Ketamine Preconditions Isolated Human Right Atrial Myocardium: Roles of Adenosine Triphosphate–sensitive Potassium Channels and Adrenoceptors

After the approval of the local medical ethics committee (Centre Hospitalier Universitaire Côte de Nacre, Caen, France), right atrial appendages were obtained during cannulation for cardiopulmonary bypass from patients scheduled to undergo routine coronary artery bypass surgery or aortic valve replacement. In all patients, anesthesia was induced with a target-controlled infusion of propofol, sufentanil or remifentanil, and pancuronium. Patients with atrial arrhythmia and those who were taking oral hypoglycemic medications were excluded from the study.

Right atrial trabeculae (one to two per appendage) were dissected and suspended vertically between an isometric force transducer (MLT0202; ADInstruments, Sydney, Australia) and a stationary stainless 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 reservoir was maintained at 34°C using a thermostatic water circulator (Polystat micropros; Bioblock, Illkirch, France). The bathing solution was bubbled with carbogen (95% O²–5% CO²), resulting in a pH of 7.40 and a partial pressure of oxygen of 600 mmHg. Isolated muscles were field stimulated at 1 Hz by two platinum electrodes with rectangular wave pulses of 5 ms in duration 20% above threshold (CMS 95107; Bionic Instrument, Paris, France).

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). At the end of the stabilization period, trabeculae were randomly assigned to one of the experimental groups summarized in figure 1. The force developed was measured continuously, digitized online at a sampling frequency of 400 Hz (PowerLab 4SP; ADInstruments), and stored on a Writable Compact Disc for analysis (Chart version 5.0.1; ADInstruments).

At the end of each experiment, the length and the weight of the muscle were measured. The muscle cross-sectional area was calculated from its weight and length assuming a cylindric shape and a density of 1. To avoid core hypoxia, trabeculae included in the study should have a cross-sectional area less than 1.0 mm², an active isometric force normalized per cross-sectional area (FoC) greater than 5.0 mN/mm², and a ratio of resting force/total force less than 0.50.

Experimental protocols are summarized in figure 1. In all groups, hypoxia was induced by replacing 95% O²–5% CO²with 95% N²–5% CO²in the buffer for 30 min, followed by a 60-min reoxygenation period (HR protocol). In the control group (n = 10), muscles were exposed to the HR protocol alone. In all other groups, a 10-min washout period preceded the HR protocol. Racemic ketamine and its S  (+) isomer were administered for 15 min at 10⁻⁶, 10⁻⁵, and 10⁻⁴m (n = 6 in each group). Mechanisms involved in ketamine-induced preconditioning were studied at 10⁻⁴m of racemic ketamine in the presence of 10⁻⁵m HMR 1098 (specific inhibitor of the sarcolemmal K^ATP(sarcK^ATP) channel opening; n = 6), 8.10⁻⁴m 5-hydroxydecanoate (5-HD; inhibitor of mitoK^ATPchannel opening; n = 6), 10⁻⁶m propranolol (β-adrenoceptor antagonist; n = 6) and phentolamine (α-adrenoceptor antagonist; n = 6). The effects propranolol (n = 6), phentolamine (n = 6), 5-HD (n = 6), and HMR 1098 (n = 6) alone were examined in separate pharmacologic control groups.

Propranolol, phentolamine, and 5-HD were purchased from ICN Pharmaceuticals (Orsay, France), and racemic ketamine and its S  (+) isomer were purchased from Sigma Aldrich (St. Quentin Fallavier, France). HMR 1098 was a gift from Aventis Pharma (Frankfurt am Main, Germany).

Data are expressed as mean ± SD. Baseline values of main mechanical parameters and values of FoC at 60 min of reperfusion were compared by univariate analysis of variance. If the F  value was significant (P < 0.05), Newman-Keuls post hoc  analysis was performed. Within-group data were analyzed over time using univariate analysis of variance for repeated measures and Newman-Keuls post hoc  analysis. All P  values were two tailed, and a P  value of less than 0.05 was required to reject the null hypothesis. Statistical analysis was performed using Statview 5 software (Deltasoft, Meylan, France).

Patients’ demographic data, preoperative treatments, and left ventricular ejection fractions are reported in table 1. Ninety-four human right atrial trabeculae were studied. There were no significant differences in control values for L^max, cross-sectional area, or FoC (table 2). Although differences were found between groups for ratio of resting force/total force, there was no muscle with a ratio of resting force/total force greater than 0.50 according to our selection criteria.

