The current results confirm previous findings that brief exposure to 70% xenon before coronary artery occlusion and reperfusion reduces myocardial infarct size in rats in vivo
In the present study, cardioprotection by xenon against ischemia-induced damage was obtained at the nonanesthetic dose of 70%. The minimum alveolar concentration value for xenon in rats is significantly higher compared to humans and has been reported to be 1.61 atmosphere15
compared to about 70% in humans.16
Thus, if anesthetic efficacy is used as a measure, and if xenon at 1.61 atm in rats is equivalent to xenon at 70% in humans, then 70% xenon in rats would correspond to about 30% in humans. Xenon is essentially chemically inert, but induced dipoles in its large electron field allow xenon to interact with amphiphilic domains of proteins and membrane.17
The precise mechanism by which this interaction produces selective biological effects remains unknown. Xenon produces minimal hemodynamic effects and may be especially suitable as an inhaled anesthetic for patients with cardiac risk factors undergoing surgery.18-20
Xenon and volatile anesthetics exert very similar actions on cardiac and neuronal signal transaction. The current results extend this observation by implicating prosurvival signaling kinases (Akt, GSK-3β
) in xenon preconditioning that have been shown to mediate cardioprotection by volatile anesthetics.1-3
Mitochondria play an essential role in the development of ischemia and reperfusion injury, but also in the protection against it. Recently, we reported that isoflurane preconditioning elicits partial mitochondrial uncoupling, an action that preserves cell viability during the conditions of metabolic challenge.11
The current results are supported, to some degree, by these previous findings. However, in the current study the RCR (a measure of mitochondrial coupling) under baseline condition was not altered in isolated ventricular mitochondria obtained from xenon-preconditioned rats. Since mitochondrial uncoupling was not observed in xenon-preconditioned mitochondria, this phenomenon may not be required for protection of mitochondria, as xenon exposure still preserved mitochondrial function after hypoxia and reoxygenation. Opening of mPTP contributes to cell death after ischemia/reperfusion injury.21-22
Ischemic and anesthetic pre- and postconditioning have been shown to inhibit mPTP opening.23-25
The current results indicate brief, intermittent xenon exposure attenuated Ca2+
-induced mPTP opening in vitro
, as demonstrated by larger amounts of Ca2+
required for dissipating Δϕm
. Collectively, these data suggest that cardioprotection by xenon occurs as a consequence of a mechanism inherent to the mitochondria, independent of the cytosolic environment.
The current findings strongly suggest that xenon preconditioning phosphorylates two key proteins known to modulate the transition state of mPTP. Activation of several endogenous cardioprotective signaling pathways has been shown to be essential for xenon-induced cardioprotection.4,6-8
Interestingly, other noble gases without anesthetic properties have also recently been demonstrated to produce cardioprotection by activating prosurvival signaling kinases and inhibiting mPTP opening in rabbits.26
The exact manner in which xenon and other anesthetic stimuli activate prosurvival signaling pathways remains unknown. The involvement of G protein-coupled receptors has been discussed27
as well as reactive oxygen species (ROS) derived from mitochondria.28
The downstream targets of many of these enzymes have yet to be fully characterized. Nevertheless, the participation of several prosurvival kinases in protection of the myocardium against ischemia has been well described. Activation of PI3K, which is blocked by wortmannin, leads to sequential phosphorylation and thereby activation of phosphoinositide-dependent kinase −1 and Akt.29
PI3K and phosphoinositide-dependent kinase −1may also activate PKC.30
Activated Akt stimulates the activity of several antiapoptotic proteins, such as Bcl-2, endothelial nitric oxide synthase and Mdm2,30
and also inhibits GSK 3β
mediates the convergence of intracellular prosurvival signaling kinases, including Akt, mammalian target of rapamycin, 70 kDa ribosomal s6 kinase, PKC and protein kinase.31
The activity of GSK-3β
is inhibited by phosphorylation through activated Akt,32
and inhibition of GSK-3β
prevents opening of mPTP.33
The precise mechanism by which phosphorylation of GSK-3β
deactivates the enzyme and prevents mPTP opening during reperfusion remains unclear. Nevertheless, our results demonstrating that xenon preconditioning increases phosphorylation of Akt and GSK-3β
may represent a link between the activation of these enzymes and protection of mitochondrial function by rendering mPTP less susceptible to Ca2+
-induced opening. Our findings, that PI3K-inhibitor wortmannin not only blocked reduction of infarct size but also reversed conservation of mitochondrial RCR, are in agreement with this assumption.
