LAUSR

Succinate dehydrogenase (SDH) is a mitochondrial enzyme that participates in both the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) as Complex II. In the TCA cycle, SDH catalyzes the oxidation of succinate to fumarate, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. Previously we developed and validated a quantitative histochemical technique to determine the maximum velocity of the SDH (SDHmax) reaction as a direct measurement of mitochondrial respiratory capacity in individual human airway smooth muscle (hASM) cells. The measurement of SDHmax was shown to provide a reliable estimate of the maximum respiratory capacity of an individual cell within a heterogeneous population of cells when compared to respirometry-based OCR measurements. In the present study we further assessed the specificity of this method by performing a targeted knockdown of the SDHB subunit in hASM cells. As a part of the catalytic core of the SDH complex, the SDHB subunit mediates electron transfer to ubiquinone and thereby regulates electron transfer by SDH. Therefore, we hypothesize that Sdhb-knockdown decreases SDHmax in hASM cells. This study was performed in hASM cells dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors with no current history of smoking or respiratory diseases. Targeted Sdhb-knockdown was generated by siRNA mediated knockdown. hASM cells were also treated with malonate (1 mM for 24 h), which is a competitive inhibitor of SDH. To measure SDHmax, the cells were exposed to a solution containing 1-methoxyphenazine methosulphate (mPMS; exogenous electron carrier), azide (cytochrome oxidase inhibitor), 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT; 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. In the quantitative histochemical procedure, the change in optical density (OD), reflecting the progressive accumulation of NBT diformazan (NBTdfz; peak absorbance of 570 nm) in the cell, was measured every 15 s over an 8-min period (a linear reaction). SDHmax was determined from the OD using the Beer-Lambert equation and expressed as mM fumarate/L cell/min. In addition, we used MitoTracker Green (200 nM) to label and image mitochondria in hASM cells to determine mitochondrial volume density and mitochondrial complexity index (MCI). Our results show that SDHmax was markedly decreased in both Sdhb-knockdown and malonate treated hASM cells indicative of loss of function of SDH. Sdhb-knockdown increased mitochondrial volume density and decreased MCI in hASM cells, indicative of increased mitochondrial fragmentation and a role of SDH in the maintenance of mitochondrial morphology. The results confirm that the measurement of SDHmax is specific to SDH. Supported by NIH grant HL157984 (GCS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.