Bhat, Sanjana Mahadev

Abstract RATIONALE: Mitochondria show cell and context-specific heterogeneity in their morphometry, determined by regulators of mitochondrial dynamics. Previously, we reported that TNFα increased maximum O2 consumption rate (OCR) and mitochondrial volume density (MVD) in human airway smooth muscle (hASM) cells. However, when normalized to mitochondrial volume, maximum OCR per mitochondrion was reduced following TNFα exposure. TNFα exposure also altered mitochondrial distribution and motility within hASM cells relative to the nuclear membrane. Although high-resolution respirometry is a valuable tool for assessing mitochondrial function, it does not account for the presence of mitochondrial structural and functional heterogeneity within cells. Therefore, a direct measurement of cellular mitochondrial function provides valuable novel information and physiological insight. Previously, we developed a confocal-based quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase (SDH) reaction (SDHmax) in single cells and observed that cellular SDHmax corresponds with MVD. Therefore, we hypothesize that SDH activity varies relative to the intracellular mitochondrial distribution within hASM cells. METHODS: Cells were dissociated from bronchiolar tissue samples collected from female and male patients with no history of smoking or respiratory disease. Isolated hASM cells were serum-deprived for 48 h and split into two treatment groups: 1) TNFα-treated (20 ng/mL, 6 h and 24 h) and 2) time-matched untreated control. Following treatments, CellLight GFP-labeled mitochondria were imaged in 3D (Z optical slice of 0.5 μm) on a Nikon Eclipse A1 laser scanning confocal system (oil-immersion ×60/1.4 NA objective). Using a concentric shell method for analysis, MVD, mitochondrial complexity index (MCI) and SDHmax were quantified relative to the nuclear membrane. SDHmax was measured using a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitroblue tetrazolium (reaction indicator). As the reaction proceeded, the change in optical density (OD), was measured every 15 s over a 10-min period. Using the Beer-Lambert equation, the change in OD was used to determine SDHmax expressed as mmol fumarate/L cell/min. RESULTS: Within each shell, SDHmax and MVD peaked in the perinuclear region and decreased toward the distal compartments of the cell in all treatment groups. When normalized to mitochondrial volume, SDHmax decreased in the perinuclear region compared to the distal compartments of the cell. Compared to control, TNFα exposure caused a significant shift in mitochondrial morphometry and function relative to the nuclear membrane. CONCLUSIONS: In summary, our data indicate that mitochondria within individual cells are morphologically heterogeneous with distinct functional properties, which is disrupted in disease.

Mitochondria show context-specific heterogeneity in their morphometry. Previously, we reported that acute TNFα exposure increased O 2 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.

Abstract Background Agricultural contaminants, including inhaled organic dust (OD) and gases, are known to cause inflammation in the lungs and the brain. We investigated the role of the potassium channel Kv1.3 in organic dust (OD)-induced neuroinflammation models. Kv1.3 channels play a multifaceted role in microglial immune modulation, cancer, neurodegenerative diseases, and constitute a potential therapeutic target. Methods We used in vivo (C57BL/6 mice), in vitro (microglial cell line, primary microglia), and ex vivo (brain slice culture) models of OD-induced neuroinflammation. A sterile OD extract (ODE) was prepared, and mice were exposed to either normal saline or ODE intra-nasally for 5 weeks (5 days/week) to simulate an occupational exposure scenario. Primary microglia were isolated from neonatal mice for total RNA sequencing (RNA-seq). The ODE-induced expression of Kv1.3 was quantified using in vitro and ex vivo models with and without PAP-1 treatments. Exposure-induced changes in cytokines and reactive species markers were measured. Using western blot, we quantified phosphorylated p38 MAPK14 (p-p38 MAPK) and NOX2. We measured the currents through Kv1.3 channels using a microglial patch-clamp assay. Results Exposure to ODE increased the expression of Kv1.3 and p-p38 MAPK in mouse microglia without affecting the Kv1.3 currents at the cell surface. Exposure increased the levels of inflammatory cytokines and NOX2. Kv1.3 inhibition with PAP-1 decreased inflammatory markers (TNF-α and IL-6), levels of Kv1.3, p-p38 MAPK, NOX2, and nitrites. Conclusion Our study revealed that pharmacological inhibition of Kv1.3 potassium channels reduces ODE-induced neuroinflammation by decreasing inflammatory and oxidative stress markers.

