The authors examined the role of adenosine triphosphate-sensitive potassium (K(ATP)) channels, adenosine A1 receptor, and alpha and beta adrenoceptors in desflurane-induced preconditioning in human myocardium, in vitro.
The authors recorded isometric contraction of human right atrial trabeculae suspended in oxygenated Tyrode's solution (34 degrees C; stimulation frequency, 1 Hz). Before a 30-min anoxic period, 3, 6, and 9% desflurane was administered during 15 min. Desflurane, 6%, was also administered in the presence of 10 microm glibenclamide, a K(ATP) channels antagonist; 10 microm HMR 1098, a sarcolemmal K(ATP) channel antagonist; 800 microm 5-hydroxy-decanoate (5-HD), a mitochondrial K(ATP) channel antagonist; 1 microm phentolamine, an alpha-adrenoceptor antagonist; 1 microm propranolol, a beta-adrenoceptor antagonist; and 100 nm 8-cyclopentyl-1,3-dipropylxanthine (DPX), the adenosine A1 receptor antagonist. Developed force at the end of a 60-min reoxygenation period was compared (mean +/- SD).
Desflurane at 3% (95 +/- 13% of baseline), 6% (86 +/- 6% of baseline), and 9% (82 +/- 6% of baseline) enhanced the recovery of force after 60 min of reoxygenation as compared with the control group (50 +/- 11% of baseline). Glibenclamide (60 +/- 12% of baseline), 5-HD (57 +/- 21% of baseline), DPX (63 +/- 19% of baseline), phentolamine (56 +/- 20% of baseline), and propranolol (63 +/- 13% of baseline) abolished desflurane-induced preconditioning. In contrast, HMR 1098 (85 +/- 12% of baseline) did not modify desflurane-induced preconditioning.
In vitro, desflurane preconditions human myocardium against simulated ischemia through activation of mitochondrial K(ATP) channels, adenosine A1 receptor, and alpha and beta adrenoceptors.
MYOCARDIAL ischemic preconditioning, i.e. , pretreatment with transient ischemia, initially referred to the reduction in infarct volume 1following a sustained ischemia, but its definition has been extended to include the beneficial effects on ischemia- and reperfusion-induced myocardial stunning. 2,3The involvement of adenosine triphosphate-sensitive potassium (KATP) channels, especially mitochondrial ones (mitoKATP), has been shown using selective antagonists or openers. 4,5Furthermore, stimulation of various sarcolemmal receptors, such as adenosine subtype 1 (A1) receptor, 6α and β adrenoceptors, 7,8and δ-opioid receptor, 9has been shown to mimic ischemic preconditioning.
A growing body of evidence indicates that volatile anesthetics may precondition the myocardium against ischemia and infarction. 10–14Isoflurane-induced preconditioning of the myocardium has been related to the activation of KATPchannels, 10,11stimulation of adenosine A1receptor, 11and mechanogated channels. 12Recently, sevoflurane and desflurane have been shown to precondition canine myocardium in vivo through activation of KATPchannels. 13,14Because we have recently shown that desflurane may induce intramyocardial catecholamines release in human myocardium in vitro , 15we tested the hypothesis that desflurane may also precondition isolated human myocardium through stimulation of α and β adrenoceptors. Furthermore, because halothane has been shown to protect rabbit 16and rat 17but not human 11myocardium against ischemia and because species differences remain a critical issue in experimental studies, we reexamined the involvement of KATPchannels and adenosine A1receptor in desflurane-induced preconditioning of human myocardium in vitro .
Materials and Methods
After approval of the local medical ethics committee (Caen, France), right atrial appendages were obtained during cannulation for cardiopulmonary bypass from patients scheduled for routine coronary artery bypass surgery or aortic valve replacement. All patients received midazolam or propofol, sufentanil, etomidate, pancuronium, and isoflurane. 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 (UC3; Gould, Cleveland, OH) 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 11 mm glucose. The jacketed reservoir was maintained at 34°C with use of 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 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 (Lmax). At the end of the stabilization period, trabeculae were randomized to experimental groups detailed below. The force developed was measured continuously, digitized at a sampling frequency of 400 Hz, and stored on a Writeable Compact Disc for analysis (MacLab; AD Instrument, Sydney, Australia).
