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From the *Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin; Department of Biology, Portland State University, Portland, Oregon; and Departments of Anesthesiology and Physiology, Cardiovascular Research Center, Medical College of Wisconsin and ||VA Medical Center Research Service, and #Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin.
Address correspondence and reprint requests to Matthias L. Riess, MD, PhD, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Address e-mail to mriess{at}mcw.edu.
Abstract
BACKGROUND: Mitochondrial (m) adenosine triphosphate sensitive potassium (KATP) channel opening has been reported to trigger and/or mediate cardioprotection by volatile anesthetics. However, the effects of volatile anesthetics on mitochondrial function are not well understood. Prevention of mitochondrial matrix volume (MMV) contraction during ischemia may contribute to cardioprotection against ischemia/reperfusion injury. We investigated whether sevoflurane increases MMV and if this increase is mediated by mKATP channel opening.
METHODS: Mitochondria from fresh guinea pig hearts were isolated and diluted in buffer that included oligomycin and ATP to inhibit ATP synthesis. Changes in MMV by diazoxide, a known mKATP channel opener, and by different sevoflurane concentrations, were measured by light absorption at 520 nm in the absence or presence of the mKATP channel blocker, 5-hydroxydecanoate.
RESULTS: Compared with control, 30–300 µM sevoflurane (approximately 0.2–2.1 vol %) increased MMV by 30%–55%, which was similar to the effect of diazoxide. These increases were blocked by 5-hydroxydecanoate. Higher sevoflurane concentration (1000 µM; 7.1 vol %), however, had no effect on MMV.
CONCLUSIONS: In clinically relevant concentrations, sevoflurane increases MMV via mKATP channel opening. Preservation of mitochondrial integrity may contribute to the cardioprotective effects of sevoflurane against ischemia/reperfusion injury. Impaired mitochondrial function at supraclinical anesthetic concentrations may explain the observed biphasic response. These findings add to our understanding of the intracellular mechanisms of volatile anesthetics as cardioprotective drugs.
Twenty years ago, volatile anesthetics were found to enhance the tolerance to myocardial ischemia1 and to improve recovery after ischemia in isolated hearts2,3 as well as in vivo.4 Since then, a growing number of studies have shown that volatile anesthetics improve postischemic mechanical function, reduce infarction in various models,5 and decrease the incidence of late cardiac events after cardiac surgery.6 In 1997, it was discovered that volatile anesthetics, administered and washed out before ischemia, trigger a cardioprotective memory effect that lasts even beyond their elimination.7–9 Because of its similarities to the previously described "ischemic preconditioning," whereby several short periods of ischemia attenuate ischemia/reperfusion injury,10 this phenomenon has been called "anesthetic preconditioning" (APC).
A variety of intracellular mechanisms are thought to be involved in cardioprotection by volatile anesthetics, since APC can be abolished by adenosine receptor antagonists,8 Gi protein inhibitors,11 reactive oxygen species scavengers,12,13 protein kinase C (PKC) inhibitors,8,14 and blockers of adenosine triphosphate (ATP) sensitive potassium (KATP) channels.9,13,15,16
The exact intracellular signaling mechanism from anesthetic exposure to attenuated ischemia/reperfusion injury is not well understood. However, the role of altered mitochondrial function has gained increasing acceptance.17 For example, Riess et al.18 reported that sevoflurane (Sev) exposure caused a reduced mitochondrial redox state in intact beating hearts, implying that Sev attenuates rather than accelerates mitochondrial respiration. Experiments in isolated cardiac mitochondria under phosphorylating (state 3) conditions confirmed a concentration-dependent attenuation of mitochondrial respiration by Sev that was independent of mitochondrial (m) KATP channel opening.19 Interestingly, accelerated respiration, i.e., mild uncoupling, as a consequence of mKATP channel opening20,21 by Sev that is blocked by mKATP channel antagonists, could be observed only under nonphosphorylating (state 4) conditions.22
The notion that mKATP channel opening uncouples oxidative phosphorylation is not supported by Garlid et al., who propose that the critical role of mKATP channel opening is to modulate mitochondrial matrix volume (MMV), specifically to attenuate matrix contraction during ischemia when ATP levels are low.23–25 We therefore sought to investigate if volatile anesthetics increase MMV and if mKATP channel opening is involved in this effect. To do so, we isolated and used intact cardiac mitochondria under state 4 conditions to examine the effect of Sev on MMV as assessed by changes in light scattering.
