Inhalation anaesthetics have been shown to induce myocardial preconditioning . Several studie ssuggest that opening of the adenosine triphosphate-regulated mitochondrial potassium (mitochondrial KATP) channels may trigger ischaemic preconditioning (IPC) by generating reactive oxygen species (ROS) [2,3]. ROS generation is also involved in anaesthetic preconditioning [4-6]. 5-hydroxydecanoate (5-HD) is a selective mitochondrial KATP channel blocker, which has been shown to attenuate the cardioprotective effects of inhalation agents when given before anaesthetic-induced preconditioning. On the other hand, inhalation anaesthetics given only during post-ischaemic reperfusion offer some protective effect against reperfusion injury of the heart .
In the present study, we used a rat heart-lung model to investigate whether administration of isoflurane or sevoflurane only during post-ischaemic reperfusion provided beneficial effects on cardiac function, metabolism and hydroxyl radical formation in hearts pretreated with a mitochondrial KATP channel blocker.
The study was approved by the Animal Care Committee of University of Yamanashi. The technique was used in an earlier study . After completed heart-lung preparation, 40 male Wistar rats were allocated to four groups of equal size using permuted blocks randomization as follows: control group: no drug, 5-HD group: 5-HD 100 μmol, 5-HD + Sevo group: 5-HD and 2.7% sevoflurane and 5-HD + Iso group: 5-HD and 1.4% isoflurane. These concentrations of the inhalational agents were considered to represent 1.0 minimum alveolar concentration (MAC) in rats [9,10].
All rats were anaesthetized with sevoflurane. After tracheostomy, intermittent positive pressure ventilation was instituted with O2 95% and CO2 5%. The chest was opened and flooded with ice-cold saline until cardiac arrest, and sevoflurane was stopped in all groups. Cannulas were inserted into the aorta, as well as the superior and inferior venae cavae. The cannula in the superior vena cava was used for monitoring of right atrial pressure.
When all cannulas had been inserted, the heart was rewarmed with saline and kept at 37°C during the experiment. As soon as the heart had been warmed, it started to beat spontaneously. The heart-lung preparation was perfused with an oxygenated solution (total volume 25 mL) containing red blood cells (from a donor rat) and Krebs-Ringer bicarbonate buffer, adjusted to a haematocrit of 25% and a pH of 7.4. Blood was pumped from the heart through a pneumatic resistance and was collected in a reservoir kept at 37°C and then returned to the inferior vena cava. In this model, only the heart and lung were perfused. Thus, cardiac output was determined by the inflow as long as the heart did not fail, and mean arterial pressure was regulated by the pneumatic resistance. The pneumatic resistance and the inflow to the inferior vena cava from the reservoir were increased gradually. All hearts were perfused initially with a cardiac output of 30 mL min−1 and a mean arterial pressure of 70 mmHg.
Heart rate (HR) was recorded with a bioelectric amplifier (AB-621G; Nihon Kohden, Japan) and cardiac output was measured with an electromagnetic blood flow meter (MFV-1200; Nihon Kohden, Japan). Arterial pressure and right atrial pressure were measured with transducers (TP101T and LPU-0.1A; Nihon Kohden, Japan). The maximal rate of left ventricular tension development (LV dP/dt max) was obtained electronically from aortic blood pressure curve . The cardiac output, HR and LV dP/dt max were measured at 0, 5 and 10 min after the start of pre-ischaemic perfusion, and at 0, 5 and 10 min after the start of post-ischaemic reperfusion.
Seven minutes after the start of perfusion, 5-HD 2.5 mmol in 0.1 mL saline was administered into the reservoir in all groups except the control group, resulting in a 5-HD concentration in the perfusate of 100 μmol L−1. This concentration of 5-HD was based on a recent study . The control group was given the same volume (0.1 mL) of saline. Ten minutes after the start of perfusion, the heart was rendered globally ischaemic for 10 min by reducing the preload and after-load to zero. One minute before the end of the ischaemic period, sevoflurane was administered in the 5-HD + Sevo group and isoflurane in the 5-HD + Iso group. After the 10 min ischaemic period, reperfusion of the heart was started. If the heart did not function within 8 min it was classified as non-recovering. The perfusate was collected 1 min before and just after the ischaemic period, and at the end of reperfusion. Figure 1 shows the experimental design.
