Preconditioning with isoflurane has been shown to confer cardioprotection via activation of mitochondrial adenosine triphosphate-sensitive K+ (mito K(ATP)) channels. However, the relative contribution of mito K(ATP) channel and non-mito K(ATP) channel mechanisms to isoflurane-mediated cardioprotection has not been investigated.
Isolated and buffer-perfused rat hearts were used. Flavoprotein fluorescence was monitored as an index for mito K(ATP) channel activity. Isovolumic left ventricular function and infarct size were measured as indices for cardioprotection.
Flavoprotein fluorescence, which was monitored as an index for mito K(ATP) channel activity, was increased by isoflurane and a known mito K(ATP) channel opener, diazoxide, in a 5-hydroxydecanoate-sensitive manner. Although flavoprotein oxidation induced by diazoxide was dissipated soon after its removal from the buffer, flavoprotein oxidation induced by isoflurane was sustained after cessation of the treatment. The sustained increase in flavoprotein oxidation was associated with a significant reduction in infarct size after 30 min of ischemia followed by 120 min of reperfusion. Although adenosine and S-nitroso-N-acetyl-penicillamine each alone did not increase flavoprotein fluorescence, nor did they confer significant cardioprotection, coadministration of adenosine and S-nitroso-N-acetyl-penicillamine with isoflurane conferred a highly significant reduction of infarct size and improvement of left ventricular function without increasing flavoprotein oxidation over isoflurane alone. The early treatment with 5-hydroxydecanoate before and during preconditioning completely reversed flavoprotein oxidation and inhibited the infarct-sparing effect of isoflurane and combined preconditioning with isoflurane, adenosine, and S-nitroso-N-acetyl-penicillamine. The late treatment with 5-hydroxydecanoate after preconditioning abolished flavoprotein oxidation and the infarct-sparing effect of isoflurane but only partially inhibited cardioprotection conferred by the combined preconditioning, despite complete abrogation of flavoprotein oxidation.
Mito K(ATP) channel activation is the essential trigger of both preconditioning with isoflurane and combined preconditioning with isoflurane, adenosine, and S-nitroso-N-acetyl-penicillamine. Mito K(ATP) channel activation is also a crucial mediator of cardioprotection afforded by preconditioning with isoflurane. However, enhanced cardioprotection conferred by combined preconditioning is mediated through both mito K(ATP) channel-dependent and -independent mechanisms.
BRIEF periods of cardiac ischemia and reperfusion exert a protective effect against subsequent, more prolonged period of ischemia, a phenomenon termed ischemic preconditioning (IPC). 1IPC-like effects have also been observed by administration of various pharmacologic agents. Volatile anesthetics, including isoflurane, have long been known as such cardioprotective agents against ischemia–reperfusion injury, and this cardioprotective effect has at least in part been attributed to opening of mitochondrial adenosine triphosphate–sensitive K+(mito KATP) channels. 2–8Despite accumulating evidence showing cardioprotection afforded by isoflurane, the exact role of mito KATPchannels in isoflurane-induced cardioprotection remains to be investigated.
Pharmacologic preconditioning (PPC) has been performed by using IPC mimetic drugs, such as adenosine, a G protein–coupled receptor agonist (GPCRA) 9,10; diazoxide, a mito KATPchannel opener 11; and nitric oxide donors. 12,13However, acute cardioprotection by single PPC with adenosine, diazoxide, or nitric oxide donors remains a controversial issue. There are a number of studies showing that PPC with each drug alone confers significant cardioprotection, 11–15but also, a considerable number of studies have failed to substantiate acute cardioprotection by PPC with single-drug administration. 16–18It is not surprising that a single PPC strategy failed to completely mimic IPC because IPC is mediated by complex signaling cascades arising from multiple triggers. Ninomiya et al. 19have demonstrated that PPC with adenosine had no acute benefit in functional protection after global ischemia, whereas administration of adenosine during IPC was capable of significantly enhancing IPC-induced protection. This observation suggests that adenosine enhancement of IPC-induced cardioprotection is elicited by robust activation of G protein–coupled receptors in concert with other, yet unidentified triggers produced by preconditioning ischemia and reperfusion. Our subsequent studies 20,21have shown that adenosine in combination with a mito KATPchannel opener and a nitric oxide donor is necessary to completely mimic IPC, and pretreatment with a Gi/o protein inhibitor, pertussis toxin, a mito KATPchannel inhibitor, 5-hydroxydecanoate (5-HD), or a nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, completely abrogated cardioprotection afforded by combined preconditioning. This observation has led us to hypothesize that GPCRA and nitric oxide are the likely cotriggers of successful preconditioning with a mito KATPchannel opener. Requirement of a GPCRA and a nitric oxide donor may also be the case for isoflurane to confer optimal cardioprotection. Patel et al. 22have demonstrated that a known GPCRA, δ opioid, enhances an infarct-sparing effect of isoflurane. Moreover, the fact that functional protection mediated by the nitric oxide donor nicorandil 23was enhanced by isoflurane, which by itself exerted no functional benefit, 24points to the conclusion that isoflurane requires coadministration with a nitric oxide donor to mediate functional protection. Therefore, we attempted to address the issue of whether coadministration with a GPCRA and a nitric oxide donor potentiates cardioprotection by preconditioning with isoflurane and whether such enhanced cardioprotection occurs solely through activation of mito KATPchannels or also through mito KATPchannel-independent mechanisms.