METHODS
All investigations conformed to the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health No. 85–23, revised 1996) and were approved by the Institutional Animal Care and Use Committee (Medical College of Wisconsin, Milwaukee, WI). Thirty milligram of ketamine and 1000 U of heparin were injected intraperitoneally into 21 albino English short-haired guinea pigs (weight 250–300 g) of either sex. Animals were decapitated 15 min later when unresponsive to noxious stimulation. After thoracotomy, the heart was immediately removed and immersed in 4°C cold isolation buffer containing the following: 200 mM mannitol, 50 mM sucrose, 5 mM KH2PO4, 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 5 mM 3-(n-morpholino)propanesulfonic acid, and 0.1% bovine serum albumin, with KOH to adjust the pH to 7.2 to mimic intracellular milieu. The atria were discarded, and the ventricles were minced into 1-mm pieces. The suspension was rinsed and homogenized for 30 s in 2.5 mL isolation buffer containing 5 U/mL protease (bacillus licheniformis), followed by another 30 s of homogenization after 10-fold dilution of the protease. Mitochondria were then isolated by differential centrifugation at 4°C as previously described.19,26 The tissue suspension was centrifuged at 8000g for 10 min to remove the protease. The resulting pellet was resuspended in 28 mL isolation buffer, and the suspension centrifuged at 700g for 10 min to remove cellular debris. The supernatant containing the mitochondrial fraction was further centrifuged at 8000g for 10 min without EGTA, and the final pellet was resuspended in 500 µL cold isolation buffer without EGTA and kept on ice. Total protein concentration was determined by the Bradford-method.27 Mitochondrial yield from one heart was sufficient for approximately 15 experiments on average. Anatomical integrity of isolated mitochondria was verified by electron microscopy in random studies. Functional integrity after isolation was verified by measuring oxygen consumption and oxidative phosphorylation. State 3/state 4 respiration yielded a suitable Respiratory Control Index of 2.5 ± 0.3 for the substrate succinate plus rotenone.
Measurements of MMV
Changes in MMV as a consequence of net salt transport and concomitant water uptake into mitochondria were measured using a well established quantitative light-scattering technique.28 This technique is based on the observation that the inverse absorbance of the mitochondrial suspension at 520 nm, when corrected for the extrapolated value at infinite protein concentration, is linearly related to MMV within three well defined osmolality regions.29,30 To obtain the rates of MMV increases, it is necessary to avoid the sharp discontinuities in the absorbance measurements. For ion fluxes leading to observable changes in light scattering, the ideal buffer osmolality is 115 mOsm, at which the outer membrane begins to rupture30 because in the more isotonic range matrix swelling is largely compensated for by intermembrane space contraction, so that total volume changes are small,29 and the light-scattering traces have only low signal-to-noise ratios. Recent concerns regarding the validity of this technique31 have been thoroughly addressed by Costa et al.28
An aliquot of the concentrated mitochondria was pipetted at t = 0 s into a cuvette containing a total of 2.5 mL buffered salt medium with the following composition: 55 mM KCl, 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, 0.1 mM EGTA, 2.5 mM KPO4, 0.5 mM MgCl2, 10 mM succinate. Total [Ca2+] was <100 nM as assessed by indo 1 fluorescence. 10 µM Rotenone (dissolved in dimethyl sulfoxide, DMSO) was added to inhibit reverse electron transfer from complex II to complex I of the electron transport chain. Oligomycin 100 µM was added to inhibit ATP synthesis by the FOF1-ATP synthase (complex V). Immediately before addition of the mitochondria, one of four different Sev concentrations (calculated final concentration 30, 100, 300, or 1000 µM; measured as 40 ± 20, 130 ± 90, 360 ± 100, and 960 ± 200 µM by high-performance liquid chromatography, respectively) or their vehicle DMSO (Con) were added to the buffer with or without the mKATP channel antagonists 5-hydroxydecanoate (5-HD; 300 µM).32 The total DMSO concentration of 0.8% has no effect on mitochondrial function.
