Abstract P133: Role of Akt and p38 in Oxidant Stress Injury and Hypothermic Protection in Murine Cardiomyocytes
Cardiac arrest is an ischemia/reperfusion (I/R) disease characterized by oxidant generation, inflammation, and cell death; and hypothermia (HT) has been shown to improve post-cardiac arrest reperfusion injury. We developed a neonatal mouse cardiomyocyte model of I/R (90 min I + 3 hr R) that demonstrates cell injury associated with increased reactive oxygen species (ROS) generation at reperfusion (as measured by DCFH). Mild HT (32°C) protects mouse cardiomyocytes from I/R injury, and we hypothesize that this protection may be related to the activation of the survival kinase Akt. The Akt inhibitor API-2 (10 ìM) reversed HT protection [32.4 ± 7.1% vs 65.7 ± 6.3% with API-2, p< .01(as measured by PI)] to cell death levels commensurate with normothermic I/R injury (60.7 ± 6.0%). Phospho-Akt (pAkt) levels declined during ischemia, and while both Ser473 and Thr308 were phosphorylated in normothermia and HT within 15 min reperfusion, HT showed an augmented level of pAkt at Thr308. Furthermore, this increase was sustained for the first 30 min of reperfusion. To further study this relationship, murine cardiomyocytes were exposed to exogenous H2O2 to mimic the oxidant stress associated with I/R. Mouse cardiomyocytes demonstrated a dose- and time-dependent activation of Akt to H2O2 that showed maximal activation of both the Ser473 and Thr308 sites within 30 min with 200 ìM H2O2. As in I/R-stimulated cells, the Thr308 site declined to near baseline levels within 1 hr while Ser473 remained elevated. Based on recent findings linking Akt and ROS with p38, we examined the effect of I/R and H2O2 on p38. Mouse cardiomyocytes demonstrated a rapid activation of p-p38 (Thr10/Tyr182) in the context of both stresses. Further, we studied the effect of the Akt inhibitor, API-2, as well as the p38 inhibitor, SB 203580, in H2O2-stimulated cells. As in I/R, API-2 blocked H2O2-induced pAkt, but this inhibitor did not have any effect on p-p38. However, when p38 activation was blocked using SB 203580, pAkt levels decreased by 2 hr. These data suggest that HT is, in part, mediated through Akt and that p38 lies upstream of Akt in the context of oxidant stress. These kinases may act as triggers for the initiation of survival pathways in cardiomyocytes to combat potential damage induced by ROS generation.