Abstract 20531: Rheb Negatively Regulates Cardiomyocyte Survival during Nutrient Starvation and Prolonged Ischemia through mTORC1 Activation and Inhibition of Autophagy
Ras homology enriched in brain (Rheb), a small GTP-binding protein, activates the mTOR complex 1 (mTORC1), thereby regulating cell growth and survival. However, the role of Rheb in mediating survival during energy stress and ischemia remains unclear in the heart. mTORC1 activity, as evaluated by phosphorylation of p70S6 kinase (S6K), was reduced in neonatal cardiomyocytes (CMs) during glucose deprivation (GD), whereas Rheb overexpression significantly restored it, suggesting that Rheb might critically regulate mTORC1 during GD. Rheb overexpression reduced cell survival (GD 18 h: Lacz 39.9±8.4% vs. Rheb 22.7±7.4%, p<0.01, n=3) and increased apoptosis (GD 10 h: 211±64% vs. Lacz, p<0.05, n=5) during GD, whereas sh-RNA-mediated knock-down of Rheb increased CM survival (GD 18 h: sh-scramble 48.1±7.9% vs. sh-Rheb 60.0±9.4%, p<0.01, n=3). Rheb overexpression significantly inhibited, whereas downregulation of Rheb significantly increased, autophagy both at baseline and during GD, as assessed by LC3II/I ratio, p62 protein accumulation and GFP-LC3 puncta. These results suggest that Rheb negatively regulates CM survival during GD, possibly through inhibition of autophagy. Indeed, Rheb overexpression significantly decreased ATP content during GD (GD 18 h: Lacz 57.9% vs. Rheb 31.5% vs. controls, p<0.05), whereas downregulation of Rheb significantly increased it (GD 18 h: sh-scramble 43.9% vs. sh-Rheb 62.9%, p<0.05). During GD, Rheb overexpression increased the unfolded protein response, including upregulation of C/EBP homologous protein (CHOP) and caspase-12 fragments. After prolonged myocardial ischemia (PMI) for 3 hours, cardiac-specific Rheb transgenic mice (Tg-Rheb) (n=5) exhibited a larger infarct size than non-transgenic mice (n=7) (60.4±19.5% vs. 41.1±9.4%, p<0.05) and a significant increase in apoptosis in the ischemic area (4.6±1.2% vs. 2.3±0.8%, p<0.05). Tg-Rheb exhibited mTORC1 activation and suppression of autophagy at baseline and after PMI. Tg-Rheb also presented a significant reduction in the ATP content during PMI (51.2±12.4% vs. 71.5±10.3% with respect to baseline, p<0.05). In conclusion, Rheb negatively regulates CM survival during nutrient starvation and PMI through mTORC1 activation and inhibition of autophagy.
- © 2010 by American Heart Association, Inc.