In additional experiments, ATP was replaced by adenosine diphosphate (ADP; 250 µM; n = 12) to facilitate mKATP channel opening. The mKATP channel opener diazoxide (30 µM; in DMSO; n = 10),33 the K+ ionophore valinomycin (1 nM; in DMSO; n = 8), or the complex III inhibitor antimycin A (100 µM; in DMSO; n = 8) were added for comparison. Mitochondria were assayed at a concentration of 100 µg of protein/mL at 25°C, and at a pH of 7.2. Sev was purchased from Abbott Laboratories, Chicago, IL; all other drugs were purchased from Sigma Chemical, St. Louis, MO.
Photon counts were measured at a wavelength of 520 nm with a temperature-controlled spectrophotometer (Photon Technology International, London, Canada) and were continuously recorded at a 2 Hz sampling frequency during continuous stirring. Samples of original photon count traces over time are shown in Figure 1. After a sharp initial decrease immediately after mitochondrial injection, the photon count increased again incrementally until it reached a plateau after approximately 1 min. The difference in photon counts between the lowest value at the beginning of the experiment (between 5 and 10 s) and 60 s later was used to calculate MMV changes.
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Experiments with mitochondria from the same heart were randomized to one of the above treatment groups with at least three controls interspersed between treatments. All MMV changes from experiments of one heart, including controls, were normalized and expressed as % change compared with the average of control experiments from the same heart.
Statistical Analysis
All data were expressed as means ± standard deviations (sd). Group data were compared by analysis of variance to determine significance (Super ANOVA 1.11 software for Macintosh from Abacus Concepts, Berkeley, CA). If F values were significant (P < 0.05), post hoc comparisons of means tests (Student-Newman-Keuls) were used to compare the groups. Differences among means were considered statistically significant when P < 0.05 (two-tailed). Unless otherwise stated, the number of experiments in each group was 12.
RESULTS
As shown in Figure 2, the K+ ionophore valinomycin caused a maximal MMV increase in our model of +92% ± 50%* (P < 0.001 vs Con; n = 8) compared with control. The mKATP channel opener diazoxide caused a MMV increase of + 52% ± 34%* (P < 0.001 vs Con; n = 10); this was abolished by the mKATP channel antagonist 5-HD (–2% ± 45%; P = 0.025 vs diazoxide; n = 7). KATP channel opening by replacing ATP with ADP led to an increase in MMV (+55% ± 41%*; P < 0.001 vs Con; n = 12) similar to the one by diazoxide. 5-HD (Fig. 3) had no significant effect on MMV (–1% ± 37%; n = 12). Inhibition of mitochondrial electron flow with the complex III inhibitor antimycin A prevented any increase in MMV (–117% ± 60%*; P < 0.001 vs Con; n = 8) when compared with controls (0% ± 36%; n = 60).
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The effect of sevoflurane on MMV was biphasic (Fig. 3). The addition of 30 µM Sev (equivalent to approximately 0.2 vol % at 37°C) and 100 µM (approximately 0.7 vol %) resulted in a +34% ± 27%* (P = 0.01 vs Con; n = 12) and a +55% ± 63%* (P < 0.001 vs Con; n = 12) MMV increase, respectively, compared with control (0% ± 36%; n = 60). In contrast, the addition of 300 µM (approximately 2.1 vol %) Sev had no added effect to increase MMV (+32% ± 42%*; P = 0.02 vs Con; n = 12) whereas the administration of 1000 µM (approximately 7.1 vol %) Sev did not result in a significant MMV change compared with control (–2% ± 26%; P = 0.025 vs 100 µM Sev; n = 12). In the presence of 5-HD (n = 12 per group), Sev did not lead to altered MMV compared with control at any concentration.