Materials and Methods
Perfusion Technique and Flavoprotein Fluorescence Measurement
Male Sprague-Dawley rats weighing 250–300 g were used in the current study. All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals 25and were approved by Animal Care Committee of Kansai Medical University (Moriguchi, Japan). The rats were anesthetized intraperitoneally with pentobarbital sodium (100 mg/kg). After thoracotomy, the heart was rapidly excised, placed in a temperature-regulated heart chamber installed in a CAF-110 intracellular ion analyzer (Japan Spectroscopic Co., Tokyo, Japan), and perfused at a constant mean pressure of 60–70 mmHg with Krebs-Henseleit bicarbonate buffer solution with the following composition: 118 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 25 mm NaHCO3, 1.2 mm KH2PO4, 1.8 mm CaCl2, and 11 mm glucose, with a pH of 7.4 at 37°C when equilibrated with a mixture of 95% O2and 5% CO2gas.
During the stabilization period, a latex balloon was inserted into the left ventricle through the left atrium to measure isovolumic left ventricular (LV) function. The balloon was filled with saline solution to produce an LV end-diastolic pressure of 5–10 mmHg at baseline, and the balloon volume was kept constant throughout the experiment. The hearts with LV developed pressure less than 80 mmHg or heart rate less than 240 beats/min at baseline were excluded from the study. LV developed pressure and heart rate were expressed as percentage of the baseline values.
Flavoprotein fluorescence of the heart was monitored as an index for mito KATPchannel activity as described previously. 20,26Briefly, fluorescence excitation was provided by a xenon lamp with a band-pass filter centered at 480 nm. The excitation light was diverted onto a circular region of the LV epicardium with a diameter of 1 cm by means of a dichroic mirror. Fluorescence then passed through an emission filter centered at 530 nm before reaching a photomultiplier. The photomultiplier output was digitized, and the fluorescence was displayed on a strip-chart recorder. At the end of each experiment, dinitrophenol (1 mm) was added to elicit full oxidation of mitochondrial flavoprotein. Data were expressed as percentage of the dinitrophenol-induced fluorescence.
First, we studied the dose–response effect of a known mito KATPchannel opener, diazoxide, and isoflurane on mito KATPchannel activity. Applied concentrations of isoflurane in the perfusate buffer (0, 0.5, 1.0, 2.0, 3.0, and 4.0%) were measured by gas chromatography with a flame ionization detector (GC14-B; Shimazu, Kyoto, Japan). The isoflurane concentrations in the sample portion were 70 ± 8% of those in the flask at all concentrations of isoflurane tested. To confirm that the observed increase in flavoprotein fluorescence was a result of mito KATPchannel activation, 5-HD (0.5 mm), a relatively selective inhibitor of mito KATPchannels, was used.
