Sieck, Gary C.

Airway inflammation occurs as a defense mechanism in response to pathogens, allergens, toxins, or pollutants. The physiological responses of airway inflammation are mediated by proinflammatory cytokines such as tumor necrosis factor alpha (TNFα), that causes airway remodeling, characterized by airway smooth muscle (ASM) hyperreactivity and proliferation. The molecular mechanism underlying TNFα-induced ASM cell proliferation is not fully understood. Previous studies from our lab demonstrated that TNFα exposure induces accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggering an ER stress response that involves autophosphorylation of inositol requiring enzyme 1α at Serine 724 (pIRE1α S724 ). pIRE1α S724 promotes alternative splicing of X-box binding protein 1 (XBP1s) mRNA. XBP1s acts as a ubiquitous transcription factor in ASM cells, targets a number of genes associated with different cellular responses. In the present study, we hypothesized that TNFα promotes XBP1s mediated transcriptional activation of the Cyclin B1, a protein essential for the control of the cell cycle, thus promoting ASM cell proliferation. Human ASM (hASM) cells were dissociated from bronchiolar tissue samples collected during lung surgery from female and male patients (49 to 61 years of age) with no history of chronic respiratory diseases or current smoking. Isolated hASM cells from the same patient were serum-deprived for 48 h and separated into two groups: 1) untreated controls, and 2) treated with TNFα (20 ng/ ml) for 6 h. To confirm the effect of TNFα on hASM cell proliferation, loss of function experiments were employed by: 1) transfecting hASM cells with a non-spliceable XBP1 mutant, and 2) treating the cells with 4μ8C, a pharmacological inhibitor for IRE1α mediated XBP1 splicing. hASM cell proliferation was measured using CyQuant cell proliferation assay. The binding site sequence of XBP1s to the Cyclin B1 promoter was identified by bioinformatic analysis and confirmed by chromatin immunoprecipitation (ChIP) assay. mRNA and protein expressions of Cyclin B1 were determined by qRT-PCR and Western blot respectively. Nuclear localization of Cyclin B1 was measured by nuclear fractionation, followed by western blot of nuclear and cytosolic fraction. Results were analyzed by paired t-tests. TNFα treatment for 6 h induced pIRE1α S724 phosphorylation and XBP1s splicing. Bioinformatic analysis revealed the presence of an XBP1s binding site on the Cyclin B1 promoter. The ChIP assay confirmed that XBP1s binds to the promoter region of the Cyclin B1 genes, and the binding affinity was increased in TNFα-treated hASM cells. Consistent with these results, we found that TNFα treatment increased both mRNA and protein expressions of Cyclin B1 in hASM cells. Nuclear localization of Cyclin B1 increased significantly in TNFα treated cells consistent with an increase in hASM cell proliferation. Inhibition of XBP1 splicing reduced the expression of Cyclin B1 in hASM cells and hASM cell proliferation. In summary, TNFα induces an ER stress response involving pIRE1α S724 phosphorylation and XBP1s splicing. In turn, XBP1s transcriptionally targets expression of Cyclin B1 that mediates hASM cell proliferation. Supported by NIH grant HL157984 This abstract was presented at the American Physiology Summit 2025 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.

Histamine appears to be intimately involved with skeletal muscle responses during and following exercise. Blocking histamine’s actions during endurance exercise training by taking common over-the-counter antihistamines resulted in diminished gains in fitness. These results indicate that exercise-induced histamine release is important in generating many of the positive adaptations to exercise training.

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 Muscle plasticity is defined as the ability of a given muscle to alter its structural and functional properties in accordance with the environmental conditions imposed on it. As such, respiratory muscle is in a constant state of remodeling, and the basis of muscle's plasticity is its ability to change protein expression and resultant protein balance in response to varying environmental conditions. Here, we will describe the changes of respiratory muscle imposed by extrinsic changes in mechanical load, activity, and innervation. Although there is a large body of literature on the structural and functional plasticity of respiratory muscles, we are only beginning to understand the molecular‐scale protein changes that contribute to protein balance. We will give an overview of key mechanisms regulating protein synthesis and protein degradation, as well as the complex interactions between them. We suggest future application of a systems biology approach that would develop a mathematical model of protein balance and greatly improve treatments in a variety of clinical settings related to maintaining both muscle mass and optimal contractile function of respiratory muscles. © 2012 American Physiological Society. Compr Physiol 2:1441‐1462, 2012.

