Abstract 3427: Foxo1-Induced Stimulation of Autophagy Contributes to Cardiac Atrophy
The Forkhead box-O (Foxo) family transcription factors mediate atrophy in both skeletal and cardiac muscles. Autophagy is a bulk degradation process, in which cytosolic proteins are sequestered by double membrane vesicles termed autophagosomes and degraded through lysosomes. Expression of Foxo1 is upregulated when the heart undergoes atrophy, such as during cardiac unloading. We hypothesized that Foxo1 increases autophagic flux, which in turn plays an essential role in mediating atrophy of cardiac myocytes. Adenovirus harboring Foxo1 (Ad-Foxo1) was transduced into neonatal rat ventricular myocytes (NRVMs). Foxo1 significantly reduced the cell size at baseline (−54%, p<0.01) and in response to phenylephrine (−61%, p<0.01) in NRVMs, consistent with the notion that Foxo1 stimulates atrophy in cardiac myocytes. Ad-Foxo1 increased LC3-II/LC3-I (3.3 fold vs Ad-LacZ, p<0.01) as well as GFP-LC3 dots (the number of dots/cell, 2.5 fold vs Ad-LacZ, p<0.05), established indicators of autophagy. Ad-Foxo1 significantly reduced protein expression of p62/SQSTM1 (−45% vs Ad-LacZ, p<0.05), a protein degraded through autophagy. Ad-Foxo1 also increased the autophagic flux, as determined by LC3-II/LC3-I (LacZ 0.69, Foxo1 6.92) and accumulation of p62/SQSTM1 (LacZ 0.57, Foxo1 1.18) in the presence of 50μM leupeptin and 500μM PMSF, lysosomal protease inhibitors. Both 3-methyladenine (3MA) and adenovirus harboring shRNA Beclin1 reduced both Foxo1-induced increases in LC3-II formation (LacZ 1.0, Foxo1 3.6, Foxo1+3MA 1.7, Foxo1+shBeclin1 1.3) and Foxo1-induced decreases in p62/SQSTM1, confirming that 3MA and shBeclin1 attenuate Foxo1-induced autophagy. Both 3MA and shRNA Beclin1 attenuated Foxo1 induced reduction in NRVM cell size (LacZ 100%; Foxo1 66%; Foxo1+3MA 81%, p<0.05; Foxo1+shBeclin1 89%, p<0.05) and the relative protein content (Foxo1+3MA vs Foxo1 p<0.05; Foxo1+shBeclin1 vs Foxo1 p<0.01), suggesting that that Foxo1-induced reduction in cell size is in part mediated by autophagy. In conclusion, these results suggest that Foxo1 stimulates autophagy in cardiac myocytes and that stimulation of the autophagy-lysosomal pathway plays an important role in mediating Foxo1-induced cardiac atrophy.