Ten minutes after the reperfusion, the recovered hearts were frozen by liquid nitrogen and freeze-dried for 5 days. Myocardial high-energy phosphates (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monohosphate (AMP)) were measured by high-performance liquid chromatography . Adenylate energy charge was calculated according to the following formula:
Lactate, pyruvate and glycogen levels were determined spectrophotometrically by standard techniques . The formation of hydroxyl radicals in the perfusate blood and heart was measured with high-performance liquid chromatography using salicylic acid. Hydroxyl radicals react with salicylic acid, yielding dihydroxybenzoic acids (DHBA) .
The data are expressed as mean ± standard deviation (SD). Differences in haemodynamic changes and the perfusate DHBA among the groups were analysed by two-way analysis of variance with repeated measures. Means of the myocardial metabolites and DHBA were compared by a Tukey-Kramer multiple comparisons test. Chi-square was used to determine the difference of the ratios of recovery hearts in the reperfusion period among the groups. P value < 0.05 was considered statistically significant.
All rats were included in analysis of haemodynamic data in the perfusion period and ratios of recovered hearts in the reperfusion period. Only hearts that recovered were included in the analysis of haemodynamic data in the reperfusion period, DHBA levels and metabolites in the myocardium.
Throughout the perfusion, there were no significant differences in HR, systolic aortic pressure, cardiac output, left ventricle (LV) dP/dt max among the groups (Table 1). Right atrial pressure in the 5-HD group was significantly higher than in the 5-HD + Sevo and the 5-HD + Iso groups before ischaemia.
Two hearts in the 5-HD group did not recover after ischaemia, but this was not statistically significant (Table 2). However, 5-HD and sevoflurane, or 5-HD and isoflurane decreased the recovery ratios.
There were no significant differences in ATP, ADP, AMP, energy charge, glycogen or lactate/pyruvate ratio among the control, 5-HD and 5-HD + Iso groups (Fig. 2). However, ATP in the 5-HD + Sevo group was significantly lower than those in the control and the 5-HD + Iso groups. ADP in the 5-HD + Sevo group was significantly higher than that in the control group. In addition, energy charge in the 5-HD + Sevo group was significantly lower than those in the control and the 5-HD groups. There were no significant differences in DHBA levels among the groups (Table 3).
We have demonstrated that the mitochondrial KATP channel blocker 5-HD caused no significant changes in haemodynamics and myocardial metabolisms during post-ischaemic reperfusion, though 2 of 10 hearts did not recover from ischaemia. It has been reported that cardioprotection induced by IPC was attenuated by administration of 5-HD administered 5 min before IPC, but not if given 10 or 30 min before IPC . 5-HD has been shown to block IPC only when administered during preconditioning, but not if given after preconditioning or during global ischaemia . These studies suggest that 5-HD would affect haemodynamics and myocardial metabolism during post-ischaemic reperfusion via attenuation of preconditioning, and that blockade of the mitochondrial KATP channel during global ischaemia would cause little effect on cardiac function. In this study 5-HD was given 3 min before global ischaemia, without preconditioning. Therefore, it is unlikely that 5-HD would induce any significant changes in haemodynamics during the post-ischaemic reperfusion.
We found that 5-HD did not alter myocardial ATP content at the end of reperfusion. This is supported by previous reports that KATP channel blockers, 5-HD and glibenclamide, given before global ischaemia had no effect on the rate of ATP synthesis in normoxic cardiac mitochondria [16,17]. Moreover, non-specific KATP channel blockade with glibenclamide prevented the arteriolar vasodilatation that occurred in response to reductions of coronary perfusion pressure [18,19], whereas mitochondrial KATP channel blockade with 5-HD had no significant effect on coronary blood flow . This is in agreement with our finding that 5-HD induced no significant changes in haemodynamics or myocardial metabolism.