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 (AF) greater than 5.0 mN/mm2, and a ratio of resting force/total force (RF/TF) less than 0.45.
In a time control group (n = 10), we measured the AF of isolated human atrial trabeculae every 10 min during 120 min.
In all other groups, ischemia—reperfusion was simulated by replacing 95% O2–5% CO2with 95% N2–5% CO2in the buffer for 30 min, followed by a 60-min oxygenated recovery period (I-R protocol).
In the control group (control; n = 11) muscles were exposed to the I-R protocol alone. Anoxic preconditioning (APC; n = 6) was induced by a 4-min anoxic period followed by a 7-min oxygenated period before the I-R protocol. 9,11The mechanisms involved in APC were studied by 15 min of pretreatment with 10 μm glibenclamide (n = 7), a nonselective KATPchannel antagonist; 10 μm HMR 1098 (n = 6), a selective sarcolemmal KATPchannel antagonist; 800 μm 5-hydroxydecaoate (5-HD; n = 7), a selective mitochondrial KATPchannel antagonist; 0.1 μm 8-cyclopentyl-1,3-dipropylxanthine (DPX; n = 6), a selective adenosine A1receptor antagonist; 1 μm propranolol (n = 6), a β-adrenoceptor antagonist; and 1 μm phentolamine (n = 6), an α-adrenoceptor antagonist. Concentrations used have been validated in previous experimental studies in human myocardium in vitro . 2,11,15,18
In the desflurane treatment groups, desflurane was delivered to the organ bath by bubbling with 95% O2–5% CO2passing through a specific calibrated vaporizer. Desflurane concentration in the carrier gas phase was measured with an infrared calibrated analyzer (Capnomac; Datex, Helsinki, Finland). After a 15-min exposure to 3% (n = 5), 6% (n = 6), and 9% (n = 5) desflurane, muscles underwent the I-R protocol. Mechanisms involved in desflurane-induced preconditioning were studied with 6% desflurane. Thus, 6% desflurane was administered after 15 min of pretreatment with 10 μm glibenclamide (n = 6), 10 μm HMR 1098 (n = 6), 800 μm 5-HD (n = 6), 10 nm DPX (n = 6), 1 μm propranolol (n = 6), and 1 μm phentolamine (n = 6).
In additional groups, the I-R protocol was performed after 15 min of pretreatment with 10 μm glibenclamide (n = 4), 10 μm HMR 1098 (n = 4), 800 μm 5-HD (n = 4), 0.1 μm DPX (n = 4), 1 μm propranolol (n = 4), and 1 μm phentolamine (n = 4).
Glibenclamide, 5-HD, DPX, and phentolamine were purchased from ICN Pharmaceuticals (Orsay, France), and desflurane was purchased from GlaxoWellcome (Marly-le-Roi, 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 AF at 60 min of reperfusion were compared by a univariate analysis of variance (ANOVA). If an F value was less than 0.05, Newman-Keuls post hoc analysis was used. Within-group data were analyzed over time using univariate ANOVA 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).
One hundred forty-two human right atrial trabeculae were studied. There were no differences in baseline values for Lmax, cross-sectional area, RF/TF, and AF among all groups (table 1).
Stability with Time of Isolated Human Atrial Trabeculae
In the time control group, AF slightly decreased with time (fig. 1). This decrease became significant at 80 min (AF: 93 ± 7% of baseline;P < 0.05). At 120 min, AF was 90 ± 10% of baseline. Resting force was not significantly modified with time and was 97 ± 10% of baseline at 120 min.
Effects of Simulated Ischemia and Reperfusion on Contractile Force of Human Right Atrial Trabeculae
Figure 1shows the time course of AF for the control group. Simulated ischemia induced a marked decrease in AF. After 30 min of simulated ischemia, AF was 14 ± 10% of baseline. The 60-min reoxygenation period resulted in a recovery of AF at 50 ± 11% of baseline (figs. 1 and 2).
