TNFα-mediated subcellular heterogeneity of succinate dehydrogenase activity in human airway smooth muscle cells

Tumor necrosis factor-α (TNFα) is a pro-inflammatory cytokine, which mediates acute inflammatory effects in response to allergens, pollutants, and respiratory infections. Previously, we reported that TNFα increased maximum O₂ consumption rate (OCR) and mitochondrial volume density (MVD) in human airway smooth muscle (hASM) cells. However, TNFα decreased maximum OCR when normalized to mitochondrial volume. In addition, TNFα altered mitochondrial distribution and motility within hASM cells. Although high-resolution respirometry is valuable for assessing mitochondrial function, it overlooks mitochondrial structural and functional heterogeneity within cells. Therefore, a direct measurement of cellular mitochondrial function provides valuable information. Previously, we developed a confocal-based quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase (SDH) reaction (SDH_max) in single cells and observed that cellular SDH_max corresponds with MVD. Therefore, we hypothesized that TNFα decreases SDH_max per mitochondrion in hASM cells. The hASM cells were treated with TNFα (20 ng/mL, 6 h, and 24 h) or untreated (time-matched control). Using three-dimensional (3-D) confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified MVD, mitochondrial complexity index (MCI) and SDH_max relative to the nuclear membrane. Within each shell, SDH_max and MVD peaked in the perinuclear compartments and decreased toward the distal compartments of the cell. When normalized to mitochondrial volume, SDH_max decreased in the perinuclear compartments compared with distal compartments. TNFα caused a significant shift in mitochondrial morphometry and function compared to control. In conclusion, mitochondria within individual cells exhibit distinct morphological and functional heterogeneity, which is disrupted during acute inflammation.<b>NEW & NOTEWORTHY</b> Mitochondria show context-specific heterogeneity in their morphometry. Previously, we reported that acute TNFα exposure increased O₂ consumption rate (OCR) and mitochondrial volume density, but decreased OCR per mitochondrion. TNFα also altered mitochondrial distribution and motility. To assess TNFα-mediated subcellular mitochondrial structural and functional heterogeneity, we used a confocal-based quantitative histochemical technique to determine the maximum velocity of succinate dehydrogenase reaction. Our findings highlight that mitochondria within cells exhibit functional heterogeneity, which is disrupted during inflammation.