AbstractSuccinate dehydrogenase (SDH) is a key mitochondrial enzyme involved in the tricarboxylic acid cycle, where it facilitates the oxidation of succinate to fumarate, and is coupled to the reduction of ubiquinone in the electron transport chain as Complex II. Previously, we developed a confocal‐based quantitative histochemical technique to determine the maximum velocity of the SDH reaction (SDHmax) in single cells and observed that SDHmax corresponds with mitochondrial volume density. In addition, mitochondrial volume and motility varied within different compartments of human airway smooth muscle (hASM) cells. Therefore, we hypothesize that the SDH activity varies relative to the intracellular mitochondrial volume within hASM cells. Using 3D confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified mitochondrial volume density, mitochondrial complexity index, and SDHmax relative to the distance from the nuclear membrane. The mitochondria within individual hASM cells were more filamentous in the immediate perinuclear region and were more fragmented in the distal parts of the cell. Within each shell, SDHmax also corresponded to mitochondrial volume density, where both peaked in the perinuclear region and decreased in more distal parts of the cell. Additionally, when normalized to mitochondrial volume, SDHmax was lower in the perinuclear region when compared to the distal parts of the cell. In summary, our results demonstrate that SDHmax measures differences in SDH activity within different cellular compartments. Importantly, our data indicate that mitochondria within individual cells are morphologically heterogeneous, and their distribution varies substantially within different cellular compartments, with distinct functional properties.

Succinate dehydrogenase (SDH) is a mitochondrial enzyme that serves a dual role in mitochondrial respiration: catalyzing the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle and transferring electrons from succinate to ubiquinone as complex II in the electron transport chain (ETC). SDH is composed of four subunits (SDHA-SDHD) and the loss of SDHB subunit, specifically, has been shown to increase oxidative stress. As a part of the catalytic core of the SDH complex, the SDHB subunit mediates electron transfer to ubiquinone. In human airway smooth muscle (hASM) cells with siRNA-mediated knockdown of SDHB, the maximum velocity of the SDH reaction (SDHmax) is decreased. Moreover, SDHB-knockdown leads to an increase in mitochondrial volume density, indicating a role of SDH in mitochondrial remodeling. However, the impact of SDHB-knockdown on mitochondrial dynamics has not been explored. Therefore, we hypothesize that SDHB-knockdown promotes mitochondrial fragmentation as part of mitochondrial remodeling in hASM cells. To investigate this hypothesis, bronchiolar tissue samples were collected in female and male patients undergoing lung surgery with no current history of smoking or respiratory diseases, the smooth muscle layer was dissected and hASM cells were dissociated. The targeted knockdown of SDHB was achieved by siRNA-mediated knockdown. Mitochondria in hASM cells were labeled with MitoTracker Green (200 nM) and imaged in 3D with a 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. Optical slices were deconvolved to enhance contrast and the MitoTracker labeling was thresholded to create binary images, which were then reconstructed in 3D. From these images, the mitochondrial complexity index (MCI) was calculated to quantify the extent of fragmentation. To identify the signaling pathway involved in mitochondrial fragmentation, the expression and phosphorylation of dynamin-related protein 1 (DRP1) was quantified by Western blot. Mitochondrial fragmentation is associated with increased phosphorylation of DRP1 at Ser616 (pDRP1s616). Furthermore, mitophagy was assessed by determining the mitochondrial expression of PINK1 and the phosphorylation of Parkin at Ser65 (pParkinS65). Mitochondrial biogenesis was also evaluated through the expression of PGC1α, NRF1/2 and TFAM. Our results show that SDHB-knockdown increased mitochondrial fragmentation (reduced MCI) in hASM cells, and increased pDRP1s616 phosphorylation and its translocation to mitochondria. SDHB-knockdown also increased mitochondrial expression of PINK1 and pParkins65 phosphorylation indicating activation of mitophagy. We also confirmed that SDHB-knockdown increased expression of PGC1α, NRF1/2 and TFAM consistent with mitochondrial biogenesis. These findings unveil the mechanisms underlying SDHB-knockdown-mediated mitochondrial remodeling. Supported by NIH grant R01-HL157984 (GCS). This is the full abstract presented at the American Physiology Summit 2024 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.

Airway inflammation is increasing worldwide. Proinflammatory cytokines mediate an adaptive mechanism to overcome inflammation-induced cellular stress. Previously, we reported that TNFα mediates hASM cellular responses, leading to increased force and ATP consumption associated with increased O2 consumption, and oxidative stress. This study indicates that TNFα induces ER stress, which induces mitochondrial fragmentation via pIRE1αS724/XBP1s mediated CDK1/5 upregulation and pDRP1S616 phosphorylation. Mitochondrial fragmentation may promote hASM mitochondrial biogenesis to maintain healthy mitochondrial pool.

