Abstract 18534: TAT-Protein Blockade of PTEN After Cardiac Ischemia is Highly Protective
Introduction: Cooling protects against cardiac ischemia/reperfusion (I/R) injury by enhancing PI3K/Akt/NOS signaling. PI3K/Akt signaling depends on binding by its regulatory subunit p85 to the catalytic subunit p110 or the inhibitory phosphatase PTEN. We studied novel TAT-p85 fusion proteins that selectively inhibit these competing p85 interactions. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-ΔPTEN p85) would enhance Akt phosphorylation to afford cardioprotection in a heart cell model of I/R. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) would abrogate protection. To further test TAT fusion proteins for the I/R injury of mouse cardiac arrest (CA), we studied whether a TAT-fused PTEN C-terminal 9 amino acid (TAT-PTEN9c) PTEN inhibitor would activate Akt in critical organs and improve survival.
Methods: A TAT-Δp110 p85 plasmid was constructed with nucleotide deletions at amino acids 478-513. The TAT-ΔPTEN p85 plasmid deleted amino acids 1-313. TAT-PTEN9c was synthesized by Anaspec. Cardiomyocytes were isolated from 1-2-day old C57BL6/J mice and exposed to up to 90 min ischemic arrest with 3 h reperfusion (I/R). Cell viability was evaluated by propidium iodide uptake and p-Akt was measured by Western blot. C57BL6 mice were subjected to an established potassium-induced CA protocol. TAT-PTEN9c (7.8 mg/kg) was given IV after return of spontaneous circulation (ROSC) (n=10) and the 2nd dose for another 5 mice was given at 60 min after ROSC.
Results: TAT-Δp110 p85 increased cell death from 44.6 +/- 2.7% to 92.5 +/- 3.4%, while TAT-ΔPTEN p85 decreased cell death caused by I/R from 47.8 +/- 1.4% to 11.9 +/- 5.3%. 30 mice were randomized to either saline or TAT-PTEN9c post-ROSC. TAT fusion proteins could be seen within heart and brain tissues within 2 minutes of IV infusion. The survival rate was significantly increased in TAT-PTEN9c treated mice compared with that of saline treated mice at 2 hrs (14/15, 93% vs. 9/15, 60%, P < 0.05) and at 4 hrs (10/15, 67% vs. 6/15, 40%, P < 0.05) after ROSC.
Conclusions: TAT fusion protein inhibition of PTEN during cardiac I/R and after cardiac arrest appear to be promising strategies for enhancing Akt activation and survival.
- © 2013 by American Heart Association, Inc.