Left ventricular function and infarct size were evaluated in the protocol as shown in figure 1, in which the hearts were subjected to 30 min of global ischemia followed by 120 min of reperfusion. In protocol 1, we compared the effect of preconditioning with 50 μm diazoxide and 1 minimum alveolar concentration (MAC) of isoflurane on mito KATPchannel activity and cardioprotection. This concentration of diazoxide was chosen because it is known to confer maximum cardioprotection with fewer nonspecific effects. 27One MAC of isoflurane in the rat 28was obtained by means of a 2% mixture of this volatile anesthetic agent in our experimental model. Diazoxide and isoflurane were administered for 25 min and were discontinued 20 min before the index ischemia. 5-HD was given 10 min before and during and after the treatment with diazoxide and isoflurane. Reperfusion was performed for 120 min to assess myocardial infarct size.
In protocol 2, we compared the cardioprotective effect of preconditioning with isoflurane, adenosine, and a nitric oxide donor, S-nitroso-N -acetyl-penicillamine (SNAP), each alone or in combination. The dose of adenosine used in the current study (10 μm) seems to be appropriate as preconditioning stimulation because this dose of adenosine has been shown to increase cardiac interstitial adenosine to a level comparable with that produced by IPC. 29SNAP at a dose of 10 μm was chosen in this experimental model because we have found that SNAP at concentrations up to 50 μm had no adverse effect on LV function. 21
In the protocol 3, we investigated the role of mito KATPchannels in cardioprotection conferred by combined preconditioning with isoflurane, adenosine, and SNAP. In this set of experiments, 5-HD was administered 10 min before and during preconditioning with isoflurane or the combined PPC as the early treatment modality or for 10 min just before 30 min of ischemia as the late treatment modality.
Isoflurane was purchased from Abbott Laboratories (Abbott Park, IL). Diazoxide, adenosine, and dinitrophenol were obtained from Sigma-Aldrich Japan (Tokyo, Japan). SNAP was purchased from Wako Pure Chemical (Osaka, Japan), and 5-HD was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). Diazoxide and SNAP were dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide was less than 0.05%.
Infarct Size Measurement
After termination of experiments, the heart was trimmed to remove the atrium, the right ventricular free wall, and the connective tissues and was sliced transversely in a plane perpendicular to the apical–basal axis into approximately 1-mm-thick slices. The slices were immersed in phosphate-buffered saline containing 2% triphenyltetrazolium chloride for 15 min at 37°C and fixed with 10% formaldehyde in 0.1 m phosphate buffer (pH 7.2) at room temperature. The brick red area was traced with the use of National Institutes of Health 1.61 image-processing software (Bethesda, MD), and each digitized image was subjected to equivalent degrees of background subtraction, brightness, and contract enhancement for improved clarity and distinctness. The areas at risk (equivalent to total LV mass), as well as the infarct zones of each slice, were calculated in terms of pixels. The infarct volume was calculated, and the sum of all slices was used to compute a ratio of percent infarct to total LV mass.
All numerical data are expressed as mean ± SEM. Statistical analysis was performed by one-way analysis of variance, followed by the Bonferroni post hoc test. P < 0.05 was considered statistically significant.
Isoflurane Provokes Flavoprotein Oxidation in a Concentration-dependent Manner
Flavoprotein fluorescence was stabilized within 15 min after Langendorff perfusion with Krebs-Henseleit bicarbonate buffer and remained stable over 120 min (not shown). Addition of diazoxide increased flavoprotein fluorescence, which reached a plateau within 5 min (fig. 2A). The increase in flavoprotein fluorescence induced by diazoxide was abolished by treatment with 0.5 mm 5-HD, which by itself did not affect baseline flavoprotein fluorescence. The dose–response study showed that 50 μm diazoxide provoked a nearly maximal increase in flavoprotein fluorescence (fig. 2B). Isoflurane increased flavoprotein fluorescence, but the increase in flavoprotein fluorescence was slower than that observed with diazoxide, taking 15–20 min to reach a plateau (fig. 2C). This flavoprotein oxidation induced by isoflurane was also sensitive to 5-HD, suggesting that mito KATPchannel activation is responsible for the increase in flavoprotein fluorescence induced by isoflurane. The amplitude of flavoprotein fluorescence was increased with increasing isoflurane concentrations in a range between 0.5 and 3.0% (fig. 2D), and further increase in isoflurane concentration reduced flavoprotein fluorescence as expressed by percent of dinitrophenol-induced flavoprotein oxidation (2% isoflurane: 60 ± 10; 3% isoflurane: 63 ± 7; 4% isoflurane: 31 ± 5).