Effects of Glibenclamide, 5-HD, HMR 1098, DPX, Propranolol, and Phentolamine on Simulated Ischemia-Reperfusion
The decease in AF induced by pretreatment with glibenclamide (AF: 94 ± 4% of baseline), 5-HD (AF: 94 ± 1% of baseline), HMR 1098 (AF: 86 ± 12% of baseline), DPX (AF: 97 ± 3% of baseline), propranolol (AF: 89 ± 4% of baseline), and phentolamine (AF: 96 ± 2% of baseline) was not different among groups. As shown in figure 1the time course of AF in the control group was not modified by 15 min of pretreatment with glibenclamide, 5-HD, HMR 1098, DPX, propranolol, and phentolamine. The recovery of AF at 60 min of reoxygenation measured in the control group (50 ± 11% of baseline) was not different from groups pretreated with glibenclamide (61 ± 7% of baseline), 5-HD (56 ± 11% of baseline), HMR 1098 (62 ± 6% of baseline), DPX (47 ± 4% of baseline), propranolol (53 ± 15% of baseline), and phentolamine (58 ± 8% of baseline).
Mechanisms of Anoxic Preconditioning on Human Right Atrial Trabeculae
Figure 2shows the time course of AF for APC group. The 4-min anoxic challenge induced a marked decrease in AF (37 ± 16% of baseline) followed by complete recovery after 7 min of reoxygenation (107 ± 5% of baseline). In the APC group, AF after 30 min of simulated ischemia was 22 ± 10% of baseline. At the end of the 60-min reoxygenated period, the recovery of AF in the APC group was significantly greater than that measured in the control group (91 ± 3 vs. 50 ± 11% of baseline;P < 0.05).
As shown in figure 3, the enhanced recovery of AF induced by APC was significantly decreased by pretreatment with glibenclamide (50 ± 11% of baseline), HMR 1098 (59 ± 15% of baseline), 5-HD (56 ± 19% of baseline), and DPX (63 ± 16% of baseline) and was no more different from the recovery of AF measured in the control group. In contrast, phentolamine (90 ± 19% of baseline) and propranolol (97 ± 6% of baseline) did not modify the enhanced recovery of AF induced by APC (fig. 3).
Direct Inotropic Effects of Desflurane
Desflurane at 3% (93 ± 2% of baseline;P < 0.05), 6% (87 ± 12% of baseline;P < 0.05), and 9% (76 ± 5% of baseline;P < 0.05) induced a dose-dependent decrease in AF. At a concentration of 6%, the desflurane-induced decrease in AF was not different among groups pretreated with glibenclamide (AF: 90 ± 7% of baseline), 5-HD (AF: 78 ± 6% of baseline), HMR 1098 (AF: 77 ± 10% of baseline), DPX (AF: 89 ± 4% of baseline), propranolol (AF: 72 ± 8% of baseline), and phentolamine (AF: 67 ± 15% of baseline). As shown in figure 4, the decrease in AF induced by 6% desflurane was significantly greater in the presence of propranolol and phentolamine.
Effects of Desflurane on Simulated Ischemia-Reperfusion
As depicted in figure 5, 15 min of exposure to desflurane at 3% (AF: 86 ± 6% of baseline), 6% (AF: 5 ± 13% of baseline), and 9% (AF: 82 ± 6% of baseline) prior to the 30-min anoxic period resulted in a significant increase in the recovery of AF after 60 min of reoxygenation as compared with the control group (AF: 50 ± 11% of baseline). Recovery of AF at 60 min of reoxygenation measured in the 3, 6, and 9% desflurane groups was not different from that measured in APC group (fig. 5).
Pretreatment with glibenclamide (60 ± 12% of baseline), 5-HD (57 ± 21% of baseline), DPX (63 ± 19% of baseline), phentolamine (56 ± 20% of baseline), and propranolol (63 ± 13% of baseline) abolished desflurane-induced enhanced recovery of AF. In contrast, pretreatment with HMR 1098 (85 ± 12% of baseline) did not modify the enhanced recovery of AF induced by desflurane (fig. 6).
