Nuffield Department of Anaesthetics, Oxford University, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, UK*Corresponding author: Department of Anesthesiology, Tang Du Hospital, The Fourth Military Medical University, Xian 710038, China
Accepted for publication: March 2, 2001
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Br J Anaesth 2001; 87: 25865
Keywords: volatile anaesthetics, isoflurane; heart, stunning; receptors, adenosine; model, rat
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The protective effects of potent inhalation anaesthetics on myocardial stunning may be attributed to free radical scavenging, preservation of energy levels, and calcium channel block.7 However, several inhalation anaesthetics were recently shown to have direct cardioprotective effects that mimic ischaemic preconditioning.813 There is increasing evidence indicating that the end-effector mediating ischaemic preconditioning is the ATP-sensitive potassium channel (KATP).14 Kersten and colleagues demonstrated that the improved mechanical function afforded by isoflurane was partially blocked by glibenclamide.15 In a subsequent study, isoflurane-induced myocardial protection was shown to been partially mediated by adenosine type-1 receptor (A1) activation.16 Ismaeil and colleagues also found that 8-(p-sulfophenyl)-theophylline (SPT, an adenosine receptor antagonist) pre-treatment eliminated the preconditioning-like effect of isoflurane.17 However, the signal transduction cascade responsible for isoflurane-induced myocardial protection has not been completely defined. Ismaeil speculated that isoflurane-induced preconditioning and ischaemia-induced preconditioning share similar mechanisms, which include activation of KATP channels and adenosine receptors.
There are three adenosine receptor subtypes in the cardio vascular system: A1, A2, and A3.18 Although ischaemic preconditioning in several species can be mimicked pharmacologically by selective adenosine A1 or A3 receptor agonists, it is currently unclear which receptor subtype is physiologically involved in mediating ischaemic preconditioning. The specific adenosine receptor subtypes involved in isoflurane-induced preconditioning also remain to be determined.
The purpose of this study was to identify whether isoflurane attenuates myocardial stunning induced by 20-min global ischaemia and if so, whether the myocardial protection of isoflurane is mediated by adenosine A1 receptors. In addition, we compared the effects of A1 receptor block given pre-ischaemia or post-reperfusion. A highly selective adenosine receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), was used to block the A1 receptor.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolated heart preparation
Adult male Wistar rats (325350 g) were anaesthetized with an intraperitoneal injection of sodium pentobarbital 60 mg kg1 and heparinized with 200 IU. The hearts were rapidly removed through a median sternotomy by transsecting all the major vessels, and arrested in ice-cooled Krebs Henseleit buffer, then perfused retrogradely through the aorta in a non-recirculating Langendorff apparatus. The KrebsHenseleit perfusion solution contained (mM) NaCl 118.7, KCl 4.7, MgSO4 1.2, KH2PO4 0.95, NaHCO3 28, CaCl2 1.3, and glucose 10. The buffer was filtered through a 5.0 µm filter, equilibrated with a mixture of 95% oxygen and 5% carbon dioxide, had a pH 7.4, and was maintained at 37°C throughout the procedure. A fluid-filled polyethylene balloon was inserted through the mitral valve into the left ventricle and connected by a polyethylene catheter to a saline-filled syringe and to a pressure transducer (Medex Medical Inc, Lancashire, UK). The volume of the balloon was adjusted to achieve an end-diastolic pressure (EDP) of 58 mm Hg, which remained unchanged throughout the experiment. All hearts were paced with right ventricular epicardial electrodes at a fixed rate of 300 beats min1 (2 ms, 5 V). Perfusion pressure was set at 100 cmH2O and maintained constant. Myocardial temperature was maintained at 37°C by submersing the heart into a water-jacketed chamber filled with KrebsHenseleit buffer. A three-way stopcock placed above the aortic root was positioned to stop flow and, thus, produce global ischaemia. Electric pacing was stopped during ischaemic periods and resumed 2 min after reperfusion. Hearts were defibrillated when necessary.
Experimental design
All experiments lasted 150 min beginning with a 10-min period of stabilizing perfusion for equilibration. Forty rats were assigned randomly to one of the following five groups (n=8). The hearts in the control groups underwent 20-min ischaemia with or without DPCPX (200 nM) treatment (Cont group and DPCPX group). In the isoflurane groups, isoflurane (1.5 MAC) was present throughout the experiment except during the ischaemic period (Iso group). In addition, DPCPX (200 nM) was administered for 10 min before ischaemia and during reperfusion in the Iso+DPCPX(pre-I) group, and only during reperfusion in the Iso+DPCPX(post-I) group. Reperfusion lasted for 120 min in all groups. Isoflurane was administered by placing an isoflurane vaporizer between the fresh gas supply and the perfusate. A gas analyser (M1025A, Hewlett Packard, San Francisco, CA, USA) continuously monitored the delivered vapour concentration. As the MAC of isoflurane in rats was 1.5%19 a concentration of 2.25% was used to obtain 1.5 MAC. DPCPX (ICN Biomedicals Inc., Aurora, Ohio, USA) was first dissolved in DMSO (dimethylsulphoxide, Sigma-Aldrich, Dorset, UK) and then the perfusate so that the final concentrations of DPCPX and DMSO in the perfusate were 200 nM and 0.025%, respectively.
