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Updated: Mar 15, 2026

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice
Published on: November 22, 2017
Restoring Pharmacologic Preconditioning in the Aging Heart: Role of Mitophagy/Autophagy
1Department of Anesthesiology, School of Medicine, Stony Brook University, New York.
Abstract:
We previously reported that pretreatment with the potent antioxidant TEMPOL improves mitochondrial function and restores preconditioning in the aging heart. Because mitophagy is implicated in cardiac preconditioning and declines with age, this study was designed to investigate how age influences mitophagy in response to preconditioning and whether TEMPOL pretreatment improves it. Old (22-24 months) rats were pretreated with or without 4-week TEMPOL and compared with young (4-6 months) untreated rats. Cardioprotection induced by isoflurane (ISO) in vivo and in isolated cardiomyocytes in vitro was assessed following ischemia/reperfusion and simulated hypoxia/reoxygenation, respectively. Mitophagy was determined by comparing the levels/subcellular locations of key mitophagic markers using Western blotting and immunofluorescence techniques. ISO preconditioned the young but not old heart in vivo and in vitro. Aging impaired ISO-induced mitochondrial accumulation of PINK1 and Parkin, as well as mitochondrial ubiquitination, and baseline and ISO-induced autophagic flux assessed by LC3 puncta, membrane associated LC3-II and p62. Pretreatment with TEMPOL improved these processes and restored ISO preconditioning. Inhibition of autophagy abolished ISO-induced protection in cardiomyocytes from young and TEMPOL pretreated old rats. Thus, antioxidant pretreatment significantly improves mitophagic response to ISO in old myocardium, which may contribute to restoration of cardioprotection in senescent animals.
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