Abstract 17859: Alternative Autophagy is the Predominant Form of Autophagy During Starvation in Hearts
Besides the conventional form of autophagy (CA), the presence of an alternative form of autophagy (AA) has been reported in embryonic fibroblasts. Lipidation of LC3, a hallmark of CA, is not involved in AA. Instead, autophagosomes are associated with Rab9. Whereas Atg7 plays an essential role in mediating CA, it is not involved in AA, which requires Ulk1 instead. We have shown previously that energy starvation (ES) induces autophagosome formation in an Atg7- and LC3-independent manner in cardiomyocytes (CMs), suggesting that AA exists in CMs. Furthermore, AA mediates survival of CMs during energy deprivation. However, the mechanism mediating AA remains unknown. After ES for 48 hours, the number of Rab9 puncta co-localized with Lamp2 pucta, which indicate autolysosomes, but not Lamp2A punca, which indicate chaperone-mediated autophagy, was significantly increased in wild type and cardiac specific Atg7-KO mice, but not in Ulk1-KO mice. In CMs expressing GFP-Rab9, glucose deprivation (GD) increased GFP-Rab9 puncta in control and Atg7 knock-down (KD) CMs, but not in Ulk1 KD CMs. These results suggest that autophagosomes associated with Rab9 are increased in CMs in response to ES. GD increased phosphorylation of AMPK at Thr172 and of Ulk1 at Ser555 in CMs. Dominant-negative-AMPK suppressed phosphorylation of Ulk1 at Ser555. In contrast, AICAR, an AMPK stimulant, increased phosphorylation of endogenous Ulk1 at Ser555. Co-immunoprecipitation experiments showed that GD increased interaction between Rab9 and Ulk1, but not between Rab9 and an Ulk1 mutant in which Ser555 cannot be phosphorylated. These results suggest that AMPK phosphorylates Ulk1 at Ser555, which induces recruitment of Rab9 to Ulk1 and autophagosome formation. GD significantly increased degradation of long-lived proteins, as determined by ubiquitin accumulation. GD-induced increases in degradation of long-lived proteins were significantly attenuated in the presence of Ulk1 KD, but not Atg7 KD. These results suggest that autophagosomes associate with Rab9, and Ulk1 phosphorylation at Ser555 is required in AA, and that Ulk1-dependent autophagy, namely AA, plays an essential role in mediating GD-induced degradation of long-lived proteins in CMs.
- © 2013 by American Heart Association, Inc.