The main results of our study are as follows: brief exposure to desflurane (3, 6, and 9%) preconditions isolated human right atrial myocardium against 30 min of simulated ischemia; mechanisms involved in desflurane-induced preconditioning are opening of mitochondrial KATPchannels and stimulation of adenosine A1receptor and α and β adrenoceptors.
Strong evidence supports the cardioprotective effects of volatile anesthetics against prolonged ischemia. Isoflurane has been shown to decrease infarct size in canine myocardium in vivo 10and to improve functional recovery of isolated human myocardium. 11Recently, sevoflurane and desflurane have been shown to exert similar cardioprotective effects in dogs. 13,14KATPchannels have been shown to play a pivotal role in mediating anesthetic-induced preconditioning. 10–14It has been suggested that activation of both sarcolemmal KATP(sarcKATP) and mitoKATPcould be involved in the cardioprotection conferred by volatile anesthetics. 14Furthermore, the participation of adenosine A1receptor stimulation and mechanogated channel activation has recently been suggested in isoflurane-induced preconditioning. 11,12The present results confirm and extend findings of Toller et al. , 14who showed that brief exposure to desflurane exerts cardioprotective effects against irreversible ischemia. Our study showed that 15 min of exposure to desflurane prior to 30 min of simulated ischemia enhanced contractile recovery of isolated human myocardium during the reoxygenation period. In addition, we showed that this effect was blocked by glibenclamide, indicating that opening of KATPchannels was implicated. Furthermore, specific blockade of mitoKATPchannels with 5-HD abolished desflurane-induced preconditioning, suggesting that opening of mitoKATPchannels is involved in desflurane-induced preconditioning. In contrast, specific blockade of sarcKATPchannels with HMR 1098 did not abolished desflurane-induced preconditioning but abolished APC. These results suggest that opening of sarcKATPis involved in APC but not in desflurane-induced preconditioning. Although current opinion favors a predominant role for mitoKATPchannels in ischemic preconditioning, there is evidence demonstrating that sarcKATPchannels are important mediators of protection during the reoxygenation phase of injury. 19,20At this time, the precise role and timing of activation of sarcKATPand mitoKATPchannels during ischemic preconditioning remains unresolved. The results of Toller et al. 14showing that both mitoKATPand sarcKATPchannels were implicated in desflurane-induced preconditioning are not in accordance with our results showing that the specific inhibition of sarcKATPchannels failed to abolished desflurane-induced preconditioning. This discrepancy may be related to major differences in experimental models. First, it should be emphasized that HMR 1098 should be an effective blocker of sarcKATPchannels at 10 μm since its IC50value has been reported at 0.8 μm. 20,21Thus, an HMR 1098 concentration of 10 μm has been used to block more than 90% of sarcKATPchannels in various experimental models. 18–21Second, Toller et al. 14measured myocardial infarct size after 60 min of coronary artery occlusion and 3 h of reperfusion, whereas we measured recovery of force of contraction in isolated myocardium after 30 min of anoxia and 60 min of reoxygenation. Recent findings suggest distinct roles of sarcKATPand mitoKATPchannels in myocardial ischemic preconditioning benefits in infarct volume and contractile recovery. 19,20Third, we administered KATPchannels blockers prior to the administration of desflurane, whereas Toller et al. 14administered KATPchannels blockers prior to and during the administration of desflurane. The timing of pharmacologic agent administration has been suggested to be critical in the preconditioning phenomenon. 7,22Fourth, interspecies differences may be a factor that helps to explain the different results. Thus, halothane has been shown to precondition rabbit 16but not rat 17and human 11myocardium, and isoflurane has been shown to precondition rabbit 12and human 11but not rat 23myocardium. Finally, differences between atrial and ventricular myocardium, influence of surgical stress, and barbiturate anesthesia in dogs cannot be ruled out.