Cellular mitochondrial function can be assessed using high-resolution respirometry that measures the O2 consumption rate (OCR) across a number of cells. However, a direct measurement of cellular mitochondrial function provides valuable 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, which participates in both the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) as Complex II. In this study, we determine the maximum velocity of the SDH reaction (SDHmax) in individual human airway smooth muscle (hASM) cells. To measure SDHmax, hASM cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT, reaction indicator). As the reaction proceeded, the change in optical density (OD) due to the reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) was measured using a confocal microscope with the pathlength for light absorbance tightly controlled. SDHmax was determined during the linear period of the SDH reaction and expressed as mmol fumarate/liter of cell/min. We determine that this technique is rigorous and reproducible, and reliable for the measurement of mitochondrial function in individual cells.

Succinate dehydrogenase (SDH) is a key enzyme in the tricarboxylic acid (TCA) cycle, as well as Complex II of the electron transport chain (ETC). In the SDH reaction, succinate is oxidized to fumarate in the TCA cycle, producing FADH2 contributing to the proton gradient that drives the reduction of ubiquinone to ubiquinol in the ETC. Thus, measurement of the maximum velocity of the SDH reaction (SDHmax) provides insight into both TCA cycle and ETC contributions to the maximum respiratory capacity of mitochondria. The goal of the present study was to use a quantitative histochemical technique, which we previously developed in skeletal muscle fibers, to quantify the SDHmax in permeabilized (45 μg/mL saponin) cardiomyocytes isolated from male Sprague-Dawley rats. In the SDH reaction, the oxidation of succinate (80 mM succinate; determined to be the maximum substrate concentration) to fumarate is coupled to the reduction of nitroblue tetrazolium (NBT; 1.5 mM) to its diformazan (NBTdfz) using 1-methoxyphenazine methosulphate (mPMS; 1 mM) as an exogenous electron carrier and azide (0.75 mM) to inhibit Complex IV. The progressive accumulation of NBTdfz as the reaction proceeds is measured by repeated densitometric (at 570 nm, the peak absorbance wavelength for NBTdfz) measurements. The optical density (OD) measurements are converted to mM fumarate using the Beer-Lambert-Bouger equation based on pathlength for light absorbance and the molar extinction coefficient for NBTdfz. Importantly, the pathlength for light absorbance must be controlled. In cross-sections of skeletal muscle fibers, pathlength was controlled by cryosectioning at 6 μm. In the present study, pathlength was controlled by optical sectioning using a confocal microscope. The isolated cardiomyocytes were imaged in 3D using a Nikon Eclipse A1 laser scanning confocal system with a ×40x/1.3 NA oil-immersion lens with an optical slice set at 0.5 μm. Changes in OD within a series of 10 optical slices were measured every 15 s over a 5 min period and expressed as mM fumarate/min using the Beer-Lambert-Bouger equation. We found that the SDH reaction was linear across 5 min, and that the change in OD was consistent throughout all 10 optical slices within a cardiomyocyte, suggesting that NBTdfz accumulates in the cytoplasm. Our results confirm that this assay of SDHmax is reproducible in cardiomyocytes and offers a single cell alternative to high-resolution respirometry. The ability to measure SDHmax in single cells is in contrast to respirometry, which only measures the average O2 consumption of aggregates of cells (isolated or in tissue). Thus, a direct measurement of SDHmax can provide valuable physiological insight into variation in mitochondrial function across a population of cardiomyocytes, e.g., as might occur with ischemic injury. Supported by the Mayo Clinic 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.

