Abstract 12698: Role of Rictor in Cardiac Cell Survival
The mammalian target of rapamycin (mTOR), acting downstream phosphatidylinositol-3-phosphate kinase (PI3K) and Akt, binds raptor to form an mTOR complex mTORC1 that activates S6 kinase-1 (S6K1) and protein synthesis. mTOR can also bind rictor to form an mTORC2 phosphorylating Akt on S473, which is additive to the PI3K-dependent activation of Akt by phosphorylation on T308. The function of mTORC2 in the heart is largely unknown. We hypothesized that, upon PI3K activation, mTORC2 promotes cell survival through Akt. Isolated cardiac myocytes were infected with adenoviruses harboring a hairpin sequence for selective RNA silencing (si) of either raptor or rictor, or with a control (ctrl) luciferase si. PI3K was stimulated by 0.1 μM insulin. Phosphorylation of Akt (P-Akt) and S6K1 (P-S6K1) were measured by immunoblotting. Apoptosis was stimulated by 5 μM chelerythrine (chel) and quantified by caspase-3 activity. Protein synthesis was measured by phenylalanine incorporation. A dose-dependent and specific knockdown of up to 90% was obtained for either raptor or rictor. Without insulin, rictor si abolished P-Akt on S473 but did not affect T308, and increased P-S6K1 by 3-fold (P<0.01 vs ctrl). Raptor si did not affect P-Akt on either residue but decreased P-S6K1 by 50% (P<0.01 vs ctrl). Insulin increased P-Akt on S473 by 5-fold and T308 by 10-fold, and P-S6K1 by 3-fold, and doubled the rate of protein synthesis (all, P<0.01 versus no insulin). Rictor si blocked insulin-mediated increase in P-Akt on S473 but also on T308, and did not further increase P-S6K1. Reciprocally, raptor si abolished insulin-mediated increase in P-S6K1 but did not affect P-Akt. Rictor si did not affect protein synthesis, despite increasing P-S6K1. Raptor si decreased protein synthesis by 50% in ctrl (P<0.05), and abolished its increase by insulin. Neither si significantly affected apoptosis in basal conditions. Apoptosis increased by 3-fold in presence of chel (P<0.01 vs no chel), it further increased by 100% with rictor si, and only by 25% upon raptor si (both, P<0.05 vs chel alone). Therefore, mTORC2 positively affects P-Akt on both S473 and T308 upon PI3K stimulation, exerts a negative feed-back on S6K1, and promotes cardiac cell survival, whereas mTORC1 mainly contributes to protein synthesis.
- © 2010 by American Heart Association, Inc.