Bhat, Sanjana Mahadev

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.

Proinflammatory cytokines such as TNFα mediate airway inflammation. Previously, we showed that TNFα increases mitochondrial biogenesis in human ASM (hASM) cells, which is associated with increased PGC1α expression. We hypothesized that TNFα induces CREB and ATF1 phosphorylation (pCREBS133 and pATF1S63), which transcriptionally co-activate PGC1α expression. Primary hASM cells were dissociated from bronchiolar tissue obtained from patients undergoing lung resection, cultured (one–three passages), and then differentiated by serum deprivation (48 h). hASM cells from the same patient were divided into two groups: TNFα (20 ng/mL) treated for 6 h and untreated controls. Mitochondria were labeled using MitoTracker green and imaged using 3D confocal microscopy to determine mitochondrial volume density. Mitochondrial biogenesis was assessed based on relative mitochondrial DNA (mtDNA) copy number determined by quantitative real-time PCR (qPCR). Gene and/or protein expression of pCREBS133, pATF1S63, PCG1α, and downstream signaling molecules (NRFs, TFAM) that regulate transcription and replication of the mitochondrial genome, were determined by qPCR and/or Western blot. TNFα increased mitochondrial volume density and mitochondrial biogenesis in hASM cells, which was associated with an increase in pCREBS133, pATF1S63 and PCG1α expression, with downstream transcriptional activation of NRF1, NRF2, and TFAM. We conclude that TNFα increases mitochondrial volume density in hASM cells via a pCREBS133/pATF1S63/PCG1α-mediated pathway.

Cellular mitochondrial function can be assessed using high resolution respirometry that measures O 2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O 2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDH max ) in isolated human airway smooth muscle (hASM) cells using 1‐methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDH max , the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil‐immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDH max was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP‐mediated disruption of the mitochondrial proton gradient does not affect SDH max , while SDH max is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDH max was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDH max can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.

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 O 2 consumption rate as well as mitochondrial biogenesis and an increase in mitochondrial volume density. However, when normalized to mitochondrial volume, maximum O 2 consumption rate per mitochondrion is reduced in hASM after TNFα exposure. Unfortunately, standard respirometry (e.g., Seahorse) measures only the averaged maximum O 2 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 (SDH max ) 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 SDH max reflects the maximum respiratory capacity of individual mitochondrion. We hypothesized that TNFα decreases SDH max 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 SDH max 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 SDH max 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 SDH max was normalized to mitochondrial volume, we found that SDH max per mitochondrion decreased in hASM cells, consistent with the reduction in maximum O 2 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.

Tumor necrosis factor α (TNFα) contributes to the pathophysiology of several inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and COVID‐19. Previously, we showed that acute (24‐h) exposure to TNFα promotes mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with an increased level of cytosolic GTPase dynamin‐related protein 1 (DRP1), a major protein involved in mitochondrial fragmentation. Phosphorylation of DRP1 at Ser616 (S616) promotes its translocation and binding to the outer mitochondrial membrane and induces mitochondrial fragmentation; however, phosphorylation of DRP1 at the Ser637 (S637) residue reverses the total process and diminishes fragmentation. The balance between S616 and S637 phosphorylation of DRP1 regulates mitochondrial dynamics. In hASM cells, exposure to TNFα triggers protein unfolding and selectively activates the endoplasmic reticulum (ER) stress pathway involving the phosphorylation of inositol‐requiring enzyme 1α (pIRE1α) and subsequent splicing of X‐box binding protein 1 (XBP1s) that acts as a potent transcriptional activator for several downstream genes. We hypothesized that TNFα mediated activation of the pIRE1α/ XBP1 ER stress pathway transcriptionally activates genes that code for proteins involved in DRP1 phosphorylation at S616 leading to mitochondrial fragmentation. hASM cells were dissociated from bronchial biopsies from patients without a history of respiratory diseases and exposed to TNFα (20 ng/ ml for 6 h). Cells were also treated with Mdivi1 (50 μM for 6 h), a small‐molecule inhibitor of mitochondrial fragmentation that targets the GTPase activity of DRP1. Bioinformatic analysis revealed the presence of putative binding sites for XBP1 on the promoter region of genes that translate into kinases (cyclin dependent kinases CDK1 and CDK5, cyclin B1), which are reported to phosphorylate DRP1 at S616. Western blot and/or immunocytochemistry were employed to quantify the expression and phosphorylation status of IRE1α, DRP1, XBP1 splicing, CDKs and cyclin B1. Mitochondrial morphology was assessed by 3‐D confocal microscopy using MitoTracker green to label mitochondria. Chromatin immunoprecipitation (ChIP) and quantitative real time PCR was done to validate XBP1 target genes. Spliced XBP1 transcriptionally activates expression of CDK1, CDK5 and cyclin B1 and subsequent phosphorylation at S616 of DRP1 without significant alteration in S637 phosphorylation. As a result, TNFα induces an increase in S616/S637 phosphorylation ratio that promotes the translocation of DRP1 from cytosol to mitochondria and mediates mitochondrial fragmentation. Inhibition of DRP1 GTPase activity by Mdivi1 ameliorates DRP1 phosphorylation at S616 and significantly reduces mitochondrial fragmentation. These results reveal the mechanisms that underlie TNFα induced ER stress and downstream mitochondrial fragmentation.