Xenon preconditioning: the role of prosurvival signaling, mitochondrial permeability transition and bioenergetics in rats

Abstract

Background: Similar to volatile anesthetics, the anesthetic noble gas xenon protects the heart from ischemia/reperfusion injury, but the mechanisms responsible for this phenomenon are not fully understood. We tested the hypothesis that xenon-induced cardioprotection is mediated by prosurvival signaling kinases that target mitochondria.

Methods: Male Wistar rats instrumented for hemodynamic measurements were subjected to a 30 min left anterior descending coronary artery occlusion and 2 h reperfusion. Rats were randomly assigned to receive 70% nitrogen/30% oxygen (control) or three 5-min cycles of 70% xenon/30% oxygen interspersed with the oxygen/nitrogen mixture administered for 5 min followed by a 15 min memory period. Myocardial infarct size was measured using triphenyltetrazolium staining. Additional hearts from control and xenon-pretreated rats were excised for Western blotting of Akt and glycogen synthase kinase 3 beta (GSK-3beta) phosphorylation and isolation of mitochondria. Mitochondrial oxygen consumption before and after hypoxia/reoxygenation and mitochondrial permeability transition pore opening were determined.

Results: Xenon significantly (P < 0.05) reduced myocardial infarct size compared with control (32 +/- 4 and 59% +/- 4% of the left ventricular area at risk; mean +/- sd) and enhanced phosphorylation of Akt and GSK-3beta. Xenon pretreatment preserved state 3 respiration of isolated mitochondria compared with the results obtained in the absence of the gas. The Ca(2+) concentration required to induce mitochondrial membrane depolarization was larger in the presence compared with the absence of xenon pretreatment (78 +/- 17 and 56 +/- 17 microM, respectively). The phosphoinositol-3-kinase-kinase inhibitor wortmannin blocked the effect of xenon on infarct size and respiration.

Conclusions: These results indicate that xenon preconditioning reduces myocardial infarct size, phosphorylates Akt, and GSK-3beta, preserves mitochondrial function, and inhibits Ca(2+)-induced mitochondrial permeability transition pore opening. These data suggest that xenon-induced cardioprotection occurs because of activation of prosurvival signaling that targets mitochondria and renders them less vulnerable to ischemia-reperfusion injury.

Figure 1

Schematic illustration of the experimental protocols used in the current investigation. Xe = xenon; Wort = wortmannin.

Figure 2

Typical chart recordings showing respiration under baseline conditions and after hypoxic stress in mitochondria isolated from control and xenon-preconditioned rats. Oxygen consumption was initiated by addition of pyruvate/malate, accelerated by addition of adenosine diphosphate (ADP) (state 3 respiration), and decelerated after all ADP was consumed (state 4 respiration). After hypoxic stress, state 3 and state 4 respiration were preserved in xenon-preconditioned mitochondria. Open circle = control; Solid circle = xenon preconditioning.

Figure 3

Effect of xenon preconditioning on isolated mitochondrial bioenergetics. Under baseline conditions, no difference was detected in state 3 and 4 respiration of control and xenon-preconditioned mitochondria. After exposure to hypoxic stress, state 3 respiration of mitochondria from control rats was significantly decreased (Panel A). Respiratory control ratio (RCR) of mitochondria from xenon-preconditioned rats was preserved after hypoxic stress, in contrast to the findings in the absence of xenon (Panel B). CON = control; Xe = xenon preconditioning (n = 7 per group). Values are mean ± sd. *Significantly (P < 0.05) different from baseline.

Figure 4

Effect of xenon preconditioning on isolated mitochondrial permeability transition pore (mPTP) opening. A typical chart recording showing mitochondrial membrane potential at state 2 with increasing calcium concentration ([Ca²⁺]) is illustrated in Panel A. The arrows denote the addition of Ca²⁺ in 5 μM increments. Depolarization was detectable by opening mPTP and releasing Ca²⁺ from mitochondrial through the mPTP with sequential Ca²⁺ loading. Xenon preconditioning delayed depolarization, indicating inhibition of mPTP opening. Open circle = control; Solid circle = xenon preconditioning. Depolarization was not observed in the presence of mPTP inhibitor cyclosporine A. Histograms summarizing the results are depicted in Panel B. CON = control; Xe = xenon preconditioning (n = 6 per group). Values are mean ± sd. *Significantly (P < 0.05) different from CON.

Figure 5

Xenon increased phosphorylation of Akt and glycogen synthase kinase 3 β (GSK-3β) in rat ventricular myocardium. Western blots are shown using antiserum against phosphorylated Akt at Ser 473 (p-Akt) and total Akt (Panel A), and against phosphorylated GSK-3β at Ser 9 (p-GSK-3β) and total GSK-3β (Panel B). Histograms depict the relative density of p-Akt and p-GSK-3β normalized to the total Akt and GSK-3β content, respectively (Panel C). CON = control; Xe = xenon preconditioning (n = 4 per group). *Significantly (P < 0.05) different from CON.