Data collection and analysis
Left ventricular pressure was monitored continuously throughout the experiment. The left ventricular pressure signal was digitized at 100 Hz (analogue-digital converter AT-MIO, National Instruments Corporation, TX, USA) and stored on the hard disk of a desktop computer. Peak left ventricular pressure (PSP), developed pressure (end-systolic minus end-diastolic, DP), end-diastolic pressure (EDP) and positive and negative LVdp/dt were obtained and processed using data acquisition and analysis software developed in our department. After a period of stabilization (10 min), ventricular mechanical function was measured as a baseline, then measurements were obtained at the following time points: just before the onset of ischaemia, 10, 20, 30, 60, 90, and 120 min after reperfusion.
Statistical analysis
All values are expressed as mean (SEM). After ischaemia, DP, LV+dp/dtmax, and LVdp/dtmin are expressed as the percentage of the values before ischaemia. Before ischaemia and during reperfusion differences in indices among groups were compared using two-way analysis of variance (ANOVA) for treatment and time with repeated measures on a time factor. If ANOVA indicated significant difference between groups, further comparisons on specific times were performed using one-way ANOVA followed by Bonferronis post-hoc test. Statistical significance was assumed at P<0.05. All analyses were performed using the SPSS 10.0 software system under Windows 98 operating system.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many studies indicate that volatile anaesthetics protect the myocardium after global or regional ischaemia, in models of reversible and irreversible tissue injury.25 20 21 Recent findings suggest that isoflurane modulates cardioprotection via a signal transduction mechanism similar to the endogenous mechanism that mediates ischaemic preconditioning, for example, through activation of adenosine A1 receptor coupled to KATP channels.9 10 1417 Indeed, Kersten and colleagues found that isoflurane directly preconditions the myocardium via activation of KATP channels, confirming and extending previous studies.9
The preconditioning protection observed with brief ischaemia seems to be mediated by the release of adenosine. Adenosine binds to its receptors (A1 and possibly A3), and, via a G-protein-linked process, increases protein kinase C (PKC) activity.22 However, the role of adenosine receptors in the modulation of the cardioprotective signal transduction during isoflurane anaesthesia has not been completely identified. Kersten showed that the cardioprotective effects of isoflurane were attenuated by pre-treatment with DPCPX in dogs16 and DPCPX, a xanthine derivative, has been shown to be 700-fold more selective for A1 than for A2 receptors in radioligand binding studies and in vitro functional assays.23 These data indicated that isoflurane-induced myocardial protection is partially mediated by A1 receptor activation. Ismaeil and colleagues found that SPT, a non-specific adenosine receptor antagonist, could block the preconditioning-like effect of isoflurane in propofol-anaesthetized rabbits; SPT returned infarct size to values comparable with those in the control group.17 Previously, Cope and co-workers demonstrated that halothane, enflurane, and isoflurane protect the heart from infarction. They also found that myocardial protection by halothane was abolished by SPT in vitro in the rabbit heart.20 It seems that isoflurane-induced preconditioning shares some of the same fundamental underlying mechanisms as ischaemia-induced preconditioning. That is the activation of adenosine A1 receptors followed by the opening of KATP through protein kinase C (PKC). However, in our study, DPCPX did not block or attenuate isoflurane-induced myocardial protection.
How can the differences between this and previous studies in respect of DPCPX be explained?
