Abstract 2: Therapeutic Hypothermia Cardioprotection During Cardiac Arrest Inhibits mTOR Kinase Signaling
Introduction: Therapeutic hypothermia (TH) is highly cardioprotective against ischemia/reperfusion (I/R) injury in our murine cardiomyocyte and mouse models of cardiac arrest, and requires Akt activation. The mechanism whereby cooling increases Akt activation is unclear. The mammalian target of rapamycin (mTOR) is an important sensor of physiological stress that may act acutely as a feedback pathway to inhibit Akt signaling. We hypothesized that TH attenuates over-activation of mTOR during I/R, and that TH cardioprotection would be mimicked by mTOR inhibition using rapamycin. We also tested whether TH would increase activation of AMP activated protein kinase (AMPK), a related stress sensor and endogenous inhibitor of mTOR.
Methods: Cardiomyocytes isolated from 1–2-day old C57BL6/J mice, were exposed to simulated ischemia (90 min) followed by reperfusion (3 h). For TH, cells were cooled from 37C to 32C during ischemia and the first hour of reperfusion. Samples were also obtained from C57BL/6 mice that underwent an 8-min cardiac arrest. After 6 min, the mice were randomized to normothermia (NT, 37C) or TH (30C) extended during CPR and for 1 h after resuscitation. Protein lysates were collected at serial timepoints for Western blot analyses.
Results: Cell death during ischemia was minimal, but accelerated during reperfusion (4% vs 39%, p<.05 n=5). TH attenuated this cell death (15% vs. 39%, p<.05 ,n= 7) and attenuated reperfusion-induced mTOR activation seen within 30 min as measured by differential phosphorylation of the mTOR targets 70S6K and eEF2. Compared to controls, TH-treated cardiomyocytes and hearts demonstrated increased p-AMPK after resuscitation, consistent with mTOR inhibition. The mTOR inhibitor rapamycin (100nM) given during I/R similarly affected 70S6K and eEF2 and significantly reduced cell death (28% vs. 41%, n=5, p<.05).
Conclusions: TH enhanced survival in both our isolated cardiomyocyte and cardiac arrest models and was associated with altered signaling events suggesting mTOR inhibition. TH cardioprotection may have direct effects on mTOR and/or increase the activity of the endogenous mTOR inhibitor AMPK. Pharamacologic strategies that mimic these TH affects could be useful to improve survival after cardiac arrest.
- © 2010 by American Heart Association, Inc.