Abstract P284: Inhibition of the Rheb/mTORC1 Pathway During Prolonged Ischemia Is Protective Through Autophagy Activation in Cardiomyocytes

Abstract

The mTOR complex 1 (mTORC1) mediates the cellular response to energy stress. However, the role of mTORC1 signaling in mediating cardiomyocyte (CM) tolerance of nutrient starvation and ischemia remains unclear. mTORC1 activity, as evaluated by the phosphorylation status of p70S6K and 4E-BP1, was decreased in CMs during glucose deprivation (GD) in vitro, and was accompanied by decreases in the activity of Rheb, a positive regulator of mTORC1 (31.7% reduction in Rheb-bound GTP, p<0.05). Exogenous Rheb was sufficient to restore mTORC1 activity, suggesting that Rheb regulates the activity of mTOR during GD. Rheb overexpression reduced CM survival during GD (42.6% reduction at 18 hours, p<0.01), whereas knock-down of Rheb increased CM survival (158.6% increase at 18 hours, p<0.05). Rheb overexpression significantly inhibited autophagy, reduced expression of autophagic genes, including Atg7, reduced the ATP content, and increased ER stress in CMs during GD. Under these conditions, restoration of autophagy through Atg7 overexpression or trehalose inhibited cell death, increased ATP content, and reduced ER stress. The activity of Rheb/mTORC1 is also inhibited during myocardial ischemia in vivo. After 3 hours of ischemia, transgenic mice with cardiac-specific and inducible Rheb overexpression (Tg-Rheb) exhibited a larger myocardial infarction (MI) (59.7% vs. 37.8%, p<0.05) and a significant reduction in autophagy compared to control mice, both of which were reversed by rapamycin, a selective mTORC1 inhibitor. Interestingly, both Rheb (+135% in Rheb-bound GTP, p<0.05) and mTORC1 (+158% in phospho-p70S6K, p<0.05) were activated in the hearts of mice with high fat diet-induced obesity. Prolonged ischemia induced less autophagy and a larger MI (60.6% vs. 37.2%, p<0.05) in the obese mice, effects which were reversed in the presence of rapamycin. However, rapamycin failed to protect the obese mice in the presence of genetic downregulation of Beclin 1 (beclin1+/− mice fed with high fat diet). In summary, inhibition of the Rheb/mTORC1 pathway during energy deprivation is protective through activation of autophagy. Rheb and mTORC1 represent potential therapeutic targets to reduce myocardial damage during prolonged myocardial ischemia in obese patients. .