Impaired autophagy: A mechanism of mitochondrial dysfunction in anoxic rat hepatocytes

Jae Sung Kim, Takashi Nitta, Dagmara Mohuczy, Kerri A. O'Malley, Lyle L. Moldawer, William A. Dunn, Kevin E. Behrns

Research output: Contribution to journalArticle

133 Scopus citations

Abstract

Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of autophagy-related protein 7 (Atg7), Beclin-1, and Atg12, autophagy regulatory proteins, was analyzed by western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding green fluorescent protein (control), Atg7, and Beclin-1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in an amino acid-free and serum-free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were coloaded with calcein and tetramethylrhodamine methyl ester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with an adenovirus expressing green fluorescent protein-microtubule-ssociated protein light chain 3 (GFP-LC3) and subjected to A/R. Calpain activity was fluorometrically determined with succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. A/R markedly decreased Atg7 and Beclin-1 concomitantly with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression, and nutrient depletion all substantially suppressed A/R-induced loss of autophagy proteins, prevented onset of the MPT, and decreased cell death after reoxygenation. Confocal imaging of GFP-LC3 confirmed A/R-induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. Conclusion: Calpain 2-mediated degradation of Atg7 and Beclin-1 impairs mitochondrial autophagy, and this subsequendy leads to MPT-dependent hepatocyte death after A/R.

Original languageEnglish
Pages (from-to)1725-1736
Number of pages12
JournalHepatology
Volume47
Issue number5
DOIs
StatePublished - May 1 2008
Externally publishedYes

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    Kim, J. S., Nitta, T., Mohuczy, D., O'Malley, K. A., Moldawer, L. L., Dunn, W. A., & Behrns, K. E. (2008). Impaired autophagy: A mechanism of mitochondrial dysfunction in anoxic rat hepatocytes. Hepatology, 47(5), 1725-1736. https://doi.org/10.1002/hep.22187