Abstract 17292: Ulk1 Plays a Crucial Role in Mitochondrial Autophagy in Cardiomyocytes

Abstract

Mitochondrial autophagy (MA) plays an important role in mediating mitochondrial (Mt) quality control. The molecular mechanism by which damaged mitochondria are removed during energy stress in cardiomyocytes (CMs) remains poorly understood. We investigated the roles of Atg7 and Ulk1, key regulators of autophagy, in mediating MA. Although cell viability, evaluated with CellTiter-Blue assays, was significantly lower (p<0.05) in Ad-shRNA-Atg7 (sh-Atg7)-transduced CMs than in Ad-shRNA-Ulk1 (sh-Ulk1)- or Ad-shRNA-Control (sh-Cont)-transduced CMs at baseline, those after glucose deprivation (GD) were significantly greater (p<0.05) in sh-Ulk1-transduced CMs than in sh-Atg7- or sh-Cont-transduced CMs. Although LC3-II and reduction of p62, markers of conventional autophagy, were indistinguishable in sh-Ulk1 and sh-Cont-transduced CMs, they were significantly suppressed in sh-Atg7-transduced CMs in comparison. GD significantly decreased (p<0.05) the amount of mtDNA and citrate synthase activity, markers of Mt mass, in sh-Cont- or sh-Atg7-transduced CMs, but not in sh-Ulk1-transduced CMs. More CMs with depolarized mitochondria were observed in sh-Ulk1-transduced CMs than in sh-Cont- or sh-Atg7-transduced CMs after GD. Thus, Ulk1 plays a more important role in mediating CM survival and Mt clearance in response to GD than Atg7. Clearance of MA through lysosomes was evaluated with mitochondria-targeted (Mito)-Keima, with a dual excitation spectrum peaking at 440 and 560 nm, corresponding to neutral and acidic pH, respectively. Knockdown of Drp1 or Beclin1 abolished the fluorescent dots with a high 560nm/440nm ratio, which represent lysosomal translocation of Mito-Keima, suggesting that the high-ratio dots represent MA. In control CMs, high-ratio dots represented 3.6±0.5 % of the area after GD, indicating stimulation of MA. Although knockdown of Ulk1 significantly reduced the area to 0.7±0.4 % (p<0.01, vs sh-Cont) after GD, that of Atg7 or p62 did not (4.9±1.3 % and 4.0±1.0 %). These results suggest that although Atg7 plays an important role in mediating autophagy at baseline, MA may take place even in the absence of Atg7 during energy stress. Furthermore, Ulk1 plays a major role in MA and CM survival in response to energy stress.