TNFα Reduces the Maximum Respiratory Capacity of Mitochondria in Human Airway Smooth Muscle Cells

Abstract

Previously, we reported that airway inflammation mediated by pro‐inflammatory cytokines such as TNFα induces hypercontractility and increased ATP consumption in human airway smooth muscle (hASM). We also found that TNFα induces an increase in maximum O2 consumption rate as well as mitochondrial biogenesis and an increase in mitochondrial volume density. However, when normalized to mitochondrial volume, maximum O2 consumption rate per mitochondrion is reduced in hASM after TNFα exposure. Unfortunately, standard respirometry (e.g., Seahorse) measures only the averaged maximum O2 consumption rate of a large number of cells. In the present study, we used a quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase reaction (SDHmax) together with mitochondrial volume density in individual hASM cells. SDH is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC), and SDHmax reflects the maximum respiratory capacity of individual mitochondrion. We hypothesized that TNFα decreases SDHmax per mitochondrion in hASM cells. hASM cells were dissociated from bronchial biopsies obtained during thoracic surgeries in patients with no history of asthma or chronic obstructive pulmonary disease. The hASM cells were treated with TNFα (20 ng/ml) or vehicle (DMSO) for 24 h, and SDHmax was measured using a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT) as the reaction indicator. As the SDH reaction proceeded, the hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using an oil‐immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The change in optical density (OD), reflecting the accumulation of NBT diformazan reaction product in the hASM cell, was measured every 15 s over a 10 min period. Based on the Beer‐Lambert equation, the slope of the change in OD was used to determine SDHmax expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the same hASM cells were also loaded with MitoTracker Green (500 nM) and imaged in 3D. We found that mitochondria were more fragmented after TNFα exposure although mitochondrial volume density increased. When SDHmax was normalized to mitochondrial volume, we found that SDHmax per mitochondrion decreased in hASM cells, consistent with the reduction in maximum O2 consumption per mitochondrion that we previously observed. Thus, the increased energetic demand induced by inflammation (TNFα) are met by mitochondrial fragmentation leading to biogenesis and an increase in mitochondrial volume density rather than an increase in mitochondrial respiratory capacity.