Sieck, Gary C.

The impact of airway inflammation is mediated by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), which underlies several respiratory diseases, including COVID-19, asthma, chronic bronchitis, and chronic obstructive pulmonary disease (COPD). In human airway smooth muscle (hASM) cells, exposure to TNFα leads to an accumulation of unfolded proteins in the ER, which triggers a homeostatic ER stress response activating a signaling cascade to restore normal function. Previously, we showed that TNFα selectively activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 (XBP1) ER stress pathway in hASM cells. Most likely, the protein unfolding in hASM cells caused by TNFα results from an increase in reactive oxygen species (ROS) formation. We hypothesize that ER stress induced by TNFα is mitigated by the ROS scavenger TEMPOL. Primary hASM cells were isolated from bronchiolar tissue samples obtained from 5 patients (both sexes included) who had no history of respiratory diseases. hASM cells were serum deprived for 48 h before treatment with 1) TNFα (20 ng/ml) for 12 h; 2) TEMPOL (500 μM) for 30 min followed by TNFα (20 ng/ml) for 12 h; and 3) untreated control for 12 h. At the end of the treatment period, hASM cells were processed for determination of protein expression through Western blotting. Activation of the ER stress response was assessed by phosphorylation of IRE1α and splicing of XBP1. Confirming our previous results, we found that 12 h TNFα exposure induced an ER stress response in hASM cells, evidence by phosphorylation of IRE1α and XBP1 splicing. Treating hASM cells with TEMPOL before exposure to TNFα mitigated the ER stress response. These results support our hypothesis and indicate that ROS scavenging prevents TNFα induced protein unfolding and triggering of the IRE1α/XBP1 ER stress pathway. R01HL 157984 (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.

Sarcopenia is defined as age-related atrophy of muscle fibers as well as a decrease in specific force (force per cross-sectional area). In the rat diaphragm muscle (DIAm), sarcopenia primarily affects type IIx/IIb fibers that generate greater specific force, have the fastest maximum shortening velocities, but display greater fatigue with repeated activation. We hypothesized that as a result of sarcopenia, maximum shortening velocity and power output of the DIAm decrease with age. DIAm strips ~2-3 mm wide were excised from 6-month and 24-month female (n=4 per age group) and male (n=4 per age group) rats. DIAm strips were suspended in a tissue chamber containing Rees–Simpson solution maintained at 26oC, with the costal margin clamped and the central tendon attached to a Cambridge Dual-Mode force/length transducer. To determine maximum isometric specific force, DIAm strips were stimulated using platinum plate electrodes with supramaximal current (~250 mA) pulses (1 ms) at 75 Hz delivered in 1-s duration train. The muscle strips were then allowed to shorten over a 30-ms period against different percentages of maximum isometric load. Based on the load/velocity relationship, the maximum shortening velocity (Vmax) and peak power output were determined. Subsequently, the muscle strip was repetitively stimulated and allowed to shorten at 30% maximum load (approximating peak power output). In this protocol, the muscle strip was stimulated at 75 Hz an generated isometric force during the initial 225 ms of the 330 ms duration train the muscle. Thereafter, the load was reduced to 30% maximum and the muscle strip was allowed to shorten for the subsequent 105 ms. Finally, the DIAm strip was stretched to ~150% its resting length (corresponding to optimal sarcomere length) and rapidly frozen in melting isopentane. Transverse serial sections of DIAm fibers were cut, and fiber types identified by immunoreactivity to specific myosin heavy chain (MyHC) isoforms. As previously observed, the cross-sectional areas of type IIx/IIb DIAm fibers and maximum isometric specific force were reduced in 24-month compared to 6-month rats confirming sarcopenia. The maximum shortening velocity of the DIAm in 24-month rats was slower than in the 6-month rats and peak power output of the DIAm was reduced in 24-month rats. The duration during which peak power output could be maintained (i.e., endurance) of the DIAm was prolonged in 24-month compared to 6-month rats (120 s vs 60 s, respectively). These results support our hypothesis that aging affects maximum shortening velocity and peak power output of the DIAm. It is important to note that endurance of the DIAm is preserved, consistent with resilience of breathing motor function in old age. Research supported by NIH grant AG44615 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.

Low frequency brain rhythms facilitate communication across large spatial regions in the brain and high frequency rhythms are thought to signify local processing among nearby assemblies. A heavily investigated mode by which these low frequency and high frequency phenomenon interact is phase-amplitude coupling (PAC). This phenomenon has recently shown promise as a novel electrophysiologic biomarker, in a number of neurologic diseases including human epilepsy. In 17 medically refractory epilepsy patients undergoing phase-2 monitoring for the evaluation of surgical resection and in whom temporal depth electrodes were implanted, we investigated the electrophysiologic relationships of PAC in epileptogenic (seizure onset zone or SOZ) and non-epileptogenic tissue (non-SOZ). That this biomarker can differentiate seizure onset zone from non-seizure onset zone has been established with ictal and pre-ictal data, but less so with interictal data. Here we show that this biomarker can differentiate SOZ from non-SOZ interictally and is also a function of interictal epileptiform discharges. We also show a differential level of PAC in slow-wave-sleep relative to NREM1-2 and awake states. Lastly, we show AUROC evaluation of the localization of SOZ is optimal when utilizing beta or alpha phase onto high-gamma or ripple band. The results suggest an elevated PAC may reflect an electrophysiology-based biomarker for abnormal/epileptogenic brain regions.

Type S and FR motor units, comprising smaller phrenic motor neurons (PhMNs) are regularly activated to perform indefatigable ventilatory requirements. By contrast, type FF motor units, comprising larger PhMNs, are infrequently activated to perform expulsive straining and airway defense maneuvers. This difference in activation history is mirrored in the mitochondrial volume density (MVD), with smaller PhMNs having higher MVD than larger PhMNs. In proximal dendrites, this trend was reversed, with larger PhMNs having higher MVD than smaller PhMNs, likely due to the maintenance requirements for the larger dendritic arbor of FF PhMNs.

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.

Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). Rat phrenic nerve-diaphragm muscle preparations were used to determine the rapid effect of BDNF on synaptic quantal release during repetitive stimulation. BDNF treatment significantly enhanced quantal release at all fiber types. BDNF increased synaptic vesicle cycling (measured using FM4-64 fluorescence uptake); conversely, inhibiting BDNF/TrkB signaling decreased FM4-64 uptake.