5-HD with sevoflurane or isoflurane markedly decreased the recovery rates. It has been reported that volatile anaesthetics administered only during post-ischaemic reperfusion have some protective effect against reperfusion injury of the heart . Therefore, we suggest that the combination of 5-HD and inhalation anaesthetics in our study induced some inhibitory effects during post-ischaemic reperfusion. Although there were no significant differences in haemodynamics of recovered hearts during post-ischaemic reperfusion among the groups, it would appear that the inhibitory mechanisms by 5-HD with sevoflurane differ from those by 5-HD with isoflurane. Administration of 5-HD with sevoflurane decreased myocardial ATP level and energy charge, whereas 5-HD with isoflurane provided no changes in myocardial metabolites in spite of its poor recovery ratio. It is unknown why the effects on myocardial metabolism by isoflurane or sevoflurane are different. Our previous report indicates that 1.0 MAC of sevoflurane or isoflurane, administered before ischaemia and during ischaemia and reperfusion, preserved myocardial metabolism . In that study, isoflurane improved myocardial ATP content, but sevoflurane did not. Isoflurane and sevoflurane reduce cardiac contractility . Therefore, the combination of isoflurane and 5-HD would have adversely affected recovery from ischaemia in rat hearts caused by a decrease in myocardial contractility. It is also likely that sevoflurane given with 5-HD would have prevented myocardial recovery from ischaemia, not only due to a decrease in myocardial contractility, but also due to the reduced myocardial ATP content and energy charge.
The hydroxyl radical is one of the most harmful ROS, which are formed in excess during oxidative stress [21,22]. For measurement of hydroxyl radical in this study, we used indirect methods determining aromatic acids hydroxylation products, DHBA. There were no significant differences in DHBA contents among the groups. Considering that 5-HD caused no significant changes in haemodynamics and myocardial metabolisms, 5-HD might have little effect on the ROS generation in our study. On the other hand, sevoflurane or isoflurane might affect the ROS generation. There are some reports showing relationship between inhalation anaesthetics and ROS. Kevin and colleagues demonstrated that sevoflurane increased ROS independent of mitochondrial KATP channel during aerobic perfusion . Tanaka and colleagues reported that mitochondrial KATP channel opened by isoflurane-induced preconditioning generated ROS, but that 5-HD given after isoflurane reduced ROS . Although it is likely that the administration of inhalation anaesthetics with 5-HD would change the generation of ROS, it did not alter the DHBA contents in the perfusate and the heart. It is possible that the administration of inhalation anaesthetics only during the reperfusion period may have influenced the production of ROS. In addition, our previous study has shown that sevoflurane and isoflurane when administered during the whole experimental period did not alter the hydroxyl radical generation in the same animal model .
The limitation of this study is that all rats were anaesthetized with sevoflurane during the heart- lung preparation. It is possible that the rat hearts could have been preconditioned by the short administration of sevoflurane. From this aspect, pentobarbital might have been a better alternative. However, it has been reported that pentobarbital may inhibit the cardioprotective effect of volatile anaesthetics . We selected sevoflurane due to its low blood/ gas partition coefficient which resulted in rapid onset and rapid offset of action. Even if the rat hearts were preconditioned by sevoflurane in the current study, it was probable that all groups were under the same conditions. The other limitation is the significant differences in right atrial pressure during the perfusion period. It is unclear why right atrial pressure in the 5-HD was higher than in the 5-HD + Sevo and the 5-HD + Iso groups. In contrast, there were no significant differences in the right atrial pressure during the reperfusion period among the groups.
In conclusion, a mitochondrial KATP channel blocker (5-HD) caused no significant changes in haemodynamics and myocardial metabolisms during post-ischaemic reperfusion. Moreover, 5-HD with sevoflurane or isoflurane did not affect hydroxyl radical generation. However, 5-HD with sevoflurane decreased recovery ratio and worsened myocardial metabolism. As for 5-HD with isoflurane, it decreased recovery ratio without changing myocardial metabolism. Although animal data cannot be extrapolated to human beings, we suggest that more attention be paid to patients treated with sulphonylurea drugs, which inhibit KATP channel opening, when they are anaesthetized with volatile anaesthetics.
The authors are indebted to Mr Koshimizu for technical assistance.