The time course of FoC for the control group and pharmacologic controls is shown in figure 2. Hypoxia induced a marked decrease in FoC (15 ± 14% of baseline after 30 min of hypoxia). In the control group, reoxygenation resulted in a partial recovery of FoC (FoC = 47 ± 10% of baseline at the end of the reoxygenation period; fig. 2).

As compared with the control group, the time course and recovery of FoC at the end of the reoxygenation period were not modified by 5-HD (51 ± 15% of baseline; not significant [NS]), HMR 1098 (49 ± 17% of baseline; NS), propranolol (46 ± 12% of baseline; NS), or phentolamine (50 ± 9% of baseline; NS).

Racemic ketamine at 10⁻⁶m (99 ± 2% of baseline; NS) at 10⁻⁵m (104 ± 6% of baseline; NS) and at 10⁻⁴m (99 ± 6% of baseline; NS) did not modify the FoC of atrial trabeculae. In contrast, S  (+)-ketamine induced a dose-dependent increase in FoC (S  (+)-ketamine at 10⁻⁶m: 103 ± 5% of baseline; NS; S  (+)-ketamine at 10⁻⁵m: 104 ± 3% of baseline; P = 0.04; S  (+)-ketamine at 10⁻⁴m: 108 ± 6% of baseline; P = 0.02). Phentolamine at 10⁻⁶m (99 ± 2% of baseline; NS) and propranolol at 10⁻⁶m (92 ± 4% of baseline; NS) did not modify FoC of atrial trabeculae. Racemic ketamine in the presence of propranolol (64 ± 14% of FoC value after propranolol; P = 0.002) but not phentolamine (92 ± 14% of FoC value after phentolamine; NS) induced a significant decrease in FoC reversed after a 10-min washout period (FoC: 90 ± 12% of baseline value).

Ketamine at 10⁻⁶, 10⁻⁵, and 10⁻⁴m, and its S  (+) isomer at 10⁻⁶, 10⁻⁵, and 10⁻⁴m enhanced the recovery of FoC at the end of the 60-min reoxygenation period as compared with the control group (table 3and fig. 3).

The enhanced recovery of FoC at the end of the reoxygenation period induced by racemic ketamine at 10⁻⁴m was abolished in the presence of 10⁻⁵m HMR 1098, 8.10⁻⁴m 5-HD, 10⁻⁶m propranolol, and 10⁻⁶m phentolamine (table 3and fig. 4).

Our study shows that a brief exposure to clinically relevant concentrations of racemic and S  (+)-ketamine preconditions isolated human myocardium as shown by the enhanced recovery of force after hypoxia–reoxygenation. Furthermore, we show that a 5-HD–dependent mechanism, the inhibition of sarcK^ATPchannel opening, and the specific blockade of α and β adrenoceptors abolished the preconditioning effect of ketamine.

Numerous studies have shown that volatile anesthetics precondition myocardium against ischemia. In clinical practice, anesthetic preconditioning has been shown to be associated with improved cardiac outcome after coronary artery bypass graft surgery.14Currently, there are few data about intravenous anesthetic preconditioning.9,15However, this could help to identify the best anesthetic association leading to the greatest cardioprotective effect against ischemia. This may be of importance in the perioperative period, when myocardial ische-mia frequently occurs in patients with ischemic heart disease, and in patients scheduled to undergo cardiac surgery with cardiopulmonary bypass.

