title: Evaluating mitochondrial autophagy in the mouse heart.
abstract: Mitochondrial autophagy plays an important role in mediating mitochondrial quality control. Evaluating the extent of mitochondrial autophagy is challenging in the adult heart in vivo. Keima is a fluorescent protein that emits different colored signals at acidic and neutral pHs. Keima targeted to mitochondria (Mito-Keima) is useful in evaluating the extent of mitochondrial autophagy in cardiomyocytes in vitro. In order to evaluate the level of mitochondrial autophagy in the heart in vivo, we generated adeno-associated virus (AAV) serotype 9 harboring either Mito-Keima or Lamp1-YFP. AAV9-Mito-Keima and AAV9-Lamp1-YFP were administered intravenously and mice were subjected to either forty-eight hours of fasting or normal chow. Thin slices of the heart prepared within cold PBS were subjected to confocal microscopic analyses. The acidic dots Mito-Keima elicited by 561nm excitation were co-localized with Lamp1-YFP dots (Pearson's correlation, 0.760, p<0.001), confirming that the acidic dots of Mito-Keima were localized in lysosomes. The area co-occupied by Mito-Keima puncta with 561nm excitation and Lamp1-YFP was significantly greater 48h after fasting. Electron microscopic analyses indicated that autophagosomes containing only mitochondria were observed in the heart after fasting. The mitochondrial DNA content and the level of COX1/GAPDH, indicators of mitochondrial mass, were significantly smaller in the fasting group than in the control group, consistent with the notion that lysosomal degradation of mitochondria is stimulated after fasting. In summary, the level of mitochondrial autophagy in the adult heart can be evaluated with intravenous injection of AAV-Mito-Keima and AAV-Lamp1-YFP and confocal microscopic analyses.
authors: Department of Cell Biology and Molecular Medicine, Rutgers-New Jersey Medical School, Newark, NJ, USA.
authors: Akihiro
authors: A
authors: Shirakabe
authors: Core Imaging Facility, Rutgers-New Jersey Medical School, Newark, NJ, USA.
authors: Luke
authors: L
authors: Fritzky
authors: Department of Cell Biology and Molecular Medicine, Rutgers-New Jersey Medical School, Newark, NJ, USA.
authors: Toshiro
authors: T
authors: Saito
authors: Department of Cell Biology and Molecular Medicine, Rutgers-New Jersey Medical School, Newark, NJ, USA.
authors: Peiyong
authors: P
authors: Zhai
authors: Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
authors: Shigeki
authors: S
authors: Miyamoto
authors: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA.
authors: Åsa B
authors: ÅB
authors: Gustafsson
authors: Departments of Medicine and Cell Biology, Wilf Family Cardiovascular Research Institute, Albert Einstein College of Medicine, Bronx, NY, USA.
authors: Richard N
authors: RN
authors: Kitsis
authors: Department of Cell Biology and Molecular Medicine, Rutgers-New Jersey Medical School, Newark, NJ, USA. Electronic address: sadoshju@njms.rutgers.edu.
authors: Junichi
authors: J
authors: Sadoshima
mesh_terms: D000818:Animals; D001343:Autophagy; D004272:DNA, Mitochondrial / Q000378:metabolism* / Q000648:ultrastructure; D000229:Dependovirus / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D052118:Lysosomal-Associated Membrane Protein 1 / Q000235:genetics; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D004272:DNA, Mitochondrial; D052118:Lysosomal-Associated Membrane Protein 1
keywords: Keima; Lamp1; Mitophagy