ABSTRACT Symmorphosis is a concept of economy of biological design, whereby structural properties are matched to functional demands. According to symmorphosis, biological structures are never over designed to exceed functional demands. Based on this concept, the evolution of the diaphragm muscle (DIAm) in mammals is a tale of two structures, a membrane that separates and partitions the primitive coelomic cavity into separate abdominal and thoracic cavities and a muscle that serves as a pump to generate intra‐abdominal (P ab ) and intrathoracic (P th ) pressures. The DIAm partition evolved in reptiles from folds of the pleural and peritoneal membranes that was driven by the biological advantage of separating organs in the larger coelomic cavity into separate thoracic and abdominal cavities, especially with the evolution of aspiration breathing. The DIAm pump evolved from the advantage afforded by more effective generation of both a negative P th for ventilation of the lungs and a positive P ab for venous return of blood to the heart and expulsive behaviors such as airway clearance, defecation, micturition, and child birth. © 2019 American Physiological Society. Compr Physiol 9:715‐766, 2019.

Abstract Striated respiratory muscles are necessary for lung ventilation and to maintain the patency of the upper airway. The basic structural and functional properties of respiratory muscles are similar to those of other striated muscles (both skeletal and cardiac). The sarcomere is the fundamental organizational unit of striated muscles and sarcomeric proteins underlie the passive and active mechanical properties of muscle fibers. In this respect, the functional categorization of different fiber types provides a conceptual framework to understand the physiological properties of respiratory muscles. Within the sarcomere, the interaction between the thick and thin filaments at the level of cross‐bridges provides the elementary unit of force generation and contraction. Key to an understanding of the unique functional differences across muscle fiber types are differences in cross‐bridge recruitment and cycling that relate to the expression of different myosin heavy chain isoforms in the thick filament. The active mechanical properties of muscle fibers are characterized by the relationship between myoplasmic Ca 2+ and cross‐bridge recruitment, force generation and sarcomere length (also cross‐bridge recruitment), external load and shortening velocity (cross‐bridge cycling rate), and cross‐bridge cycling rate and ATP consumption. Passive mechanical properties are also important reflecting viscoelastic elements within sarcomeres as well as the extracellular matrix. Conditions that affect respiratory muscle performance may have a range of underlying pathophysiological causes, but their manifestations will depend on their impact on these basic elemental structures. © 2013 American Physiological Society. Compr Physiol 3:1533‐1567, 2013.

Abstract The mammalian neuromuscular junction (NMJ) comprises a presynaptic terminal, a postsynaptic receptor region on the muscle fiber (endplate), and the perisynaptic (terminal) Schwann cell. As with any synapse, the purpose of the NMJ is to transmit signals from the nervous system to muscle fibers. This neural control of muscle fibers is organized as motor units, which display distinct structural and functional phenotypes including differences in pre‐ and postsynaptic elements of NMJs. Motor units vary considerably in the frequency of their activation (both motor neuron discharge rate and duration/duty cycle), force generation, and susceptibility to fatigue. For earlier and more frequently recruited motor units, the structure and function of the activated NMJs must have high fidelity to ensure consistent activation and continued contractile response to sustain vital motor behaviors (e.g., breathing and postural balance). Similarly, for higher force less frequent behaviors (e.g., coughing and jumping), the structure and function of recruited NMJs must ensure short‐term reliable activation but not activation sustained for a prolonged period in which fatigue may occur. The NMJ is highly plastic, changing structurally and functionally throughout the life span from embryonic development to old age. The NMJ also changes under pathological conditions including acute and chronic disease. Such neuroplasticity often varies across motor unit types. © 2022 American Physiological Society. Compr Physiol 12: 3731–3766, 2022.

Autophagy impairments have been implicated in various aging conditions. Previous studies in cervical motor neurons show an age-dependent increase in the key autophagy proteins LC3 and p62, reflecting autophagy impairment and autophagosome accumulation. Chloroquine is commonly used to inhibit autophagy by preventing autophagosome–lysosome fusion and may thus emulate the effects of aging on the neuromuscular system. Indeed, acute chloroquine administration in old mice decreases maximal transdiaphragmatic pressure generation, consistent with aging effects. We hypothesized that chloroquine alters diaphragm muscle neuromuscular junction (NMJ) morphology and increases denervation. Adult male and female C57BL/6 × 129J mice between 5 and 8 months of age were used to examine diaphragm muscle NMJ morphology and denervation following daily intraperitoneal injections of chloroquine (10 mg/kg/d) or vehicle for 7 days. The motor end-plates and pre-synaptic terminals were fluorescently labeled with α-bungarotoxin and anti-synaptophysin, respectively. Confocal microscopy was used to assess pre- and post-synaptic morphology and denervation. At diaphragm NMJs, chloroquine treatment decreased pre-synaptic volume by 12% compared to the vehicle (p < 0.05), with no change in post-synaptic volume. Chloroquine treatment increased the proportion of partially denervated NMJs by 2.7-fold compared to vehicle treatment (p < 0.05). The morphological changes observed were similar to those previously reported in the diaphragm muscles of 18-month-old mice. These findings highlight the importance of autophagy in the maintenance of the structural properties at adult NMJs in vivo.