Evaluating mitochondrial autophagy in the mouse heart

Akihiro Shirakabe, Luke Fritzky, Toshiro Saito, Peiyong Zhai, Shigeki Miyamoto, Åsa B. Gustafsson, Richard N. Kitsis, Junichi Sadoshima

Research output: Contribution to journalArticle

7 Citations (Scopus)

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 561 nm 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 561 nm excitation and Lamp1-YFP was significantly greater 48 h 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.

Original languageEnglish (US)
Pages (from-to)134-139
Number of pages6
JournalJournal of Molecular and Cellular Cardiology
Volume92
DOIs
StatePublished - Mar 1 2016

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Autophagy
Fasting
Dependovirus
Mitochondria
Lysosomes
Mitochondrial DNA
Cardiac Myocytes
Intravenous Injections
Quality Control
Electrons
Control Groups
Proteins

Keywords

  • Keima
  • Lamp1
  • Mitophagy

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

Cite this

Shirakabe, A., Fritzky, L., Saito, T., Zhai, P., Miyamoto, S., Gustafsson, Å. B., ... Sadoshima, J. (2016). Evaluating mitochondrial autophagy in the mouse heart. Journal of Molecular and Cellular Cardiology, 92, 134-139. https://doi.org/10.1016/j.yjmcc.2016.02.007

Evaluating mitochondrial autophagy in the mouse heart. / Shirakabe, Akihiro; Fritzky, Luke; Saito, Toshiro; Zhai, Peiyong; Miyamoto, Shigeki; Gustafsson, Åsa B.; Kitsis, Richard N.; Sadoshima, Junichi.

In: Journal of Molecular and Cellular Cardiology, Vol. 92, 01.03.2016, p. 134-139.

Research output: Contribution to journalArticle

Shirakabe, A, Fritzky, L, Saito, T, Zhai, P, Miyamoto, S, Gustafsson, ÅB, Kitsis, RN & Sadoshima, J 2016, 'Evaluating mitochondrial autophagy in the mouse heart', Journal of Molecular and Cellular Cardiology, vol. 92, pp. 134-139. https://doi.org/10.1016/j.yjmcc.2016.02.007
Shirakabe A, Fritzky L, Saito T, Zhai P, Miyamoto S, Gustafsson ÅB et al. Evaluating mitochondrial autophagy in the mouse heart. Journal of Molecular and Cellular Cardiology. 2016 Mar 1;92:134-139. https://doi.org/10.1016/j.yjmcc.2016.02.007
Shirakabe, Akihiro ; Fritzky, Luke ; Saito, Toshiro ; Zhai, Peiyong ; Miyamoto, Shigeki ; Gustafsson, Åsa B. ; Kitsis, Richard N. ; Sadoshima, Junichi. / Evaluating mitochondrial autophagy in the mouse heart. In: Journal of Molecular and Cellular Cardiology. 2016 ; Vol. 92. pp. 134-139.
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