Airway inflammation is the central driver behind the pathogenesis of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and the very recent pandemic outbreak of COVID-19. Previous studies reported that proinflammatory cytokine TNFa plays a significant role in airway inflammation by inducing hyper-reactivity of airway smooth muscle (ASM) and airway remodeling. TNFa also increases ASM force generation and ATP hydrolysis that requires energy. The excess energy demand is met by an increase in the level of PGC1a protein, mitochondrial biogenesis and subsequently increased mitochondrial volume density. Based on these previous findings, we hypothesized that TNFa promotes CREB phosphorylation at a specific serine 133 residue (pCREBS133) thereby transcriptionally activating PGC1a expression leading to mitochondrial biogenesis and increased mitochondrial volume density. In the study, human ASM cells were dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors (34 to 71 years of age) with no history of respiratory diseases or current smoking. The dissociated ASM cells were serum-deprived for 48 h before experimentation. The ASM cells from each donor were separated into two groups: 1) time-matched untreated controls, and 2) TNFa treated (20 ng/ml for 6 h). Expression of CREB, pCREBS133, PCG1a, VDAC (an indicator of mitochondrial volume), and downstream signaling molecules (NRFs, TFAM), which regulate transcription and replication of the mitochondrial genome, were determined by quantitative real-time PCR (qPCR) and/or Western blot analysis. The binding of pCREBS133 to the PGC1a promoter was confirmed by bioinformatics analysis and chromatin immunoprecipitation (ChIP) assay. Mitochondria within ASM cells were labeled using MitoTracker green and imaged by 3D confocal microscopy for the assessment of mitochondrial volume density. Mitochondrial biogenesis was quantified by mitochondrial DNA (mtDNA) copy number relative to nuclear DNA using qPCR. Results were analyzed by one-way analysis of variance (ANOVA). Human ASM cells treated with TNFa showed increased pCREBS133 phosphorylation without any significant change in total CREB. The ChIP assay using a CREB-specific antibody revealed that pCREBS133 binds to the promoter of PGC1a and TNFa treatment further increased the binding affinity. Consistent with these findings, TNFa treatment increased PCG1a expression in ASM cells, and subsequently activated its downstream signaling pathway revealed by increased expression of its target genes, NRF1, NRF2 and TFAM. TNFa-induced activation of the PCG1a signaling pathway in ASM cells resulted in increased mitochondrial biogenesis as indicated by increased mitochondrial DNA copy number and increased mitochondrial volume density. Supported by NIH grant HL157984 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.

Mitophagy is a regulated multistep process of selective removal of dysfunctional/damaged mitochondria by autophagy and is critical for the regulation of mitochondrial quality control in many physiological processes. The best characterized pathway of mitophagy involves PTEN-induced kinase 1 (PINK1) and E3-ubiquitin ligase Parkin. Upon mitochondrial depolarization, PINK1 stabilizes on the outer mitochondrial membrane (OMM) and recruits Parkin. After recruitment, PINK1 phosphorylates Parkin at Ser65, thus activating Parkin which promotes the ubiquitination of OMM proteins resulting in the subsequent lysosomal degradation of the mitochondria. Previously, we reported that tumor necrosis factor α (TNFα), a proinflammatory cytokine produced during inflammatory airway diseases, increases force generation and ATP hydrolysis, and results in increased mitochondrial fragmentation in human airway smooth muscle (hASM) cells. We also showed that TNFα induces PGC1α signaling, thus promoting mitochondrial biogenesis and increased mitochondrial volume density. In the present study, we hypothesize that TNFα induces mitophagy to promote selective clearance of dysfunctional mitochondria, thus maintaining a healthy mitochondrial pool to meet the increased energy demand in hASM cells. Experiments were 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. The hASM cells were serum deprived for 48 h and the phenotype was confirmed prior to experimentation. hASM cells were separated into three 6-h treatment groups: 1) TNFα (20 ng/mL), 2) FCCP (5 μM; positive control), and 3) untreated controls. The mitochondria and lysosomes in hASM cells were labelled with MitoTracker green (200 nM) and LysoTracker red (100 nM), respectively and 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 3D images were used to determine the coefficient for colocalization of mitochondria with lysosomes, lysosomal volume density, mitochondrial volume density, and mitochondrial complexity index (MCI). Protein expression and phosphorylation status of PINK1, Parkin, phospho-Parkin, VDAC (voltage-dependent anion channel), and LAMP1 (Lysosomal-associated membrane protein 1) were quantified by western blot. We observed that TNFα and FCCP treatment increased lysosomal volume density and mitochondrial colocalization with lysosomes in hASM cells, indicating the formation of mitophagosomes, characteristic of mitophagy. Consistent with previous findings, TNFα treatment increased mitochondrial volume density and decreased MCI, indicative of increased mitochondrial fragmentation. In addition, TNFα treatment increased the expression and mitochondrial translocation of PINK1 and Parkin. The results suggest that TNFα activates PINK1/Parkin-mediated mitophagy in hASM cells. Supported by NIH grants HL157984 (GCS) and HL105355 (OARR) 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.