Abstract P135: Hypothermia Regulation of Akt and p53 Results in Increased Survival in a Cardiomyocyte Model of Ischemia/Reperfusion and Cardiac Arrest
While therapeutic hypothermia appears to be highly protective against ischemia/reperfusion (I/R) injury associated with cardiac arrest, its mechanism is not entirely known. We have previously shown in a cellular model of cardiac arrest, that I/R injury results in apoptotic cell death and that hypothermia attenuates caspase-mediated apoptosis and cell death. In this study, we hypothesized that hypothermia protection involves modulation of two critical regulators of apoptosis, Akt and p53. Using our established model of in vitro I/R injury (Ischemia 70 min/Reperfusion 3 hours) in chick cardiomyocytes, western blot analysis demonstrated that phospho(Ser473)Akt (pAkt-Ser473) and phospho(Thr308)Akt (pAkt-Thr308) decreased from baseline levels to near undetectable levels during ischemia and 15 minutes into reperfusion. In contrast, total p53 levels went from low baseline levels to elevated levels as early as 15 minutes into reperfusion. Hypothermia (25°C) during the last 10 minutes of ischemia and into the first hour of reperfusion decreased cell death >50%, restored contractile activity, significantly increased pAkt-Ser473 and pAkt-Thr308, and reduced total p53 levels. Hypothermia mediated cardioprotection following I/R was reversed with the Akt inhibitor API-2 (10ìM). Akt can bind to and phosphorylate mdm2, activating its inhibitory actions on p53 function; blockade of mdm2 function can precipitate pro-apoptotic functions of p53. Therefore, we utilized the mdm2 inhibitor, nutlin-3, to examine the potential involvement of this pathway in I/R-induced cardiomyocyte apoptosis and hypothermic protection. We found that like Akt inhibition, nutlin-3 (1ìM) abrogated the protective effects of hypothermia and increased I/R-mediated cell death. These findings suggest that hypothermia induced cardioprotection following cardiac arrest and I/R is mediated by changes in the Akt/p53 balance, subsequently altering apoptosis and cell death. More importantly, these findings demonstrate that hypothermia activates specific cell signaling cascades that may be amenable to pharmacological intervention.