First, the animal model may be an important factor. We used the model of the isolated buffer-perfused rat heart to investigate direct effects of isoflurane on myocardial ischaemia/reperfusion injury. Compared with in vivo studies, that identified the role of adenosine receptors in isoflurane-induced myocardial protection, our model excludes several effects of isoflurane that may be important in the in vivo situation of ischaemia and reperfusion, such as haemodynamic and humoral effects, sympathetic nervous system activity, or activation of neutrophils.24 25 Furthermore, the species difference should also be considered. It has been found that adenosine A1 receptor activation did not appear to be involved in ischaemic preconditioning in the rat, although it has been implicated in the rabbit, the dog, and the pig. In isolated working rat hearts, Cave and colleagues found that a 5-min period of perfusion with adenosine (10, 50, and 100 µM) followed by a 5-min adenosine-free perfusion period failed to increase the recovery of function after a subsequent 20-min period of global ischaemia.26
Second, the concentration of DPCPX used in the present study was 200 nM according to previous experiments. To determine whether the concentration of DPCPX at 200 nM was enough to block the adenosine A1 receptor in rat heart, Hara and co-workers examined the effects of DPCPX (100 nM) on the negative chronotropic action induced by a high concentration of adenosine (50 µM).27 The results showed that DPCPX completely prevented the decrease in heart rate. Therefore, concentration of DPCPX used in our study has the ability to block the adenosine A1 receptors. In Kerstens study, which showed that isoflurane-induced myocardial protection of stunned myocardium is partially mediated by A1 receptor activation, the dosage of DPCPX was 0.8 mg kg1. But Yao and co-workers previously demonstrated that a higher dose of DPCPX (1 mg kg1, intravenously) exacerbated myocardial stunning and caused significantly greater reduction in %SS during reperfusion when compared with dogs receiving the drug vehicle alone.28
Third, although it is well established that adenosine A1 receptor activation preconditions the heart against infarction, it remains controversial whether A1 receptor activation can also precondition against stunning. Studies using multiple 5-min coronary occlusions interspersed with 10 min of reperfusion have yielded conflicting results, with one study suggesting that adenosine receptor block prevents the preconditioning effects of the first occlusion and another suggesting that it does not.28 29 The mechanism of stunning may be different from myocardial infarction; adenosine receptors could be important in infarction, but we cannot necessarily implicate them in myocardial stunning.
A number of hypotheses regarding the mechanism of myocardial stunning have been proposed since the 1980s, most of which have been subsequently abandoned. At present, the two favoured theories regarding the pathogenesis of myocardial stunning are the oxyradical hypothesis and the calcium hypothesis.30 Volatile anaesthetics have been shown to affect free radical activity. Tanguay and colleagues showed a beneficial effect of halothane, isoflurane, and enflurane on free radical-induced impairment of cardiac function in isolated rabbit hearts. Halothane completely inhibited the initial burst of hydroxyl production during early reperfusion.31 However, the effects of anaesthetics on free radicals are inconclusive. On the other hand, volatile anaesthetics affect Ca2+ movements during the cardiac cycle. Ca2+ flux into and out of the heart cell is mediated primarily by Ca2+ channels and the Na+/Ca2+ exchanger. Haworth and colleagues found that halothane, isoflurane, and enflurane inhibit both Na+/Ca2+ exchange and Ca2+ channels at concentrations relevant to anaesthesia, although they exhibit differences in potency and number of sites of action. At 1.5 MAC, halothane inhibits Ca2+ channels more than Na+/Ca2+ exchange, whereas enflurane inhibits Na+/Ca2+ exchange more than Ca2+ channels. Isoflurane inhibits both systems equally.32 The negative inotropic effect of isoflurane on rat papillary muscle has been investigated by Lynch and Frazer,33 suggesting isoflurane also affects Ca2+ channels in the rats.
The observation that there is an initial recovery of function immediately after reperfusion, followed by a subsequent decline at 30 min, supports the occurrence of additional injury in the initial phase of reflow. Both free radicals and calcium overload may contribute to this phenomenon. Accordingly, the better functional recovery in the isoflurane groups compared with control group could be ascribed to the inhibition of the production of free radicals or the prevention of calcium overload by isoflurane. Furthermore, physiological studies of the mechanism of stunning point to a lesion at the level of the myofilaments. Toda found that isoflurane increased submaximum Ca2+ activated force development of the contractile proteins in skinned rabbit femoral arterial strips.34 This isoflurane-induced increase in force is blocked by inhibition of Ca2+-independent PKC and suggests that isoflurane may increase the activity of -PKC, a prominent calcium-independent PKC isoform and one that has been implicated as a critical mediator of ischaemic preconditioning. Thus, we can speculate that PKC is also involved in the isoflurane-induced myocardial protection. Although such a hypothesis may be highly plausible, direct evidence of activation of any specific PKC isoform by isoflurane has yet to be demonstrated and would represent an important goal of future research.
