Abstract 5554: FoxO1 Stimulates Autophagic Flux Through Upregulation of the Small GTP Binding Protein Rab7

Abstract

We have shown previously that FoxO1, a member of the FoxO transcription factor family, is upregulated by nutrient starvation and cardiac unloading. FoxO1 in turn promotes autophagy, thereby mediating survival and regression of hypertrophy in cardiac myocytes. The current investigation addressed the molecular mechanism by which FoxO1 promotes autophagy in cardiac myocytes. In order to evaluate formation of both autophagosomes and autolysosomes, cultured cardiac myocytes were transduced with adenovirus harboring mRFP-GFP-LC3. Adenoviral mediated overexpression of FoxO1 increased accumulation of both GFP-LC3 dots (1.5 fold vs Ad-LacZ) and mRFP-LC3 dots (2 fold vs Ad-LacZ, p<0.01), which was further enhanced in the presence of protease inhibitors(PI) (1.5 fold vs Ad-LacZ+PI, p<0.01). FoxO1 overexpression caused greater increases in mRFP-LC3 dots than GFP-LC3 dots, suggesting that FoxO1 enhances autolysosome formation more strongly than autophagosome formation, thereby stimulating autophagic flux. Rab7 is a small GTP binding protein which plays an important role in mediating maturation of late autophagic vacuoles. mRNA expression of Rab7 is strongly upregulated in cardiac myocytes (4.7 fold, p<0.05) and hearts (2.5 fold) with FoxO1 overexpression. Overexpression of Rab7 was sufficient to stimulate autolysosome formation and autophagic flux in cultured cardiac myocytes, as evidenced by accumulation of LC3-II (2.8 fold, p<0.05) and mRFP-LC3 dots (2.3 fold, p<0.05), and decreased p62 (−40%, p<0.05). Lentivirus mediated knockdown of Rab7 (LV-sh-Rab7) attenuated FoxO1- induced autophagy, as indicated by LC3-II accumulation (Ad-Foxo1WT 1.9, Ad-Foxo1WT + LV-sh-Rab7 0.11 vs Ad-LacZ, p<0.05) and p62 expression levels (Ad-Foxo1WT 0.49, Ad-Foxo1WT + LV-sh-Rab7 0.84 vs Ad-LacZ, p<0.05), suggesting that Rab7 mediates FoxO1-induced autophagy in cardiac myocytes. In summary, FoxO1 stimulates autophagosome/autolysosome formation and autophagic flux in cardiac mycoytes, accompanied by upregulation of Rab7. Rab7 plays an essential role in mediating FoxO1-induced stimulation of autophagy, possibly by stimulating autophagosome -lysosomal fusion in cardiac myocytes.