Mahadev Bhat, Sanjana

In human airway smooth muscle cells, TNFα induces reactive oxygen species (ROS) generation and promotes endoplasmic reticulum (ER) stress and mitochondrial fragmentation. Importantly, treatment with the ROS scavenger tempol effectively attenuated TNFα-induced oxidative stress, suppressed ER stress and mitochondrial fragmentation, and improved the intrinsic respiratory capacity and functional efficiency of individual mitochondria. These findings identify oxidative stress as a central mediator linking TNFα signaling to ER stress and mitochondrial fragmentation, highlighting tempol as a potential therapeutic strategy.

Abstract Age‐associated muscle weakness and atrophy of limb muscles, termed sarcopenia, is a major factor in the morbidity of the elderly. It is becoming increasingly recognized that a substantial contribution to the sarcopenic phenotype arises from motor neuron (MN) death in old age and subsequent muscle denervation. In human and rodent models, mitochondrial dysfunction is a leading culprit in both muscle and MN deterioration with age. In other motor pools within the Fischer 344 (F344) aging rat model, we showed that sarcopenia is selective to type IIx/b muscle fibers. We also showed a loss of larger MNs and denervation of the IIx/b fibers, which together comprise more fatigable fast (type FF) motor units. This selective vulnerability of larger MNs and type IIx/b muscle fibers to sarcopenia is further reflected by reductions in oxidative capacity of IIx/b fibers, as assayed by determining the maximum velocity of the succinate dehydrogenase reaction (SDHmax). Here, we hypothesize similar changes will occur in F344 tibialis anterior (TA) muscle from young (6‐months) and old (24‐months) groups. We also predict lumbar MN death in young and old F344 rats. We found selective atrophy of type IIx/b fibers in the TA from old rats. In old age, SDHmax was reduced but only in IIx/b TA fibers. These muscle observations were concomitant with the death of larger lumbar MNs in old age. This study indicates the remarkable selectivity of type FF TA motor units to age‐associated perturbations and sarcopenia.

The study establishes the presence of a robust mitophagic response to acute mitochondrial depolarization in hASM cells mediated through the canonical PINK1-Parkin pathway. By integrating quantitative mitochondrial labeling and biochemical analyses, we demonstrate increased mitochondrial damage and remodeling, accompanied by mitochondrial PINK1 accumulation and Parkin activation, resulting in ubiquitin-dependent signaling and lysosomal engulfment. These findings identify mitophagy as a critical adaptive mechanism for maintaining mitochondrial quality control in airway smooth muscle cells during metabolic stress.