Although ketamine may be used for induction of anesthesia in high-risk patients because it maintains hemodynamic variables, its effect on ischemia–reperfusion remains poorly studied and has led to controversial results. Haessler et al.  16have shown that ketamine/xylazine-based anesthesia resulted in an infarct volume comparable to that of isoflurane- or pentobarbitone-based anesthesia in rabbits undergoing 30 min of coronary occlusion followed by 3 h of reperfusion. In contrast, Cope et al.  17showed that ketamine/xylazine-based anesthesia resulted in an infarct volume comparable to that measured with pentobarbital- or propofol-based anesthesia but twice as large as that measured with volatile anesthetic–based anesthesia. Two recent studies suggested that ketamine may have a stereoselective inhibitory effect on ischemic preconditioning.1,2These authors also showed that a bolus administration of racemic ketamine or its R  (−) isomer before 30 min of myocardial ischemia did not modify infarct volume after 2 h of reperfusion in rabbit heart2or contractile function after 1 h of reperfusion in isolated rat heart.1In contrast, in rabbits anesthetized with a single injection of ketamine/xylazine followed by a continuous infusion of propofol, ischemic preconditioning was more effective in reducing the infarct volume than a 15-min administration of isoflurane at 0.5 minimal alveolar concentration.18Finally, Zaugg et al.  3showed that ketamine did not modify rat cardiomyocyte viability after simulated ischemia–reperfusion but that ketamine stereoselectively affected diazoxide-induced mitoK^ATPchannel opening. Several hypothesis may explain the discrepancies observed between these studies. First, Haessler et al.  studied rabbits anesthetized with α-chloralose, the main metabolite of which has been shown to precondition isolated rat myocytes and to potentiate the diazoxide-induced opening of mitoK^ATPchannels.3Second, xylazine associated with ketamine could be a confounding factor because it may limit preconditioning through xylazine-induced hyperglycemia and decrease in postsynaptic sympathetic activity.19,20Third, Molojavyi et al.  1anesthetized rats with halothane, the effect of which could not be totally excluded. Furthermore, these authors also suggested that isolated rat heart is not a suitable model to study anesthetic preconditioning because they did not observe isoflurane-induced preconditioning.8Fourth, the mode and duration of administration of ketamine and the concentration tested are not comparable between studies. Our results showing that racemic and S  (+)-ketamine precondition isolated human myocardium are in contrast with those previously reported in rat1,3and rabbit2myocardium. However, as discussed above, differences between species, experimental model, and concentrations of ketamine tested could explain this discrepancy.

Increasing evidence suggests that adrenoceptors are important triggers of ischemic and pharmacologic myocardial preconditioning.11,12,15We have previously shown that desflurane preconditioning was mediated, at least in part, via α- and β-adrenoceptor stimulation.13The current study also shows that ketamine may precondition isolated human myocardium, at least in part, via α- and β-adrenoceptor stimulation. It should be noted that the blockade either of α or β adrenoceptors completely abolished ketamine-induced preconditioning. The main hypothesis that could explain our result is that the preconditioning phenomenon is a graded response, not an “all-or-none” response. A threshold must be reached by a stimulus to trigger the preconditioning cascade.21Therefore, the blockade of α or β adrenoceptors may be sufficient to decrease the stimulus below the threshold required to trigger the preconditioning cascade. The stimulation of adrenoceptors by ketamine is dependent on species4–7but participates in the direct inotropic effect of ketamine in isolated human myocardium.7The origin of ketamine-induced adrenoceptor stimulation remains incompletely understood but has been related to inhibition of neuronal (i.e. , extracardiac and intrinsic cardiac neurons) and extraneuronal (nonneuronal cardiac adrenergic cells) catecholamine uptake.22 

The preconditioning signal triggered by α and β adrenoceptors may involve multiple actors of the adrenoceptor signal transduction pathway. Therefore, β-adrenoceptor stimulation leads to the activation of cyclic adenosine monophosphate-protein kinase A–dependent pathway, which has recently been shown to mediate ischemic preconditioning in dog myocardium.23α-Adrenoceptor stimulation activates phospholipases C and D, leading to the formation of inositol triphosphate, which induces release of Ca²⁺from the sarcoplasmic reticulum and diacylglycerol, which, in turn, activates protein kinase C, the crucial role of which is well known in ischemic and anesthetic preconditioning.10,15Finally, it has been suggested that a diversity of signaling pathways all seem to converge to inhibit glycogen synthase kinase 3β, regulating the mitochondrial permeability transition pore complex activity, which is a key component of cardioprotection.24 

