Fluorescence-based quantification of mitochondrial damage in human airway smooth muscle cells

Mitochondrial quality control is essential for maintaining cellular homeostasis by balancing the removal of damaged mitochondria (mitophagy) with the generation of new mitochondria (mitochondrial biogenesis). A key feature of mitochondrial damage is loss of mitochondrial membrane potential (ΔΨ_m), which initiates mitophagy, enabling effective mitochondrial clearance. Although an array of tools exists to assess mitochondrial damage (depolarization), many rely on acute, nonphysiological depolarization or provide semiquantitative measures of mitochondrial damage, limiting their ability to resolve intact versus damaged mitochondria within heterogeneous mitochondrial networks. Therefore, in the present study, we developed and validated an imaging-based assay to quantify intact mitochondria in human airway smooth muscle (hASM) cells using dual-fluorescence labeling. This approach combines a ΔΨ_m-dependent (MitoTracker Red FM) dye with a ΔΨ_m-independent label [CellLight mitochondria-green fluorescent protein (GFP)]. Dual-labeled mitochondria in untreated hASM cells exhibited ∼10% nonoverlap between the two fluorescence signals, indicating the presence of damaged (depolarized) mitochondria in homeostatic conditions. Dose- and time-dependent treatment with the mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) induced loss of ΔΨ_m, confirmed by tetramethylrhodamine methyl ester (TMRM), and resulted in a marked reduction in fluorescence overlap, volume of intact mitochondria, and increased mitochondrial fragmentation. Complementary analysis using the redox-sensitive reporter pMitoTimer was performed, where a shift in fluorescence signal from green to red is indicative of increased mitochondrial oxidative stress and rate of mitochondrial turnover. Together, these findings validate the dual-labeling strategy as a quantitative method to distinguish intact from damaged mitochondria in situ and as a useful tool for studying mitochondrial quality control, potentially translatable to various cell and disease models.NEW & NOTEWORTHY We introduce an imaging-based approach to quantitatively distinguish intact from damaged mitochondria within heterogeneous mitochondrial networks using fluorescent labels that exhibit different sensitivities to mitochondrial membrane potential. By combining membrane potential-independent CellLight GFP label with membrane potential-dependent MitoTracker Red, this method sensitively quantifies basal and stress-induced mitochondrial damage in hASM cells. This assay provides a practical and interpretable metric of mitochondrial integrity that complements existing methods that measure mitochondrial membrane potential and oxidative stress.