Recent work shows that cooling protection after mouse cardiac arrest and cardiomyocyte ischemia is mediated by Akt activation. The PI3K p85 subunit can either augment or inhibit Akt activation depending on its binding to p110 or PTEN respectively. To further clarify the role of PI3K p85 in cardioprotection, we studied novel TAT-p85 fusion proteins that selectively inhibit PI3K p85 binding. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-ΔPTEN p85) would enhance Akt phosphorylation to afford cardioprotection. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) would decrease Akt phosphorylation and abrogate cardioprotection. Microscopy and Western blot analysis demonstrated that TAT fusion protein was transduced into cardiomyocytes within 5 min and remained more than 2 h. Inhibition of PI3K/Akt by TAT-Δp110 p85 significantly increased cell death from 44.6±2.7% to 92.5±3.4% after simulated ischemia and reperfusion. By contrast, PTEN inhibition using TAT-ΔPTEN p85 decreased cell death to 11.9±5.3%, a similar level of cardioprotection seen with past cooling studies. Additional studies with the small molecule PTEN inhibitor VO-OHpic confirmed that PTEN inhibition was highly protective against cell death induced by ischemia and reperfusion. We conclude that blockade of p85-PTEN interaction and PTEN inhibition may be promising strategies for rescuing the heart from ischemia and reperfusion injury.