Abstract 19326: Cardiac-specific Deletion of Ulk1, but not Atg7, Impairs Mitophagy in the Heart During Ischemia

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The removal of damaged mitochondria by mitophagy is essential for maintaining the quality of mitochondria in the heart during stress, such as ischemia. Activation of mitophagy, its underlying mechanism, and its functional significance have not been clearly demonstrated in the heart during ischemia. We generated cardiac-specific transgenic mice expressing Mito-Keima, a pH-sensitive indicator (Tg-Mito-Keima), where acidic dots of Mito-Keima were induced in the heart in response to TAT-Beclin 1 (20 mg/kg), a peptide known to induce autophagy. In order to investigate the roles of Atg7 and Ulk1, key regulators of autophagy, in mediating mitophagy, we crossed wild-type (WT), cardiac-specific atg7 knockout (atg7cKO), or cardiac-specific ulk1 knockout, (ulk1cKO) mice with Tg-Mito-Keima. Then, Tg-Mito-Keima, Tg-Mito-Keima-atg7cKO, and Tg-Mito-Keima-ulk1cKO mice were subjected to 48 hours of starvation (STV) or ischemia. We quantified the dots with a high 560nm/440nm ratio, which indicate lysosomal translocation of Mito-Keima, namely mitophagy. In the heart sections from Tg-Mito-Keima mice, high-ratio dots represented 0.8±0.3 % of the area at baseline, and STV upregulated this area to 2.2±0.1 % (p<0.05), indicating the stimulation of mitophagy. Although knockout of ulk1 (Tg-Mito-Keima-ulk1cKO) significantly reduced the area to 0.9±0.2 % (p<0.05) during STV, that of atg7 (Tg-Mito-Keima atg7cKO) did not (2.0±0.2 %), suggesting that mitophagy takes place during STV in an Ulk1-dependent but Atg7-independent fashion. Mitophagy was transiently activated during ischemia, peaking at 30 minutes after coronary artery ligation in Tg-Mito-Keima (3.0±0.2 %, p<0.05) and Tg-Mito-Keima-atg7cKO (2.7±0.2 %, p<0.05) mice. However, ischemia-induced mitophagy was significantly blunted in Tg-Mito-Keima-ulk1cKO (1.6±0.1 %). ulk1cKO mice displayed significantly greater infarction (34.9±4.3 %, p<0.05) than WT mice (19.7±2.8 %) during 2 hours of ischemia, but atg7cKO mice did not, suggesting that Ulk1 protects the heart against ischemia by regulating mitophagy. These results suggest that Ulk1 rather than Atg7 is essential for the induction of mitophagy during energy stress in the heart and that Ulk1 protects the heart by promoting mitophagy.

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