It has been proposed that the injury responsible for myocardial stunning consists of ischaemic and reperfusion injury, with reperfusion injury respresenting the larger component. However, at the same time, ischaemic injury and reperfusion injury are not independent entities; any intervention that attenuates the severity of the ischaemic injury, such as adenosine, calcium antagonists, KATP channel openers, will also, indirectly, attenuate the severity of the subsequent reperfusion injury.30 The primary aim of administration of DPCPX to the perfusate before and after ischaemia was to investigate the potentially different role of A1 receptor in ischaemic vs reperfusion injury. We found that the adenosine receptor antagonist, DPCPX, does not abolish the beneficial effects of isoflurane, whether administered before or after ischaemia. This indicates that A1 receptors are not involved in the isoflurane-induced myocardial protection, vis-à-vis ischaemic injury or reperfusion injury.
We cannot completely rule out that better recovery of DP, LV+dp/dtmax, LVdp/dtmin and EDP during reperfusion resulted from the effect of isoflurane on myocardial metabolism. However, Mattheussen and co-workers found that depressed cardiac function during exposure to halothane and isoflurane did not correlate with myocardial ATP content, suggesting that ATP metabolism during ischaemia was not influenced by halothane or isoflurane.35
Myocardial protection should be considered in a variety of clinical settings such as thrombolysis, percutaneous balloon angioplasty and after periods of cardiac arrest during cardiac surgery with cardiopulmonary bypass. If clinical investigations could show that isoflurane protects against myocardial stunning, this may influence the choice of anaesthetic agent for cardiac surgery.
In conclusion, our results show that isoflurane enhances the post-ischaemic functional recovery of the isolated rat heart undergoing 20 min ischaemia when used throughout the experiment. Block of A1 receptors whether before or after ischaemia does not abolish the beneficial effects of isoflurane. We conclude that A1 receptors in rat heart are not involved in the processes of isoflurane-induced myocardial protection, in either the ischaemic or reperfusion phases.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 Warltier DC, Al-Wathiki MH, Kampine JP, Schmeling WT. Recovery of contractile function of stunned myocardium in chronically instrumented dogs is enhanced by halothane or isoflurane. Anesthesiology 1988; 69: 55265[ISI][Medline]
3 Kanaya N, Kobayashi I, Nakayama M, Fujita S, Namiki A. ATP sparing effect of isoflurane during ischaemia and reperfusion of the canine heart. Br J Anaesth 1995; 74: 5638
4 White JL, Myers AK, Analouei, Kim YD. Functional recovery of stunned myocardium is greater with halothane than fentanyl anaesthesia in dogs. Br J Anaesth 1994; 73: 2149[Abstract]
5 Gross GJ, Kersten JR, Warltier DC. Mechanisms of postischemic contractile dysfunction. Ann Thorac Surg 1999; 68: 1898904
6 Coetzee A, Skein W, Genade S, Lochner A. Enflurane and isoflurane reduce reperfusion dysfunction in the isolated rat heart. Anesth Analg 1993; 76: 6028[Abstract]
7 Ross S, Foëx P. Protective effects of anaesthetics in reversible and irreversible ischaemia-reperfusion injury. Br J Anaesth 1999; 82: 62232
8 Kersten JR, Schmeling TJ, Hettrick DA, Pagel PS, Gross GJ, Warltier DC. Mechanism of myocardial protection by isoflurane. Role of adenosine triphosphate-regulated potassium (KATP) channels. Anesthesiology 1996; 85: 794807[ISI][Medline]
9 Kersten JR, Schmeling TJ, Pagel PS, Gross GJ, Warltier DC. Isoflurane mimics ischemic preconditioning via activation of KATP channels. Anesthesiology 1997; 87: 36170[ISI][Medline]
10 Toller W, Montgomery MW, Pagel PS, Hettrick DA, Warltier DC, Kersten JR. Isoflurane-enhanced recovery of canine stunned myocardium. Anesthesiology 1999; 91: 1322
11 Boutros A, Wang J, Capuano C. Isoflurane and halothane increase adenosine triphosphate preservation, but do not provide additive recovery of function after ischemia, in preconditioned rat hearts. Anesthesiology 1997; 86: 10917[ISI][Medline]
12 Novalija E, Fujita S, Kampine JP, Stowe DF. Sevoflurane mimics ischemic preconditioning effects on coronary flow and nitric oxide release in isolated hearts. Anesthesiology 1999; 91: 70112[ISI][Medline]