Isoflurane but Not Diazoxide Induces Sustained Activation of Mito KATPChannels
Although both diazoxide and isoflurane increased flavoprotein fluorescence, a major difference in the pattern of flavoprotein fluorescence between these two agents was observed after cessation of the treatment. The increase in flavoprotein fluorescence induced by diazoxide was dissipated soon after its removal from the buffer, whereas that induced by isoflurane was sustained after cessation of the treatment (fig. 3).
Preconditioning with Isoflurane but Not with Diazoxide Confers Cardioprotection
We compared the effect of preconditioning with diazoxide to that of isoflurane on cardioprotection (protocol 1). Preconditioning with 50 μm diazoxide did not significantly reduced infarct size compared with control (P = 0.201;fig. 4). In contrast, preconditioning with isoflurane was effective in significantly reducing infarct size.
Baseline LV function was not significantly different in all groups. We compared LV function 45 min after reperfusion because maximum recovery of LV systolic function was observed between 30 min and 60 min after reperfusion in all the hearts. LV function was not significantly improved by preconditioning with diazoxide (table 1). Isoflurane was also without a significant effect on the recovery of LV function.
Treatment with 5-HD by itself had no significant effect on infarct size and postischemic recovery of LV function. However, the infarct-sparing effect of preconditioning with isoflurane was abolished by 5-HD, suggesting that activation of mito KATPchannels is involved in the infarct-sparing effect mediated by preconditioning with isoflurane.
Combined Preconditioning with Isoflurane, Adenosine, and SNAP Potentiates Cardioprotection without Enhancement of Flavoprotein Oxidation
We investigated whether cardioprotection afforded by preconditioning with isoflurane can be enhanced by coadministration with adenosine and SNAP (protocol 2). In contrast to preconditioning with isoflurane, preconditioning with adenosine and SNAP alone did not significantly reduce infarct size compared with control (P = 0.274 and P = 0.225, respectively;fig. 5). Coadministration of isoflurane with adenosine but not SNAP significantly improved LV function (table 2) and enhanced the infarct-sparing effect of preconditioning with isoflurane. Moreover, only preconditioning with isoflurane in combination with adenosine and SNAP significantly enhanced postischemic recovery of LV function and reduced infarct size over preconditioning with isoflurane alone.
Neither adenosine nor SNAP by itself significantly affected baseline flavoprotein fluorescence (fig. 6). Isoflurane significantly increased flavoprotein oxidation as described before. However, isoflurane in combination with adenosine and SNAP did not further increase in flavoprotein oxidation over isoflurane alone during and after treatment.
Early Treatment with 5-HD Completely Inhibits but Late Treatment with 5-HD Only Partially Inhibits Cardioprotection Afforded by Combined Preconditioning with Isoflurane, Adenosine, and SNAP
Mito KATPchannels may act as a trigger of cardioprotection conferred by combined preconditioning with isoflurane, adenosine, and SNAP. The trigger role of mito KATPchannels was tested by administration with 5-HD 10 min before and during the combined preconditioning (protocol 3). As was the case for preconditioning with isoflurane alone, the early treatment modality abolished the infarct-sparing effect (fig. 7) and inhibited improvement of LV function conferred by combined preconditioning (table 3). Because powerful cardioprotection afforded by combined preconditioning was not correlated with the magnitude of flavoprotein oxidation, it was thought that mito KATPchannels might not be a sole mediator of the combined preconditioning. The mediator role of mito KATPchannels was tested by late treatment with 5-HD. The late treatment with 5-HD by itself had no effect on the recovery of LV function and infarct size. Although the late treatment with 5-HD completely inhibited the infarct-sparing effect conferred by preconditioning with isoflurane, the same treatment exerted significant but only partial inhibition against enhanced postischemic recovery of LV function and infarct size reduction afforded by combined preconditioning with isoflurane, adenosine, and SNAP. Either the early treatment or the late treatment with 5-HD abolished flavoprotein oxidation induced by combined preconditioning with isoflurane, adenosine, and SNAP (fig. 8).