Xenon has been shown to exert substantial organo-protective properties in the brain and in the heart.34
To our knowledge the protective of effect of xenon on other organs has not been studied yet. Considering some of the mechanistic similarities with ischemic preconditioning, it is feasible to assume that other organs, such as the kidney, may also be protected by xenon from ischemic injury. However, it seems that some of the xenon-induced effects are organ-specific, for example the antagonism of aspartate receptors35
in neurons. Thus, ultimately only future experiments will be able to answer the question whether other organs besides brain and heart are protected by xenon.
Xenon-induced, volatile anesthetic-induced and ischemic preconditioning have all been shown to produce a strong cardioprotective effect in the rat heart in vivo
, and overall, the mechanistic similarities of those protective strategies are striking. Particularly, prosurvival signaling pathways are shared by all of these strategies, including the involvement of PKC isoforms and downstream kinases, such as p38 and ERK1/2 MAPK. Nevertheless, little is known specifically about the mechanism of xenon preconditioning, and some differences have been pointed out. For example, c-Jun N-terminal kinase
MAPK activation has been shown to play a role in ischemic preconditioning,36
but not in xenon-induced preconditioning,8
and its role in APC has not been studied. A microarray study revealed differences between APC versus ischemic preconditioning in transcripts predominantly related to biosynthesis and apoptosis where ischemic preconditioning elicited a postischemic gene expression profile closer to unprotected myocardium than preconditioning with a volatile anesthetic.37
However, a similar study has not been performed for xenon-induced preconditioning. In our study, we confirmed the participation of signaling kinases similarly to ischemic and APC, but also found subtle differences in the effect of the preconditioning agent on mitochondrial function as described above. This is interesting since a direct effect of the preconditioning agent on mitochondria has been suggested to be involved in the triggering of prosurvival pathways, for example through ROS.38
Therefore, future studies will be needed to further investigate the direct effect of xenon on mitochondrial bioenergetics, including ROS production. However, mitochondrial function remained preserved after ischemic stress on mitochondria isolated from xenon-preconditioned rats, similarly as previously reported for isoflurane-induced preconditioning. Thus, in summary, xenon-, anesthetic- and ischemic-induced preconditioning certainly share mechanistic similarities, in particular in regard to signaling pathways, but they also exhibit potentially significant differences in the triggering phase.
The current results should be interpreted within the constraints of several possible limitations. Isolated mitochondria used for assessing mitochondrial function before and after hypoxia and reoxygenation injury constitute an artificial system deprived of a normal cellular environment that most certainly influences their function under control conditions. Nevertheless, this model allowed us to address the question of whether the signaling pathways induce a “protective” memory effect within mitochondria. The experiments were conducted in barbiturate-anesthetized, acutely instrumented rats. Whether similar results also occur in other animal species or humans is unknown. A xenon dose-response relationship was not examined with the current investigation. Whether longer periods of xenon exposure produce relatively larger reductions in infarct size, phosphorylation of Akt or GSK-3β
, or protection against Ca2+
-induced mPTP opening will require additional study to ascertain. We did not measure further variables of myocardial function, such as left ventricular developed pressure and left ventricular end-diastolic pressure or cardiac output. In order to explain the observation that, in spite of decreased infarct size in the xenon group, we did not find differences in mean arterial blood pressure between control and xenon groups, we speculate that a decrease in cardiac output was paralleled by an increase in systemic vascular resistance in the controls. Sympathetic nerve activity is activated to compensate for an attenuation of contractility, leading to tachycardia and increasing afterload. Myocardial infarct size is determined primarily by the size of the area at risk and the extent of coronary collateral perfusion. The area at risk expressed as a percentage of total LV mass was similar between groups in the current investigation, and coronary collateral blood flow is minimal in rats.39
Thus, differences in collateral perfusion among groups probably did not account for the observed results in rats, but coronary collateral blood flow was not specifically measured. Coronary venous oxygen tension was not directly measured nor was myocardial oxygen consumption calculated. Rate-pressure product, an index of myocardial oxygen consumption, was similar among groups during coronary occlusion, suggesting that the ischemic burden was not responsible for differences in infarct size in rats pretreated with xenon compared with those that did not receive the noble gas. We used a 30 min LAD occlusion to produce myocardial infarction in rats. Whether xenon produces cardioprotection after more prolonged periods of ischemia is unknown.
In summary, the current results indicate that xenon preconditioning reduces myocardial infarct size, phosphorylating Akt and GSK-3β, inhibiting Ca2+-induced opening of mPTP and preserving mitochondrial function. The data suggest that xenon-induced cardioprotection occurs as a consequence of activation of prosurvival signaling that targets mitochondria and renders them less vulnerable to ischemia-reperfusion injury.