13 Hawaleshka A, Jacobsohn E. Ischemic preconditioning: mechanisms and potential clinical applications. Can J Anaesth 1998; 45: 67082[Abstract]
14 Kersten JR, Pagel PS, Gross GJ, Warltier DC. Activation of adenosine triphosphate-regulated potassium channels. Anesthesiology 1998; 88: 495513[ISI][Medline]
15 Kersten JR, Lowe D, Hettrick DA, Pagel PS, Gross GJ, Warltier DC. Glyburide, a KATP channel antagonist, attenuates the cardioprotective effects of isoflurane in stunned myocardium. Anesth Analg 1996; 83: 2733[Abstract]
16 Kersten JR, Orth KG, Pagel PS, Mei DA, Gross GJ, Warltier DC. Role of adenosine in isoflurane-induced cardioprotection. Anesthesiology 1997; 86: 112839[ISI][Medline]
17 Ismaeil MS, Tkachenko I, Gamperl AK, Hickey RF, Cason BA. Mechanisms of isoflurane-induced myocardial preconditioning in rabbits. Anesthesiology 1999; 90: 81221[ISI][Medline]
18 Hill R, Oleynek J, Hoth C. Cloning expression and pharmacological characterization of rabbit adenosine A1 and A3 receptors. J Pharmacol Exp Ther 1997; 280: 1228
19 Mazze RI, Rice SA, Baden JM. Halothane, isoflurane and enflurane MAC in pregnant and nonpregnant female and male mice and rats. Anesthesiology 1985; 62: 33941[ISI][Medline]
20 Cope DK, Impastato WK, Cohen MV, Downey JM. Volatile anaesthetics protect the ischemic rabbit myocardium from infarction. Anesthesiology 1997; 86: 699709[ISI][Medline]
21 Cason BA, Gamperl AK, Slocum RE, Hickey RF. Anesthetics-induced preconditioning. Previous administration of isoflurane decreases myocardial infarct size in rabbits. Anesthesiology 1997; 87: 118290[ISI][Medline]
22 Lynch C III. Anesthetic preconditioning, not just for heart. Anesthesiology 1999; 91: 6068[ISI][Medline]
23 Lohse MJ, Klotz KN, Lindenborn-Fotions J, Reddington M, Schwabe U, Olsson RA. 8-Cyclopentyl-1, 3-dipropylxanthine (DPCPX) a selective high affinity antagonist radioligand for A1 adenosine receptors. Naunyn-Schmiedebergs Arch Pharmacol 1987; 336: 20410[ISI][Medline]
24 Lynch C III. Differential depression of myocardial contractility by halothane and isoflurane in vivo. Anesthesiology 1986; 64: 62031[ISI][Medline]
25 Kovalski C, Zahler S, Becker BF. Halothane, isoflurane, and sevoflurane reduce postischemic adhesion of neutrophils in the coronary system. Anesthesiology 1997; 86: 18895[ISI][Medline]
26 Cave A, Collins C, Downey J, Hearse D. Improved functional recovery by ischaemic preconditioning is not mediated by adenosine in the globally ischaemic rat heart. Cardiovasc Res 1993; 27: 6638[ISI][Medline]
27 Hara A, Abiko Y. Protective effects of hypoxia on mechanical and metabolic changes induced by hydrogen peroxide in rat hearts. Am J Physiol 1995; 268: H61420
28 Yao Z, Gross GJ. Gibenclamide antagonizes adenosine A1 receptor-mediated cardioprotection in stunned canine myocardium. Circulation 1993; 88: 23544[Abstract]
29 Bunch FT, Thornton J, Cohen MV, Downey JM. Adenosine is an endogenous protectant against stunning during repetitive ischemic episodes in the heart. Am Heart J 1992; 124: 14406[ISI][Medline]
30 Bolli R, Marban E. Molecular and cellular mechanisms of myocardial stunning. Physiol Rev 1999; 70: 60963
31 Tanguay M, Blaise G, Dumont L, Beique G, Hollman C. Beneficial effects of volatile anaesthetics on decrease in coronary flow and myocardial contractility induced by oxygen-derived free radivals in isolated rabbit hearts. J Cardiovasc Pharmacol 1991; 18: 86370[ISI][Medline]
32 Haworth RA, Coknur AB. Inhibition of sodium/calcium exchange and calcium channels of heart cells by volatile anaesthetics. Anesthesiology 1995; 82: 125565[ISI][Medline]
33 Lynch C III, Frazer MJ. Depressant effects of volatile anesthetics upon rat and amphibian ventricular myocardium: insights into anesthetic mechanism of action. Anesthesiology 1989; 70: 51122[ISI][Medline]
34 Toda H, Su JY. Mechanism of isoflurane-induced submaximum Ca2+-activated force in rabbit skinned femoral arterial strips. Anesthesiology 1998; 89: 73140[ISI][Medline]
35 Mattheussen M, Rusy BF, Aken HV, Flameng W. Recovery of function and adenosine triphosphate metabolism following myocardial ischemia induced in the presence of volatile anesthetics. Anesth Analg 1993; 76: 6975[Abstract]