The current study compared the efficacy of preconditioning with isoflurane to that of a mito KATPchannel opener, diazoxide; GPCRA; adenosine; and a nitric oxide donor, SNAP, all of which are known to be mimetic of IPC. The results showed that preconditioning with isoflurane but not with diazoxide, adenosine, or SNAP conferred significant cardioprotection. The inability of preconditioning with diazoxide, adenosine, or SNAP alone to confer significant cardioprotection contradicts many former studies that showed significant cardioprotection by preconditioning with each drug alone. 11–15The mechanism underlying the discrepant observation remains unclear. Although the statistical power with respect to the infarct-sparing effect of these drugs may be increased when the sample size is increased, such a statistical power would not substantially affect the conclusion that a cardioprotective effect of each drug alone is modest compared with that afforded by IPC. Alternatively, drug concentrations are possible explanations. We have tested a preconditioning effect of only single doses of drugs and did not attempt to undertake a dose–response study. We used 50 μm diazoxide, a concentration known to confer maximum cardioprotection with less nonspecific effects. 27This concentration of diazoxide was found to cause nearly maximum activation of flavoprotein oxidation, indicating that mito KATPchannels were fully activated by this concentration of diazoxide. The doses of adenosine and SNAP seem to be lower than those used in other studies. However, this dose of adenosine was found to increase its interstitial concentration up to several times higher than that observed in IPC. 29In addition, we have demonstrated in a previous study 20that five-times higher doses of adenosine and SNAP than in the current study could not provoke significant flavoprotein oxidation and cardioprotection when each was used alone. Therefore, the inability of these drugs to put the heart into the preconditioned state may not solely be attributed to lower drug doses. The rationale for using lower doses of drugs is to elicit only a specific effect while avoiding unwanted side effects, which may be meritorious in the clinical setting of myocardial protection.
Although PPC with adenosine or SNAP was unable to confer cardioprotection, coadministration of these drugs with isoflurane dramatically potentiated cardioprotective effects conferred by isoflurane. We have previously demonstrated that preconditioning with adenosine, diazoxide, or SNAP alone was not capable of mediating cardioprotection, even at a higher dose than that which was used in this study, but the combination of these drugs conferred powerful cardioprotection that was as effective as IPC. 20,21Moreover, cardioprotective signaling as evaluated by protein kinase C (PKC)-ε activity was increased only by IPC and the combined preconditioning. These observations suggested that enhanced cardioprotection by the combined preconditioning was not simply due to an additive effect but was a synergistic effect through integration of distinct signals arising from each drug to robust cardioprotective signaling. The current study also points to the conclusion that subthreshold inputs of cardioprotective signaling provoked by adenosine and SNAP can augment isoflurane-induced cardioprotection.
We have studied the difference in the potency of cardioprotection between isoflurane and other known triggers of IPC with respect to the activity of mito KATPchannels. Because there is currently no direct means to measure mito KATPchannel activity in the intact heart and cardiomyocytes, we 21,26and others 30have adopted flavoprotein oxidation as an indirect index for mito KATPchannel activity. Using this technique, we showed in the current study that isoflurane and diazoxide increase flavoprotein oxidation in a concentration-dependent and a 5-HD–sensitive manner. A significant increase in flavoprotein fluorescence occurred with isoflurane concentrations ranging between 0.5 and 4%, but this effect was maximal at the concentrations between 2 and 3%, indicating that mito KATPchannels were fully activated by the isoflurane concentration used in the preconditioning study. The increase in flavoprotein oxidation induced by diazoxide and isoflurane was abolished in the presence of 5-HD, which did not affect flavoprotein fluorescence at baseline and flavoprotein oxidation induced by dinitrophenol, 20suggesting that mito KATPchannel activation may be responsible for flavoprotein oxidation induced by diazoxide and isoflurane. A similar dose–response effect of volatile anesthetics on mito KATPchannels has been observed in isolated cardiomyocytes. 31