DISCUSSION
The present study shows for the first time that (a) Sev causes an increase in MMV; (b) this effect is most prominent at a lower concentration range; and (c) this effect can be blocked by 5-HD, which suggests that it is mediated by mKATP channel opening.
Cardioprotection by KATP channel openers is well established.21,24 Garlid et al. provided the first evidence to support a role for mitochondrial, rather than sarcolemmal, KATP channel opening in cardioprotection.33 However, it is still unclear if mKATP channel opening acts as a trigger, distal effector or both, of ischemic and/or pharmacologic myocardial protection. As a trigger, mKATP channels would open before ischemia and lead to activation of downstream signaling pathways such as reactive oxygen species generation,34,35 PKC translocation,14,34,36 and others. In contrast, as an effector, these signaling pathways would contribute to mKATP channel opening during nonphosphorylating conditions such as ischemia, and thereby afford cardioprotection.
There is considerable disagreement about the exact mechanism by which mKATP channel opening modulates mitochondrial function. Liu et al., for example, have argued that channel opening would accelerate electron transfer, lead to a net mitochondrial oxidation, and mildly uncouple oxidative phosphorylation.20,21 Consequently, measuring a more oxidized mitochondrial redox state with autofluorescent markers has become a popular tool to assess mKATP channel opening in isolated cardiomyocytes.20,36,37
Alternatively, Garlid et al. argue that the critical effect of mKATP channel opening is the regulation of MMV, an example of which is attenuation of MMV contraction during ischemia.24,38 A decrease in membrane potential during ischemia decreases MMV, which may lead to decreased and less efficient electron transport and ATP synthesis.39 K+ influx through mKATP channels and concomitant uptake of weak acids and water by osmotic forces33 would counteract this volume decrease and help maintain a constant MMV. Better preservation of mitochondrial integrity would permit a more efficient energy transfer between mitochondria and cellular ATPases.40 Furthermore, a K+ influx large enough to cause significant uncoupling would be expected to lead to massive MMV swelling and rupture of the mitochondrial inner membrane under phosphorylating conditions.24,28 Thus, uncoupling by mKATP channel opening would not occur, and the fact that accelerated electron transport and net oxidation by mKATP channel openers has been observed may be explained by artificial study conditions.24,41
These differences emphasize the importance of the mitochondrial energetic state when mitochondrial channels are studied.26,42 For example, under state 4 conditions, as in our study, mitochondrial respiration is impeded indirectly by inhibiting protons to reenter the mitochondrial matrix via complex V, due to a shortage of ADP to be phosphorylated and/or by direct blockade of complex V by oligomycin. Under those conditions, any passage of cations, as e.g., K+ through mKATP channels, results in mildly increased electron transport and O2 consumption. Differences in study conditions may therefore explain the finding of mitochondrial oxidation by volatile anesthetics in isolated resting myocytes that were performed in or cultured in substrate-free solutions.20,36,37 In fact, we found that sevoflurane led to a concentration-dependent increase in reduced NADH18 in intact beating hearts, suggesting decreased respiration and oxidation under state 3 conditions. This was confirmed by the finding of attenuated mitochondrial respiration in isolated cardiac mitochondria under phosphorylating state 3 conditions.19 By altering the study conditions to state 4, however, we were able to demonstrate a mild increase in respiration that was blocked by mKATP channel antagonists.22 In other words, opening of mKATP channels by volatile anesthetics may indeed occur, but simply has no directly measurable effect in decreasing mitochondrial redox potential or increasing mitochondrial respiration under normal phosphorylating state 3 conditions.
