Abstract 2277: Therapeutic Hypothermia Protects Against Reperfusion Injury via Mitochondrial Oxidant Signaling and Preconditioning Signaling Pathways
Background: The protective mechanism of hypothermia remains largely unknown. As reactive oxygen species (ROS) are implicated in the signaling of preconditioning protection, we investigated whether such ROS may help trigger hypothermia protection.
Methods: Using a chick cardiomyocyte model of ischemia/reperfusion (I/R), we studied oxidant generation during I/R, with and without a protective intra-ischemic cooling protocol (i.e. IC, ischemia extended for 10 min to allow cooling to 25oC before reperfusion, with 1h reperfusion at 25oC and then rewarming to 37oC). Cell viability was assessed by propidium iodide, and intracellular oxidants monitored using dihydroethidium (DHE, sensitive to superoxide, O2−), 2′,7′-dichlorofluorescin diacetate (DCFH, sensitive to H2O2/hydroxyl radical), and 4,5-diaminofluorescin diacetate (DAF, sensitive to nitric oxide, NO).
Results: The ROS profile at reperfusion was altered by hypothermia in a pattern similar to that seen in our prior preconditioning studies. Superoxide generation was increased by IC and associated with protection (cell death 14.3 ± 0.7% vs. 49.7 ± 3.4%, P<0.001) and contractile recovery. Exogenous superoxide dismutase (PEG-SOD 200 U/ml) given during IC abrogated both the O2− increase and cardioprotection. DCFH oxidation was also augmented in IC, and could be attenuated by the mitochondrial inhibitor stigmatellin (1μM) and the mitochondrial anion channel inhibitor 4,4′-diisothiocyano-2,2′-stilbene (DIDS, 200 μM). The IC related ROS increase was associated with later increases in NO generation after 15 min of reperfusion compared to I/R, an effect abrogated by the NO synthase inhibitor L-NAME (200 μM). L-NAME or PKCϵ inhibiting peptide (5 μM) attenuated the NO increase and reversed hypothermia protection. Finally, exogenous H2O2 (100 μM) given during the first 10 min of normothermic reperfusion also increased NO generation and conferred protection.
Conclusions: Hypothermia is a post-conditioning strategy that protects against I/R injury via oxidant signaling pathways similar to those of preconditioning. Such signaling is mediated via mitochondrial ROS generation and release of superoxide through anion channels, activation of PKC and NO synthase, and NO-mediated protection.