Abstract Rationale Normal lung aging is characterized by progressive structural and functional decline that reduces respiratory efficiency. Aging is associated with excessive reactive oxygen species production and impaired homeostatic pathways that maintain mitochondrial quality control, involving mitophagy (removal of damaged mitochondria) and mitochondrial biogenesis (generation of new mitochondria). In human airway smooth muscle (hASM), reduced mitochondrial quality control can lead to the accumulation of damaged mitochondria, compromising ATP production and decreasing cellular viability. Maintaining healthy mitochondria involves a balance between mitophagy and mitochondrial biogenesis. A key regulator of this balance is PARIS, normally a target of the PINK1/pParkinS65 mitophagy pathway. PARIS transcriptionally inhibits PGC1α and NRF2 expression, key regulators of mitochondrial biogenesis and redox homeostasis. Furthermore, NRF2 can upregulate PINK1 expression, enhancing mitophagy. Therefore, we hypothesize that aging involves unmitigated mitochondrial dysfunction due to accumulation of PARIS and an imbalance between the removal of damaged mitochondria (mitophagy) and mitochondrial biogenesis. Methods hASM cells were dissociated from bronchiolar tissue samples from patients with no history of smoking or respiratory diseases and cultured (≤3 passages). Prior to experimentation, hASM cells from young (≤40 yrs) and old (≥70 yrs) patients (female and male) were serum deprived and phenotyped. In both age groups, targeted siRNA-mediated knockdown of PARIS was also performed. The extent of mitochondrial damage in hASM cells was determined by changes in mitochondrial membrane potential (Ψm) using TMRM (250 nM), and the extent of co-labeling of MitoTracker Red (200 nM) and CellLight GFP. Mitochondrial turnover was quantified by confocal imaging of hASM cells transfected with pMitoTimer. Mitophagy was determined by PINK1 recruitment to mitochondria, pParkinS65 and ubiquitin (pUbS65) phosphorylation. Total PARIS and pParkinS65-ubiquitinated PARIS (by immunoprecipitation) levels were determined. Mitochondrial biogenesis was determined by PGC1α and NRF2 mRNA and protein expression and mitochondrial DNA (mtDNA) copy number. Results In hASM cells from older patients, there was extensive mitochondrial damage, decreased PINK1 recruitment to mitochondria and decreased pParkinS65 and pUbS65 phosphorylation. Total PARIS accumulation increased due to decreased PARIS ubiquitination. Also, PGC1α and NRF2 expression, and mtDNA copy number decreased, likely due to PARIS-mediated inhibition. Knockdown of PARIS increased PGC1α and NRF2 expression, thereby promoting mitochondrial biogenesis and NRF2-mediated antioxidant responses and increasing PINK1 expression, thereby activating mitophagy. Conclusion Aging results in impaired mitochondrial quality control in hASM cells. The PARIS-NRF2 axis presents a promising target for therapeutic intervention to mitigate oxidative damage, maintain mitochondrial homeostasis and improve lung function with aging. This abstract is funded by: HL157984 (GCS) and AG44615 (GCS)

