Therapeutic hypothermia cardioprotection via Akt- and nitric oxide-mediated attenuation of mitochondrial oxidants

Abstract

TL. Therapeutic hypothermia cardioprotection via Akt-and nitric oxide-mediated attenuation of mitochondrial oxidants. Am J Physiol Heart Circ Physiol 298: H2164-H2173, 2010. First published April 9, 2010 doi:10.1152/ajpheart.00994.2009.-Therapeutic hypothermia (TH) is a promising cardioprotective treatment for cardiac arrest and acute myocardial infarction, but its cytoprotective mechanisms remain unknown. In this study, we developed a murine cardiomyocyte model of ischemia-reperfusion injury to better determine the mechanisms of TH cardioprotection. We hypothesized that TH manipulates Akt, a survival kinase that mediates mitochondrial protection by modulating reactive oxygen species (ROS) and nitric oxide (NO) generation. Cardiomyocytes, isolated from 1-to 2-day-old C57BL6/J mice, were exposed to 90 min simulated ischemia and 3 h reperfusion. For TH, cells were cooled to 32°C during the last 20 min of ischemia and the first hour of reperfusion. Cell viability was evaluated by propidium iodide and lactate dehydrogenase release. ROS production was measured by 6-carboxy-2=,7=-dichlorodihydrofluorescein diacetate and mitochondrial membrane potential (⌬⌿m) by 5,5=,6,6=-tetrachloro-1,1=,3,3=-tetraethylbenzimidazoly-carbocyanine iodide (JC-1). Phospho (p)-Akt (Thr308), p-Akt (Ser473), and phosphorylated heat shock protein 27 (p-HSP27) (Ser82) were analyzed by Western blot analysis. TH attenuated reperfusion ROS generation, increased NO, maintained ⌬⌿m, and decreased cell death [19.3 Ϯ 3.3% (n ϭ 11) vs. 44.7 Ϯ 2.7% (n ϭ 10), P Ͻ 0.001]. TH also increased p-Akt during ischemia before reperfusion. TH protection and attenuation of ROS were blocked by the inhibition of Akt and NO synthase but not by a cGMP inhibitor. HSP27, a regulator of Akt, also exhibited increased phosphorylation (Ser82) during ischemia with TH. We conclude that TH cardioprotection is mediated by enhanced Akt/HSP27 phosphorylation and enhanced NO generation, resulting in the attenuation of ROS generation and the maintenance of ⌬⌿m following ischemia-reperfusion.ischemia-reperfusion; reactive oxygen species; cardiomyocyte DESPITE INITIAL resuscitation, many cardiac arrest patients die within hours after the return of spontaneous circulation (ROSC) (12). This ischemia-reperfusion (I/R) injury is likely related to significant increases in tissue oxidant stress seen within minutes of cardiopulmonary resuscitation (CPR) and ROSC (19). Recently, it has been demonstrated that significant levels of oxidant stress in the heart following cardiac arrest originate from the mitochondria within minutes of reperfusion (11). In isolated cardiomyocytes, simulated I/R induces mitochondrial reactive oxygen species (ROS) generation, contractile dysfunction, mitochondrial release of cytochrome c, caspase activation, opening of the mitochondrial permeability transition pore, and ultimately cardiomyocyte death (4, 29, 40).To date, therapeutic hypothermia (TH) is the only treatment currently known to improve survival in the clinical postcardiac arrest setting (27). Such cool...

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