Maintenance of MMV under nonphosphorylating conditions23 has been implicated as an important component of cardioprotection by mKATP channel opening.24,25 However, no study has investigated if volatile anesthetics affect MMV favorably and if this could, in fact, be another way of linking cardioprotection by volatile anesthetics to mKATP channel opening. In this regard, the results from our study provide new insights into the intracellular mechanisms of Sev as a cardioprotective drug: they link positive MMV changes by Sev via opening of mKATP channels to the cardioprotection by Sev-induced mKATP channel opening. Preliminary results from experiments on isolated rat heart mitochondria (data not shown) not only confirmed these findings but also showed that the sevoflurane-induced increase in MMV was not blocked by the selective PKC
inhibitor peptide V1-2.43 This suggests a direct effect of Sev on mKATP channels rather than via activation of adjacent PKC.44
Our findings also suggest that mKATP channel opening by Sev may depend on an anesthetic concentration that is not so high as to also depress mitochondrial electron transport and oxidative phosphorylation.19 High concentrations of all hydrophobic drugs inhibit mitochondrial respiration45 and subsequently MMV swelling, which explains the observed biphasic response when higher Sev concentrations are used. This is supported by the pronounced decrease in MMV by complete electron transport chain inhibition with antimycin A, as shown here and by others.23 This biphasic response contrasts with the observations that APC appears to be more effective at higher concentrations,18,46 which may, in fact, limit its clinical applicability47 secondary to pronounced cardiodepressant side effects. It is, however, important to note that this is true for anesthetic exposure under phosphorylating conditions, i.e., before ischemia, followed by complete washout of the anesthetic before the onset of nonphosphorylating conditions, i.e., ischemia. Interestingly, Obal et al.14 also observed PKC phosphorylation and translocation in rat hearts only at low anesthetic concentrations. Cardioprotection by volatile anesthetics present during nonphosphorylating conditions such as ischemia may, therefore, follow a different profile and require a more modest dosing.
The present study has limitations. We chose to study cardiac isolated mitochondria outside the myocyte, heart, and whole organism to eliminate outside influences. This, however, cannot substitute for in situ conditions. For example, possible changes in intracellular, i.e., extramitochondrial, pH, or ion concentrations caused by volatile anesthetics cannot be considered, since these are predetermined by the buffer composition. Furthermore, isolation of mitochondria in low K+- medium as used in our study contracts MMV due to loss of K+ salts and water23; however, initiation of mitochondrial respiration in this medium has been shown to reverse this MMV contraction.23,24 We also acknowledge that interpolating conclusions from our experiments under nonphosphorylating conditions to more physiologic phosphorylating conditions remains challenging. Nevertheless, it is sometimes unavoidable to create conditions that allow the measurement of variables, e.g., MMV changes by mKATP channel opening that would otherwise not be measurable. Despite new knowledge, our study also introduces new questions that need to be addressed in further projects.
In summary, this report demonstrates that Sev, depending on its concentration, differentially increases MMV in cardiac isolated mitochondria via mKATP channel opening. Maintenance of normal MMV may contribute to cardioprotection during ischemia at lower anesthetic concentrations. Higher concentrations, however, may not add to this protection.
ACKNOWLEDGMENTS
The authors thank Samhita S. Rhodes, PhD (Postdoctoral Research Fellow), Amadou K.S. Camara, PhD (Associate Professor), Mohammed Aldakkak, MD (Postdoctoral Research Fellow), Srinivasan G. Varadarajan (Assistant Professor), and James S. Heisner, BS (Research Technologist, all Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin) for their valuable contributions to this study.
Footnotes
Accepted for publication December 3, 2007.
Supported, in part, by grant No. HL58691 (to Dr. Stowe), No. HL 67842 and No. 36573 (to Dr. Garlid) from the National Institutes of Health (Bethesda, MD), and by grant No. 0355608Z (to Dr. Stowe) from the American Heart Association (Dallas, TX).
REFERENCES
(PKC) in isoflurane-induced cardioprotection. Br J Anaesth 2005;94:166–73This article has been cited by other articles:
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  A. D. T. Costa and K. D. Garlid Intramitochondrial signaling: interactions among mitoKATP, PKC{varepsilon}, ROS, and MPT Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H874 - H882. [Abstract] [Full Text] [PDF]
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) and 60 s later (
). Con: control experiment; Sev: 100 µM sevoflurane; Sev + 5-HD: 100 µM sevoflurane and 300 µM 5-hydroxydecanoate, a mitochondrial KATP channel antagonist; Val: 1 nM valinomycin, a K+ ionophore.
