Abstract 17799: Downregulation of Drp-1 Inhibits Mitophagy and Causes Mitochondrial Dysfunction in Hearts
We have shown previously that tomoxifen-inducible cardiac specific Dynamin-related protein 1 (Drp-1) KO mice exhibit cardiac dysfunction at baseline. Although it is believed that mitochondrial fission mediated by Drp-1 is required for mitochondrial turnover and quality control by mitophagy, this has not yet been demonstrated in cardiomyocytes (CMs) in a convincing manner. Electron microscopy showed mitochondrial mass ratio in Drp-1 KO hearts was significantly greater than in controls (3.57 ± 1.38 vs 1.18 ± 0.31, P<0.05). Mitochondrial ATP content was significantly lower (0.70 ± 0.07 vs 1.03 ± 0.10, P<0.05), while mitochondrial swelling was significantly greater (% decrease in absorbance; 8.01 ± 1.99 vs 2.01 ± 0.58, P<0.05) in Drp-1 KO hearts than in controls. These results suggest that inhibition of fission causes mitochondrial dysfunction. Transduction of CMs with short hairpin RNA for Drp-1 (sh-Drp-1) prevented glucose deprivation (GD)-induced mitochondrial fission and showed low ATP content and increased mitochondrial swelling, indicating that, similar to in vivo experiments, Drp-1 kockdown in CMs prevents mitochondrial fission and causes mitochondrial dysfunction. We also evaluated mitophagy, using mitochondria-targeted Keima fluorescence. Keima has a bimodal excitation spectrum peaking at 440 and 586 nm, corresponding to the neutral and acidic pH states, respectively. Autolysosome maturation can be monitored by measuring transition of Keima. Fluorescent dots with high ratios of 586/440, indicating mitophagy, were significantly increased after GD in sh-scramble transduced myocytes (GD: 28.6±4.2, control 2.3±1.0 dots/cell, p<0.01), but not in sh-Drp-1 transduced CMs (GD: 2.4±1.8, control: 3.2±1.6 dots/cell, not significant), suggesting that GD stimulates mitophagy but that this is inhibited when Drp-1 is downregulated. Electron microscopic analyses also revealed that autophagosomes containing mitochondria were detected 48 hours after starvation in control mice, but not in Drp-1 KO mice (2.6±1.3, 0±0 per field, p<0.05). These results demonstrate that inhibition of mitochondrial fission via downregulation of Drp-1 inhibits mitophagy and effective removal of damaged mitochondria.
- © 2013 by American Heart Association, Inc.