The results of this investigation demonstrate for the first time that brief, repetitive exposure to helium, neon, or argon protects myocardium against irreversible ischemia injury. Helium and neon have been shown to produce convulsions, but do not cause anesthesia in rats during extreme hyperbaric conditions (84.6 ± 22.2 atm and 91.3 ± 7.0 atm, respectively) (8). In contrast to helium and neon, argon caused an anesthetic effect at 27.0 ± 2.6 atm, but the concentration of argon (70%) used in the current investigation is equivalent to a minimum alveolar concentration value of approximately 0.026 (8). Thus, the current data indicate that helium, neon, and argon produce cardioprotection independent of an anesthetic effect. The magnitude of cardioprotection produced by the nonanesthetic noble gases was similar to that previously reported for 1.0 minimum alveolar anesthetic concentration isoflurane in pre- and postconditioning experiments in an identical rabbit model of prolonged coronary artery occlusion and reperfusion (12,26,27). Decreases in infarct size caused by three 5-min cycles of 70% helium, neon, and argon were also modestly less than those observed with ischemic preconditioning resulting from three 5-min cycles of ischemia and reperfusion, although dose-response relationships to helium, neon, and argon were not performed. These data with nonanesthetic noble gases are consistent with previous findings demonstrating that volatile anesthetic-induced preconditioning does not provide a similar degree of cardioprotection when compared with ischemic preconditioning (6).
Pretreatment with wortmannin, PD 098059, and rapamycin abolished helium-induced reductions in myocardial necrosis, implicating roles for PI3K, Erk1/2, and p70s6K, respectively, in cardioprotection by the gas. These results with nonanesthetic noble gases support previous findings demonstrating the cardioprotective effects of xenon (7). Pretreatment with 70% xenon before ischemia reduced infarct size in rats (from 51% to 28% of the left ventricular area at risk) by activating the ε isoform of protein kinase C and its downstream targets p38 MAPK (2), MAPK-activated protein kinase-2 (5), and heat shock protein 27 (5). More recently, roles for mitochondrial adenosine triphosphate-regulated potassium (KATP) channels, phosphotidylinositol-dependent kinase-1 (a protein that is immediately upstream to PI3K in the signaling pathway), and Erk1/2 were also implicated in xenon preconditioning (3,30), suggesting that prosurvival signaling may play an important role in xenon-induced cardioprotection. The current results confirm and extend these findings by demonstrating that helium-induced reductions in infarct size are also mediated by several of these prosurvival signaling kinases. Xenon reduced infarct size when the gas was administered solely during early reperfusion in rabbits, suggesting that the anesthetic gas was capable of producing postconditioning as well (1). Whether other nonanesthetic gases are capable of mimicking this xenon-induced postconditioning phenomenon is unknown. However, based on the results implicating RISK cascade proteins in preconditioning by helium, such a contention appears highly plausible, and is being investigated by our laboratory.
The current results also demonstrate for the first time that preservation of myocardial integrity by helium is abolished by atractyloside, a selective opener of mPTP, indicating that helium-induced cardioprotection is mediated by inhibition of mPTP opening in vivo. An important interaction between mPTP and mitochondrial KATP channels through adenine nucleotide translocase (31,32) has been suggested in pharmacologic preconditioning [e.g., diazoxide (15), desflurane (25)] and isoflurane-induced postconditioning (26). The mitochondrial KATP channel-dependence of xenon preconditioning was recently reported (3), and the reductions in myocardial necrosis produced by brief, intermittent administration of helium that occur via inhibition of mPTP opening in the current investigation are consistent with these previous findings, based on the suspected interaction between the mitochondrial KATP channel and mPTP. The mechanisms by which helium preconditioning inhibits mPTP opening during early reperfusion remain to be fully elucidated. However, PI3K and Erk1/2 signaling pathways activated by helium regulate mitochondrial permeability transition by inhibiting caspase formation and glycogen synthase kinase-3β activity, favorably affecting pro- versus antiapoptotic protein balance, and producing nitric oxide through activation of endothelial nitric oxide synthase within the cardiac myocyte (10,23,33).
