Abstract 10123: Modulation of Ischemia/Reperfusion Cardioprotection by TAT-p85 Fusion Proteins
Introduction: Recent work shows that mouse cardiac arrest and heart cell ischemia cause tissue PI3K-Akt deactivation, while mild cooling reactivates this survival kinase pathway with cardioprotection that is reversed by genetic deletion of Akt. To enhance PI3K-Akt reactivation after cardiopulmonary resuscitation (CPR), we developed novel TAT-p85 fusion proteins designed to enter tissues rapidly during CPR and activate Akt (p-Akt). We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-ΔPTEN p85) would enchance p-Akt cardioprotection. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) would decrease p-Akt and abrogate protection.
Methods: A TAT-Δp110 p85 plasmid was constructed by deletion of nucleotides encoding amino acids 478-513. The TAT-ΔPTEN p85 plasmid deleted amino acids 1-313. A TAT-GFP plasmid produced fluorescence-tagged TAT proteins for measuring tissue penetration kinetics. TAT proteins were expressed in E. Coli and purified. Cardiomyocytes were isolated from 1-2-day old C57BL6/J mice and exposed to 90 min ischemia and 3 h reperfusion (I/R). Cell viability was evaluated by propidium iodide uptake and p-Akt was measured by Western blot. Intravenous and intraperitoneal administration of TAT-GFP proteins in C57BL6/J mice was used to measure the timing of tissue protein delivery.
Results: TAT protein transduction in heart and brain was diffuse and occurred within 5 min after intravenous delivery. In cardiomyocytes, maximal transduction occurred by 30 min using a 300 nM dose, and lasted more than 2 h. TAT-Δp110 p85 blocked p-Akt at both S473 and T308 sites in a dose-dependent fashion, increasing cell death after I/R. By contrast, p-Akt was increased by pretreatment with TAT-ΔPTEN p85 and by the PTEN chemical inhibitor VO-OH. These PTEN inhibition strategies were highly cardioprotective, resulting in significant reductions in I/R heart cell death from 45.3 ± 5.2% to 19.3 ± 2.6% (p<0.01).
Conclusions: We conclude that p85 mutants fused with a TAT protein transduction domain can enter multiple critical tissues within minutes of administration. TAT-ΔPTEN p85 with resulting PTEN inhibition may be a promising strategy to mimic and enhance protective cooling by more rapidly reactivating tissue Akt after CPR.
- © 2011 by American Heart Association, Inc.