate 10^–5 M, suggesting that etomidate does not modify the beneficial effects of hypoxic PC on FoC.

Etomidate has become one of the most commonly used drugs for induction of anesthesia in patients with compromised cardiovascular function.1–3 This is mainly related to the small or negligible cardiovascular effects of this drug compared with other anesthetics.13,14 At clinical concentrations, etomidate has been shown to be devoid of negative myocardial inotropic effects in failing and nonfailing human myocardium.5 At supratherapeutic concentrations, etomidate has been shown to exert a negative inotropic effect attributed to decreased availability of calcium6 or direct inhibition of the calcium current.14

Despite extensive literature documenting the minimal effects of etomidate on myocardial contractility, there is little known about its effect during myocardial ischemia and reperfusion. To our knowledge, only one experimental study reported the effects of etomidate on myocardial function during acute ischemia in dogs, in vivo.8 These authors reported that a 30-min infusion of increasing concentrations of etomidate did not alter regional myocardial function during a 50% flow reduction in a distal branch of the left descending coronary artery. However, etomidate was administered 30-min after the beginning of ischemia, and the reperfusion phase was not studied. Our study showed that etomidate did not modify the FoC of isolated human atrial myocardium when given before and during hypoxia and reoxygenation. We can thus assume that etomidate did not trigger myocardial pre- or postconditioning signaling pathways. This is in accordance with the results of Zaugg et al. who showed that etomidate (10^–4 M) did not affect isolated rat cardiomyocytes viability after 60 min of ischemia.10 Additionally, these authors have shown that etomidate had no effect on diazoxide-induced mitochondrial adenosine triphosphate-sensitive potassium channels, which are an important signaling pathway for ischemic and anesthetic-induced myocardial preconditioning.11,9 Similarly, etomidate in clinical concentrations did not modify upregulation of human neutrophil adhesion to coronary endothelium after myocardial ischemia in contrast to ketamine, thiopental, midazolam, and propofol that all reduce this response.15

We further found that etomidate at 10^–5 M did not alter the beneficial effect of hypoxic PC on myocardial function. Ischemic preconditioning has been shown to be a potent protective mechanism against myocardial ischemia and reperfusion both in experimental models and in humans during coronary artery bypass graft surgery, reducing the extent of myocardial ischemic injury and improving myocardial function.9,16 Ischemic preconditioning can be triggered and mediated by a variety of signaling pathways including protein kinases A and C and mitogen-activated protein kinases such as Akt and Erk1/2.9 In Xenopus oocytes, Yun et al.17 suggested that etomidate may decrease protein kinase C activity. However, signaling pathways in Xenopus oocytes over-expressing glutamate transporter may not be representative of protein kinase C activity in cardiomyocytes. Etomidate has been shown to activate phosphorylation of the mitogen-activated protein kinase Erk1/2 through activation of _2B-adrenoceptors.18 However, at the present time, _2B-adrenoceptors have not been identified in the myocardium.19 Finally, it has been suggested that etomidate may inhibit the mitochondrial respiratory chain at the complex I level.20 However, complex III and not complex I have been suggested to be involved in myocardial ischemic preconditioning.21 Regardless, our findings do suggest that etomidate will not interfere with those pathways necessary for ischemic preconditioning.

Our study should be interpreted within the constraints of several limitations. The effects of anesthetics drugs, diseases, or medical treatments received by the studied patients before obtaining the atrial specimens cannot be excluded. However, the patients included in this study are representative of those patients in whom etomidate may be used for induction of anesthesia. Opioids have been shown to possess myocardial preconditioning effects and, thus, their potential modifying effects on our results cannot be excluded.22 Importantly, our investigation included a control group that would be equally affected by any of these potentially modifying factors. We used a 30-min hypoxic period to simulate myocardial ischemia. Our experimental design that used human atrial specimens would not allow for induction of myocardial ischemia via temporary coronary artery occlusion. It has been shown that anoxia is as effective as ischemia in inducing myocardial preconditioning.23 The concentration of etomidate in the organ bath and in atrial trabeculae was not measured. Consequently, the concentrations tested in our study are assumed to be equivalent to the concentrations used in the tissue bath. Our experiments were performed at 34°C, which may have decreased the protective effects of myocardial hypoxic PC.24 However, hypoxic PC was effective in the present model, and moderate hypothermia may occur during surgical procedures. Finally, the number of trabeculae included in the experimental groups has not been calculated based on a power analysis. Thus, a statistical type 2 error may not be totally excluded.

In conclusion, in isolated human atrial myocardium, we showed that etomidate did not modify myocardial function after hypoxia-reoxygenation. Further, the beneficial effects of hypoxic myocardial PC on myocardial contractility were not altered in the presence of etomidate.

Footnotes

Accepted for publication May 7, 2008.

Supported by the Université de Caen Basse Normandie and Centre Hospitalier Universitaire de Caen.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hanouz, J.-L. Articles by Gérard, J.-L. Search for Related Content PubMed PubMed Citation Articles by Hanouz, J.-L. Articles by Gérard, J.-L. Related Collections Cardiovascular Mechanisms Preclinical Pharmacology Pharmacology