Our results suggest that sarcK^ATPchannel opening is involved in ketamine-induced preconditioning because pretreatment with HMR1098 abolished the protective effect of ketamine. First, it should be emphasized that HMR 1098 is a specific blocker of sarcK^ATPchannels (IC⁵⁰= 0.8 μm).25An HMR 1098 concentration of 10 μm has been used to block more than 90% of sarcK^ATPchannels in various experimental models. Our result is in contrast with previous results showing that racemic ketamine may inhibit sarcK^ATPchannel activity in rat ventricular myocytes.26However, in that study, the activity of sarcK^ATPchannels was measured in nonphysiologic conditions, i.e. , adenosine triphosphate–free bath solution, pinacidil-induced activation of K^ATPchannels, isolated nonbeating myocytes, and patch clamp techniques that modify K^ATPchannel environment. Our study also showed that a 5-HD–dependent mechanism (inhibition of mitoK^ATPchannel opening, inhibition of respiratory chain complexes) was involved in ketamine-induced preconditioning. Zaugg et al.  3reported that ketamine did not alter flavoprotein oxidation in rat myocytes and that R  (−)- but not S  (+)-ketamine abolished diazoxide-induced flavoprotein oxidation. However, it has been suggested that it is no longer possible to assume that diazoxide and flavoprotein oxidation specifically indicate the involvement of mitoK^ATP.27Further studies are needed to determine the precise effects of ketamine on cardiac sarcK^ATPand mitoK^ATP.

Mitochondrial K^ATPchannels play a major role in ische-mic preconditioning, but recent studies have also shown that sarcK^ATPchannels are essential. Therefore, ischemic preconditioning cannot be conferred in transgenic mice with targeted deletion of Kir6.2, the pore-forming subunit of cardiac sarcK^ATPchannel.28Similarly, Toller et al.  29have shown that, in dogs, desflurane-induced preconditioning was dependent on both sarcK^ATPand mitoK^ATPchannels. However, in isolated human myocardium submitted to hypoxia–reoxygenation, we have shown that inhibition of sarcK^ATPchannels opening attenuated desflurane-induced recovery of contractile force.14Two main hypothesis may explain, at least in part, these discrepancies. First, the relative importance of sarcK^ATPor mitoK^ATPchannels during preconditioning may depend on species. The rapid heart rate of small animals may magnify the importance of sarcK^ATPchannels.28Second, recent findings suggest distinct roles of sarcK^ATPand mitoK^ATPchannels in myocardial ischemic preconditioning benefits on infarct volume and contractile recovery.30Third, different signaling pathways may be initiated depending on conditions and molecules tested. Further studies are required to determine the relative participation and the precise timing of activation of sarcK^ATPand mitoK^ATPchannels during ischemic and pharmacologic preconditioning.

Our results must be interpreted within the constraints of several possible limitations. First, the effects of anesthetics drugs, diseases, or treatments received by the patients cannot be totally ruled out. Therefore, patients taking oral hypoglycemic medications were excluded from the study. Importantly, we have previously reported that preoperative treatment such as β-adrenergic blocking drugs do not mask desflurane-induced adrenoceptor stimulation.13Theoretically, the use of opioids during anesthesia of patients included in this study could have preconditioned the appendage. However, comparisons have been made with control experiments. Nevertheless, a superimposed effect of opioids used during surgical procedures cannot be ruled out.31Second, rather than the true ischemia obtained by coronary occlusion, we used a 30-min anoxic superfusion to simulate ischemia. However, it has been shown, in various experimental models, that anoxia is as effective as ischemia in inducing preconditioning.32Third, we measured postischemic contractile function recovery but not the infarct size. However, it has been shown that the improved recovery of contractile function produced by preconditioning was proportional to reduced infarct size.33In addition, our results, as well as previous ones, showed that this model provides a useful tool to study the mechanisms involved in ischemic preconditioning in human myocardium.13Fourth, our results could have been affected by advanced age of the patients. Aging has been shown to impair ischemic and sevoflurane myocardial preconditioning.34Nevertheless, there was no difference in age between groups, and several studies have shown that ischemic preconditioning may be restored in aged patients and animals by food restriction or physical activity.35Fifth, our experiments were performed at 34°C, which may decrease the K^ATPchannel sensitivity36and the effect of preconditioning.37However, studies in isolated human right atrial myocardium must be performed at 34°C to obtain a stable isometric contraction during several hours. Furthermore, during surgical procedures, moderate hypothermia may occur in patients. Finally, because there is no protein in Tyrode’s solution, the concentrations of ketamine tested in our study are free concentrations. However, taking into account the 20% protein binding of ketamine, the range of concentrations tested included the free plasmatic concentrations of ketamine encountered in clinical anesthesia.38 

In conclusion, ketamine and its S  (+) isomer precondition isolated human right atrial myocardium. Ketamine-induced preconditioning involves, at least in part, K^ATPchannels and stimulation of α and β adrenoceptors.