Mitochondria are organelles essential for maintaining energetic support and cellular homeostasis. In phrenic motor neurons (PhMNs), mitochondria support the high level of activity necessary to sustain neuromotor control of the diaphragm muscle during breathing. Previously, we observed that small PhMNs recruited during breathing have higher mitochondrial volume density (MVD) compared to large PhMNs that are recruited infrequently during higher force airway clearance behaviors. In support, we also found that the maximum velocity of the succinate dehydrogenase activity (SDHmax) was higher in small PhMNs compared to large PhMNs. However, total mitochondrial volume was comparable across PhMNs of varying size. This suggests that the total ATP demands of PhMNs do not vary with size. The distribution of mitochondria likely reflects the demands of ATP consumption. In airway smooth muscle cells, mitochondria are not homogeneously distributed, with a higher density in the perinuclear region. In addition, mitochondria appear to be localized in close proximity to the endoplasmic reticulum (ER) most likely to support cytosolic and mitochondrial Ca 2+ regulation. The ER is also clustered in the perinuclear compartment of cells. Perinuclear clustering of mitochondria most likely reflects the higher ATP consumption close to the nucleus. We hypothesized that MVD is higher in the perinuclear region of PhMNs compared to more distal compartments. PhMNs of young (6 month), female and male Fisher 344 rats were retrogradely labeled by intrapleural injection of Alexa 488-conjugated cholera toxin B (CTB). Three days later, mitochondria were labeled by subdural injection of MitoTracker Red CMXRos. Spinal cord tissue was collected following transcranial perfusion with 4% paraformaldehyde, sectioned longitudinally at 70 µm, and mounted onto slides. Mosaics of labeled PhMNs and mitochondria were acquired in 3D (Z optical slices of 0.5 μm) using a 60x Plan Apo oil-immersion objective (NA 1.40). Using a concentric (10 mm) shell method for analysis, MVD was quantified relative to the distance relative to the nuclear membrane of PhMNs. Our results show a disproportionately higher MVD within the perinuclear region compared to more distal regions of PhMNs. This perinuclear distribution of mitochondria was found across PhMNs of varying size. These results suggest that energetic requirements of nucleus and ER are major drivers of mitochondrial biogenesis. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Mitochondrial quality control is maintained by coordinated turnover mechanisms involving mitophagy (removal of damaged mitochondria) and mitochondrial biogenesis (generation of new mitochondria). Disruption of either one or both processes results in the accumulation of damaged mitochondria, oxidative stress and subsequently impaired cellular function. In human airway smooth muscle (hASM) cells, such impairment compromises sustained ATP production required for contractile responses. A key regulator of this balance is PARIS (ZNF746), a transcriptional repressor of PGC1α. Accumulated PARIS binds upstream to the promoter of PGC1α and interferes with the recruitment of transcriptional activators, resulting in decreased PGC1α transcription and reduced mitochondrial biogenesis. In addition, PARIS is implicated in the disruption of cellular antioxidant defense mechanisms due to its direct interaction with NRF2 transcriptional complex. Under basal conditions, PARIS levels are tightly regulated by post-translational modifications such as ubiquitination and phosphorylation. As a target of PINK1/pParkinS65 mediated mitophagy, pParkinS65 binds to PARIS, catalyzing its ubiquitination and proteasomal degradation, thereby alleviating its transcriptional repression of PGC1α and NRF2 and promoting mitochondrial biogenesis. Furthermore, evidence indicates that NRF2 transcriptionally regulates PINK1 expression during oxidative stress and amplifies mitophagy. Therefore, we hypothesize that mitochondrial turnover mechanisms can be regulated by targeting PARIS. In the present study, primary hASM cells were isolated from bronchiolar tissue of 6 patients with no history of chronic pulmonary disease or smoking. The hASM cells were phenotyped and separated into two treatment groups: 1) FCCP (1 µM, 6 h; positive control), a proton ionophore that induces mitochondrial depolarization (damage); 2) untreated control. We observed that FCCP induced extensive mitochondrial membrane depolarization, assessed by changes in mitochondrial membrane potential (Ψm) using TMRM (250 nM). Increased mitochondrial damage was observed with FCCP, measured by the extent of co-labeling of MitoTracker Red FM (200 nM) and CellLight Mitochondria-GFP. Mitochondrial turnover was also quantified by confocal imaging of hASM cells transfected with pMitoTimer, a fluorescent reporter protein in which fluorescence shifts over time from green (Excitation/Emission-488 nm/518 nm) to red (Excitation/Emission-543 nm/572 nm) when oxidized due to mitochondrial stress. Ratiometric analysis of pMitoTimer revealed significant shift from green to red fluorescence with FCCP and accumulation of pure red fluorescent puncta indicative of damaged mitochondria. FCCP increased PINK1 recruitment to mitochondria and increased pParkinS65 and ubiquitin (pUbS65) phosphorylation. Total PARIS accumulation decreased due to increased PARIS ubiquitination, as measured by immunoprecipitation. As a result, PGC1α and downstream NRF2 mRNA and protein expression, as well as mtDNA copy number were increased, indicative of increased mitochondrial biogenesis. Bioinformatics analysis and chromatin immunoprecipitation (ChIP) assay using an NRF2-specific antibody revealed that NRF2 binds to the promoter of PINK1, with FCCP further increasing the binding, thereby increasing PINK1 expression and enhancing mitophagy. Together, these findings identify the PARIS-NRF2 axis as a promising target to mitigate oxidative stress and maintain mitochondrial homeostasis in hASM cells. Supported by NIH grants HL157984 (GCS) and AG44615 (GCS) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

We introduce an imaging-based approach to quantitatively distinguish intact from damaged mitochondria within heterogeneous mitochondrial networks using fluorescent labels that exhibit different sensitivities to mitochondrial membrane potential. By combining membrane potential-independent CellLight GFP label with membrane potential-dependent MitoTracker Red, this method sensitively quantifies basal and stress-induced mitochondrial damage in hASM cells. This assay provides a practical and interpretable metric of mitochondrial integrity that complements existing methods that measure mitochondrial membrane potential and oxidative stress.

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.

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.