The precise mechanisms by which chemically inert noble gases activate endogenous signal transduction pathways to exert myocardial protection are unknown. It has been proposed that intraatomic dipole formation within the outer electron shells (4d105s25p6) of the relatively large xenon atom may account for its interaction with biologically active molecules (34). Such a hypothesis appears to be highly unlikely when helium is considered, because this atom contains only two electrons that are tightly bound within a stable 1s2 orbital configuration (35). It is equally unclear how noble gases are capable of selectively activating some signaling kinases while leaving others unaffected (30). Nevertheless, the current and previous data with helium and xenon (7), respectively, provide compelling evidence that brief, intermittent administration of noble gases are capable of exerting important beneficial effects against ischemia-reperfusion injury by activating several signaling kinases known to mediate other forms of pharmacologic and ischemic pre- and postconditioning (10).
The current results must be interpreted within the constraints of several potential limitations. Three cycles of brief administration of 70% helium-, neon-, or argon-30% O2 interspersed with 70% nitrogen–30% oxygen before prolonged coronary artery occlusion and reperfusion were used as preconditioning stimuli in the current investigation. Dose- or time-response relationships to noble gases were not performed, nor were studies conducted to determine whether more prolonged time periods between noble gas discontinuation and the onset of ischemia also provide cardioprotection (i.e., duration of “memory” period). These investigations are currently being conducted in our laboratory. A 30-min coronary artery occlusion was used so as to produce myocardial infarction. Whether brief exposure to noble gases also produces cardioprotection after more prolonged periods of coronary artery occlusion is unknown. Activation of PI3K, Erk1/2, and p70s6K produced by helium mediated reductions in myocardial necrosis in the current investigation. These signaling pathways and their proposed putative mPTP end-effector have also been strongly implicated in decreases in apoptotic cell death during reperfusion. We did not examine the role of apoptosis in helium-induced cardioprotection, and further investigation will be required to ascertain whether reductions in apoptosis also mediate salvage of myocardium by noble gases.
Wortmannin, PD 098059, and rapamycin have been shown to be selective inhibitors of PI3K, Erk1/2, and p70s6K, respectively, at the doses used in the current investigation. Nevertheless, dose-response relationships to these selective inhibitors were not performed, and the possibility that these drugs may have inhibited other protein kinases involved in myocardial protection cannot be completely excluded from the analysis. The results also require qualification because the actions of helium with or without atractyloside pretreatment on mPTP channel activity in isolated mitochondrial were not examined. Nevertheless, the current pharmacologic data strongly suggest an important role for mPTP inhibition in helium-induced preconditioning. Myocardial infarct size is determined primarily by the size of the left ventricular area at risk and the extent of coronary collateral perfusion. The ratio of the area at risk to left ventricular mass was similar among groups, and rabbits have minimal coronary collateral blood flow (36). Thus, it is unlikely that differences in collateral perfusion among groups are responsible for the observed results, but coronary collateral blood flow was not specifically quantified. Helium, neon, and argon did not cause systemic hemodynamic effects nor did administration of helium affect heart rate, mean arterial blood pressure, or rate-pressure product in wortmannin-, PD 098059-, or rapamycin-pretreated rabbits. Thus, the beneficial actions of the nonanesthetic noble gases occurred independent of hemodynamic effects. Nevertheless, the results should be qualified because coronary venous oxygen tension was not measured, nor was myocardial oxygen consumption calculated. The results also require qualification because we did not specifically examine the biochemical actions of helium on PI3K-Akt, Erk1/2, and p70s6K phosphorylation, nor did we measure the activity of these kinases in rabbit myocardium before or after ischemia and reperfusion. Western blotting was also not performed to confirm the selective blockade of PI3K-Akt, Erk1/2, and p70s6K using wortmannin, PD 098059, and rapamycin, respectively, and this also represents a limitation. Finally, the current results implicating a role for PI3K, Erk1/2, p70s6K, and mPTP in helium-induced cardioprotection were obtained in barbiturate-anesthetized rabbits. Whether similar results occur in other animal species or humans is unknown.
In summary, the current results demonstrate that brief, intermittent administration of helium, neon, or argon before prolonged coronary artery occlusion and reperfusion protects myocardium against infarction in barbiturate-anesthetized, acutely instrumented rabbits. The findings indicate that selective inhibition of PI3K, Erk1/2, and p70s6K blocks cardioprotection by helium. The results further demonstrate that mPTP opening abolishes helium-induced preconditioning against infarction in vivo. The ability to briefly administer a noble gas without anesthetic properties as a therapeutic agent before a predictable episode of ischemia (e.g., percutaneous coronary intervention, cardiopulmonary bypass) may have important clinical ramifications, but further research will be required to test this intriguing hypothesis.
The authors thank David A. Schwabe, BSEE (Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin), for technical assistance.
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