1. Ismaeil MS, Tkachenko I, Gamperl AK et al
. Mechanisms of isoflurane
-induced myocardial preconditioning in rabbits. Anesthesiology
2. Pain T, Yang XM, Critz SD et al
. Opening of mitochondrial
K(ATP) channels triggers the preconditioned state by generating free radicals. Circ Res
3. Carroll R, Gant VA, Yellon DM. Mitochondrial
K(ATP) channel opening protects a human atrial-derived cell line by a mechanism involving free radical generation. Cardiovasc Res
4. Mullenheim J, Ebel D, Frassdorf J et al
preconditions myocardium against infarction via release of free radicals. Anesthesiology
5. Tanaka K, Weihrauch D, Kehl F et al
. Mechanism of preconditioning by isoflurane
in rabbits: a direct role for reactive oxygen species. Anesthesiology
6. Novalija E, Varadarajan SG, Camara AK et al
. Anesthetic preconditioning: triggering role of reactive oxygen and nitrogen species in isolated hearts. Am J Physiol Heart Circ Physiol
7. Schlack W, Preckel B, Stunneck D, Thamer V. Effects of halothane, enflurane, isoflurane
and desflurane on myocardial reperfusion injury
in the isolated rat heart. Br J Anaesth
8. Kashimoto S, Tsuji Y, Kumazawa T. Effects of halothane and enflurane on myocardial metabolism during postischaemic reperfusion in the rat. Acta Anaesthesiol Scand
9. Mazze RI, Rice SA, Baden JM. Halothane, isoflurane
, and enflurane MAC in pregnant and nonpregnant female and male mice and rats. Anesthesiology
10. Kashimoto S, Furuya A, Nonaka A et al
. The minimum alveolar concentration of sevoflurane
in rats. Eur J Anaesthesiol
11. Nonaka A, Kashimoto S, Nakamura T, Kumazawa T. Effects of intravenous anaesthetics on function and metabolism in the isolated rat heart-lung preparation. Eur J Anaesthesiol
12. Wynants J, Van Belle H. Single-run high-performance liquid chromatography of nucleotides, nucleosides, and major purine bases and its application to different tissue extracts. Anal Biochem
13. Kevelaitis E, Oubenaissa A, Peynet J et al
. Preconditioning by mitochondrial
ATP-sensitive potassium channel openers: an effective approach for improving the preservation of heart transplants. Circulation
14. Bergmeyer HU. [New values for the molar extinction coefficients of NADH and NADPH for the use in routine laboratories (author's translation)] Z Klin Chem Klin Biochem
15. Floyd RA, Henderson R, Watson JJ, Wong PK. Use of salicylate with high pressure liquid chromatography and electrochemical detection (LCED) as a sensitive measure of hydroxyl free radicals in adriamycin treated rats. J Free Radic Biol Med
16. Fryer RM, Eells JT, Hsu AK et al
. Ischemic preconditioning in rats: role of mitochondrial
K(ATP) channel in preservation of mitochondrial
function. Am J Physiol Heart Circ Physiol
17. Eells JT, Henry MM, Gross GJ, Baker JE. Increased mitochondrial
K(ATP) channel activity during chronic myocardial hypoxia: is cardioprotection mediated by improved bioenergetics? Circ Res
18. Komaru T, Lamping KG, Eastham CL, Dellsperger KC. Role of ATP-sensitive potassium channels
in coronary microvascular autoregulatory responses. Circ Res
19. Chen Y, Traverse JH, Zhang J, Bache RJ. Selective blockade of mitochondrial
K(ATP) channels does not impair myocardial oxygen consumption. Am J Physiol Heart Circ Physiol
20. Oguchi T, Kashimoto S, Yamaguchi T et al
. Comparative effects of halothane, enflurane, isoflurane
on function and metabolism in the ischaemic rat heart. Br J Anaesth
21. Braughler JM, Hall ED. Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation. Free Radic Biol Med
22. Hall ED, Braughler JM. Central nervous system trauma and stroke. II. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. Free Radic Biol Med
23. Kevin LG, Novalija E, Riess ML et al
exposure generates superoxide but leads to decreased superoxide during ischemia and reperfusion in isolated hearts. Anesth Analg
24. Tanaka K, Weihrauch D, Ludwig LM et al
adenosine triphosphate-regulated potassium channel opening acts as a trigger for isoflurane
-induced preconditioning by generating reactive oxygen species. Anesthesiology
25. Kashimoto S, Kume M, Ikeya K, Kumazawa T. Effects of sevoflurane
on free radical formation in the post-ischaemic reperfused heart. Eur J Anaesthesiol
26. Kohro S, Hogan QH, Nakae Y et al
. Anesthetic effects on mitochondrial
ATP-sensitive K channel. Anesthesiology
Keywords:© 2006 European Society of Anaesthesiology
ANAESTHETICS INHALATION; sevoflurane; isoflurane; MITOCHONDRIA; potassium ATP channels; POTASSIUM CHANNELS; mitochondrial; 5-HYDROXYDECANOIC ACID; MYOCARDIAL REPERFUSION INJURY