Abstract 7: Therapeutic Hypothermia Attenuates Mitochondrial Reactive Oxygen Species (ROS) Generation by Enhanced Akt Signaling during Murine Cardiomyocyte Ischemia/Reperfusion (I/R)
Introduction: Therapeutic hypothermia (TH) is a highly promising cardioprotective treatment for cardiac arrest and acute myocardial infarction, but its cytoprotectve mechanisms of action remain unknown. Prior work in our chick cardiomyocyte ischemia/reperfusion (I/R) model suggests that mitochondrial reactive oxygen species (ROS) contribute to I/R injury, and TH attenuates this injury by enhanced cardioprotective nitric oxide signaling. Using a murine cardiomyocyte I/R model, we hypothesized that TH is affected by Akt, a survival kinase that regulates both nitric oxide signaling and mitochondrial protection.
Methods: Cardiomyocytes, isolated from 1–2-day old C57Bl6/J mice, were exposed to 90 min simulated ischemia and 3 h reperfusion. For TH, cells were cooled to 32oC within 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′-dichloro-dihydrofluorescein diacetate and mitochondrial membrane potential by JC-1. Akt and HSP27 were analyzed by Western Blot.
Results: TH significantly attenuated ROS generation within the first hour of reperfusion (6.8±0.6 to 2.4±0.5 a.u, n=5, p<0.01), an effect similarly achieved with the mitochondrial complex III inhibitor stigmatellin (50 nM, n=4, p<0.05). TH attenuation of ROS was associated with increased cell survival [19.3±3.3% (n=11) vs 44.7±2.7% (n=10) p<0.001], and improved mitochondrial membrane potential recovery. These effects were blocked by the Akt inhibitor API-2 and the nitric oxide synthase (NOS) inhibitor L-NAME. Compared to normothermic end-ischemia, TH induced increases in pAkt Thr308 and pAkt Ser473 within 30 min reperfusion. pHSP27 Ser82, a target associated with Akt activation and mitochondrial protection against cytochrome c release, was also significantly increased by TH reperfusion compared to end-ischemia. Both Akt and HSP27 phosphorylation were blocked by API-2.
Conclusion: Akt signaling is necessary for TH cardioprotection in a murine cardiomyocyte I/R model. TH may attenuate cardiomyocyte mitochondrial ROS and injury by Akt-related targets that include NOS and HSP27.