The opening of KATPchannels has been shown to be an important mediator of ischemic and pharmacologic preconditioning. 3–6Initially, it has been proposed that the decrease in action potential duration induced by opening of sarcKATPresulted in better preservation of energy stores and suppression of deleterious downstream events, such as Ca2+overload. However, a lack of correlation between the extent of action potential shortening and the reduction of infarct size has been shown. 24Furthermore, cardioprotection conferred by KATPchannel openers has been shown to occur on quiescent myocardium. 18The participation of mitoKATPin ischemic preconditioning has been supported by numerous studies using specific antagonists or openers. 4,5,18The mechanism of mitoKATP-induced cardioprotection may involve alterations in mitochondrial Ca2+handling, the optimization of energy production, and modulation of reactive oxygen species during ischemia or reperfusion. The precise role and importance of sarcKATPand mitoKATPduring ischemic preconditioning remain unresolved. At the present time, the influence of volatile anesthetics on KATPchannel function has been poorly studied. Recently, Roscoe et al. 11showed that isoflurane does not modify sarcolemmal KATPactivation, whereas halothane partially blocked it in isolated human myocytes. These findings suggest that cardioprotective effects of isoflurane do not implicate a direct effect on sarcKATPchannel function but rather an effect on mitoKATPand upstream intermediates, such as G protein-coupled receptors and PKC. Thus, it has been shown that volatile anesthetic-induced cardioprotection was attenuated by adenosine A1receptor antagonist 11,16and PKC inhibitors. 25In addition, it has recently been shown that activation of Giproteins was implicated in isoflurane-induced preconditioning. 26Further studies are needed to determine the precise effects of volatile anesthetics on KATPchannels and signaling pathways leading to the preconditioned state.
This is the first study showing that specific blockade of adenosine A1receptors with DPX abolishes the desflurane-enhanced postischemic recovery of force. Previous results showed that cardioprotection conferred by isoflurane was mediated through activation of adenosine A1receptors, 11,16suggesting a role of adenosine in volatile anesthetic-induced preconditioning. However, further studies are required to elucidate the mechanisms through which volatile anesthetics interact with adenosine receptors.
Our findings show that specific blockade of α and β adrenoceptors abolishes the desflurane-enhanced postischemic contractile function recovery. These results strongly suggest that stimulation of α and β adrenoceptors plays a role in desflurane-induced preconditioning. In contrast to other volatile anesthetics, desflurane has been reported to induce sympathetic activation in healthy volunteers 27but also to release intrinsic store of catecholamines in isolated rat 28and human 15myocardium. A growing body of evidence suggests that stimulation of α1adrenoceptors could mediate ischemic preconditioning in human myocardium. 7,29However, Loubani et al. 7showed that activation of α1adrenoceptors before ischemia is protective but is detrimental during ischemia. Recently, involvement of the β-adrenergic signal transduction pathway in ischemic preconditioning has been suggested, 30and isoproterenol has been shown to precondition isolated rat heart through activation of PKC. 8
The main advantage of isolated human preparations in studying myocardial preconditioning is that the effect of variable myocardial collateral flow, which may occur in in vivo models, could be eliminated. However, 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 eliminated. Therefore, patients taking oral hypoglycemic medications were excluded from the study. Furthermore, we have previously reported that preoperative treatment, such as β-adrenergic blocking drugs, do not mask desflurane-induced adrenoceptor stimulation. 15The use of isoflurane and opioids during anesthesia of patients included in this study could have theoretically precondition the appendage. However, in vitro studies were initiated at least 90 min after removal of the atrial appendage. Most importantly, comparisons have been made with control experiments. Nevertheless, a superimposed effect of opioids or isoflurane used during the surgical procedure cannot be ruled out. Second, rather than the true ischemia obtained by coronary occlusion, we used 30 min of anoxic superfusion to simulate ischemia. However, it has been shown in various experimental models that anoxia is as effective as ischemia in inducing preconditioning. 31Third, we measured postischemic contractile function recovery but not infarct size. However, it has been shown that the improved recovery of contractile function produced by preconditioning was proportional to reduced infarct size. 32In 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. 2,3,11Fourth, our experiments were performed at 34°C, which may have decreased KATPchannel sensitivity 33and the effect of preconditioning. 34However, during surgical procedures, moderate hypothermia may occur in patients.
In conclusion, desflurane exerts a cardioprotective effect in anoxic-challenged isolated human right atrial myocardium. This effect involves, at least in part, mitoKATPchannels, and stimulation of adenosine A1receptor and α and β adrenoceptors.