The increase in flavoprotein oxidation induced by diazoxide reached a plateau within 5 min, whereas that induced by isoflurane was a slow process, taking 15–20 min to reach a plateau. Moreover, the increase in flavoprotein oxidation induced by isoflurane was sustained after cessation of the treatment, while that induced by diazoxide was dissipated soon after its removal from the buffer. The sustained increase in flavoprotein oxidation induced by preconditioning with isoflurane but not with diazoxide was associated with significant reduction of infarct size. Therefore, there was correlation between flavoprotein oxidation at the time of index ischemia and cardioprotection. These findings provide an explanation for the discrepant results regarding the efficacy of cardioprotection between preconditioning with diazoxide and isoflurane and imply that the mechanism of activation of mito KATPchannels may be different between these two agents. Because isoflurane is a lipophilic substance that partitions into membranes, it presumably takes longer than diazoxide to be fully expelled from tissues. A more intriguing hypothesis is that additional triggers of preconditioning besides mito KATPchannels may be involved in isoflurane-induced sustained activation of mito KATPchannels. Accumulating evidence suggests that isoflurane not only activates mito KATPchannels, but also activates other signaling cascades. Isoflurane has been shown to induce protection of human myocardium from anoxic injury via adenosine A1receptors and KATPchannels. 32Involvement of G protein–coupled receptors in isoflurane-induced cardiac preconditioning was further suggested by the fact that the blockade of Gi/o proteins by pertussis toxin abolished the infarct size–limiting effect of isoflurane in the canine heart. 33Furthermore, another volatile anesthetic agent, halothane, has been shown to directly interact with G protein–coupled receptors. 34Isoflurane is also capable of producing nitric oxide in the mouse brain. 35Finally, the cardioprotective effect of volatile anesthetics was found to be dependent on PKC. 36Although mito KATPchannels could be opened by mito KATPchannel openers without PKC activation, a priming effect of PKC activation on enhanced mito KATPchannel opening has been demonstrated, 30and this mechanism may also be crucial in the sustained activation of mito KATPchannels that was attributed to the “acute memory” of cardioprotection afforded by isoflurane. 6Therefore, it is likely that isoflurane provokes sustained activation of mito KATPchannels even after cessation of the treatment, presumably through activation of multiple signaling pathways that converge on PKC activation, as has been proposed by Zaugg et al. 36
The current study investigated the role of mito KATPchannels in cardioprotection afforded by isoflurane and combined preconditioning with isoflurane, adenosine, and SNAP. The early treatment with 5-HD completely abolished the infarct size–limiting effect of isoflurane and the combined preconditioning, indicating that mito KATPchannels are the essential trigger of preconditioning with isoflurane and the combined preconditioning. However, although cardioprotection conferred by preconditioning with isoflurane was completely abrogated by the late treatment with 5-HD, that afforded by the combined preconditioning was only partially inhibited by the late treatment with 5-HD, which abolished flavoprotein oxidation mediated by isoflurane and the combined preconditioning. These observations suggest that cardioprotection afforded by isoflurane is mediated through mito KATPchannel activation, while the combined preconditioning–mediated cardioprotection occurs through both mito KATPchannel-dependent and -independent mechanisms. The evidence that the combined preconditioning conferred superior cardioprotection over isoflurane alone is consistent with a recent study 17showing that the KATPchannel opener bimakalim alone had no infarct size–limiting effect, whereas the simultaneous administration of adenosine and bimakalim resulted in a marked decrease in infarct size, suggesting a synergism between adenosine and KATPchannels in the combined preconditioning–mediated cardioprotection. Taken together, our study reinforces the hypothesis that a synergistic interaction of multiple triggers, including mito KATPchannels, GPCRA, and a nitric oxide donor, promotes signaling pathways that culminate in robust cardioprotection through mito KATPchannel-dependent and -independent mechanisms. Whether these cardioprotective mechanisms are promoted by PKC or in concert with other signaling pathways remains to be determined.
The current study showed that preconditioning with isoflurane in combination with low doses of adenosine and SNAP, which by themselves had no significant benefit in cardioprotection, significantly enhanced cardioprotection over preconditioning with isoflurane alone. The powerful cardioprotection mediated by the combined PPC technique is not simply additive to isoflurane of which cardioprotection may be ascribed solely to mitochondrial KATPchannel activation, but is synergistic through both mitochondrial KATPchannel-dependent and -independent mechanisms. Therefore, the current study suggests that exogenous addition of a GPCRA and a nitric oxide donor represents a reasonable approach to enhance cardioprotection afforded by isoflurane. However, clinical evidence has been lacking as to whether combined preconditioning is in fact necessary to confer superior cardioprotection over volatile anesthetic agents alone despite the fact that volatile anesthetic agents are often used in conjunction with GPCRAs such as adenosine and norepinephrine and nitric oxide donors such as nitroglycerine and sodium nitroprusside in cardiac surgery. Future clinical trials are warranted to address this issue.