Abstract 18406: Ulk1 Induces Mitochondrial Autophagy Through Rab9-dependent Phagophore Formation in Cardiomyocytes (Best of Basic Science Abstract)
Rationale: Mitochondrial quality control mediated by mitochondrial autophagy (MA) is essential for cardiac homeostasis. Although the role of the Parkin-Pink1 pathway in MA is well established, recent reports have uncovered the existence of unconventional autophagy in mediating MA in embryonic reticulocytes and fibroblasts.
Objective: To investigate the molecular mechanism of MA in cardiomyocytes (CMs) during energy stress.
Methods and Results: MA in CMs was evaluated with mitochondria-targeted (Mito)-Keima, which fluoresces with a high 560nm/440nm ratio after lysosomal translocation. In control CMs, high ratio dots represented 1.1±0.3 % of the area in cells at baseline, and glucose deprivation (GD) upregulated this area to 6.7±0.6 % (p<0.01), indicating stimulation of MA. Although knockdown of Ulk1 reduced the area to 1.3±0.1 % (p<0.01) during GD, that of Atg7 did not (6.6±0.8 %), suggesting that MA takes place during GD in Ulk1-dependent but Atg7-independent fashion. Since Ulk1-dependent alternative autophagy utilizes membrane trafficking from the trans-Golgi to form autophagosomes, we examined the effect of Brefeldin A (BFA), an inhibitor of the membrane trafficking, on MA. Treatment with BFA reduced the high-ratio dot area to 0.4±0.1 % (p<0.01) during GD without affecting the protein level of LC3-II, suggesting that MA induced by GD is mainly mediated by alternative autophagy. BFA did not suppress the Parkin-induced area of high-ratio dots, suggesting that GD-induced MA is distinct from Parkin-dependent MA. GFP-Rab9 formed puncta during GD. These puncta were upregulated in the presence of Bafilomycin, whereas they were downregulated by the knockdown of Ulk1 or Beclin1. The puncta merged with the acidic dots of Mito-Keima during GD. Treatment with Bafilomycin, which inhibits the fusion of autophagosomes with lysosomes, increased the puncta merged with neutral Mito-Keima during GD. RFP-Rab9 did not co-localize with GFP-LC3. These results indicate that Rab9 induces formation of autolysosomes or autophagosomes in MA independently of LC3. Knockdown of Rab9 reduced the area of Mito-Keima high-ratio dots to 3.3±0.4% during GD (p<0.01).
Conclusions: Ulk1 regulates Rab9-dependent phagophore formation and induces MA during energy stress in CMs.
Author Disclosures: T. Saito: None. J. Sadoshima: None.
- © 2015 by American Heart Association, Inc.