Mitophagy is a selective autophagic process that eliminates damaged mitochondria, which is essential for mitochondrial quality control and cellular homeostasis. The most extensively characterized mitophagy pathway involves PTEN-induced kinase 1 (PINK1) and E3 ubiquitin ligase Parkin. Upon mitochondrial depolarization, PINK1 stabilizes on the outer mitochondrial membrane (OMM), where it recruits and phosphorylates Parkin at serine 65 (pParkinS65), activating its E3 ligase activity. Active pParkinS65 initiates the ubiquitination (Ub) of OMM proteins resulting in the engulfment and lysosomal degradation of damaged (depolarized) mitochondria. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, is widely used to experimentally induce mitochondrial depolarization and initiate PINK1-Parkin-dependent mitophagy; however, mitophagic responses to FCCP vary across cell types. In the present study, we hypothesized that, in human airway smooth muscle (hASM) cells, FCCP-induced mitochondrial depolarization activates the PINK1-Parkin-mediated mitophagy pathway, culminating in the clearance of damaged mitochondria. We observed that exposing hASM cells to 1 µM FCCP for 6 h induced mitochondrial depolarization and a decrease in the volume of intact mitochondria. This mitochondrial depolarization triggered the accumulation of PINK1 in the mitochondria, which mediated phosphorylation of pParkinS65 and an increase in pUbS65 proteins. Confocal imaging of labeled mitochondria and lysosomes demonstrated increased colocalization of mitochondria with lysosomes, and mitophagic flux was confirmed using a pH-sensitive mitochondrial reporter mKeima. Collectively, these findings demonstrate that FCCP robustly activates the canonical PINK1-Parkin mitophagy pathway in hASM cells, providing mechanistic insight into mitochondrial quality control, with potential relevance to airway diseases characterized by mitochondrial dysfunction and altered hASM function.
