Sieck, Gary C.

Airway inflammation is a key aspect of asthma and is associated with airway smooth muscle (ASM) hyperreactivity as well as airway remodeling. Proinflammatory cytokines, such as TNFα mediate this airway inflammatory response. Previously, we found that TNFα induces an increase airway smooth muscle (ASM) force and ATP hydrolysis. In response, TNFα may also induce an increase in ATP production in ASM. This could be achieved by either an increase in mitochondrial function and/or mitochondrial biogenesis. We previously found that TNFα decreases mitochondrial Ca 2+ influx. Since dehydrogenase enzymes in the TCA cycle are Ca 2+ dependent, this would tend to reduce mitochondrial function in response to agonist stimulation. We also found that TNFα increases mitochondrial fragmentation, an initial step in mitochondrial biogenesis. We hypothesized that TNFα increases mitochondrial biogenesis and decreases mitochondrial function in human ASM. Human ASM cells were isolated from lung specimens incidental to patient surgery. The patients were normal as evaluated by a clinical pathologist (no history of asthma or COPD). hASM cells were exposed to TNFα (20 ng/ml) for 24 h. To examine mitochondrial biogenesis, protein expression of porin (an indicator of mitochondrial density) and PGC1α (a regulator of mitochondria biogenesis) were determined by Western blot analysis. In addition, the relative number of copies of human mitochondrial DNA (mtDNA) was determined by PCR and normalized to nuclear DNA. Mitochondrial function was assessed by measuring O 2 Consumption Rate (OCR – using a Seahorse Bioscience XF24 Analyzer), succinate dehydrogenase (SDH) activity (using a quantitative histochemical technique) and mitochondrial membrane potential – (DY m – using TMRM and confocal microscopy). OCR and SDH activity measurements were normalized to mitochondrial volume density. Results were analyzed by one‐way ANOVA with post hoc analysis when justified. We found that TNFα increases mitochondrial biogenesis as indicated by an increase in porin and PGC1α protein expression in hASM and an increase in mtDNA copy numbers. We also found that TNFα decreases mitochondrial membrane potential in hASM cells, and also decreases OCR and SDH activity when normalized for the increase in mitochondrial volume density induced by TNFα. In a normal homeostatic response, an increase in hASM force and ATP hydrolysis (energy demand) would be met by increased mitochondrial ATP production. Our results indicate that TNFα disrupts this normal homeostatic response by reducing mitochondrial Ca 2+ influx and thereby reducing mitochondrial TCA activity. However, TNFα increase the number of mitochondria to compensate the decrease in mitochondrial TCA activity. Support or Funding Information Supported by NIH grant HL126451 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Airway inflammation is a key trigger for asthma, and pro‐inflammatory cytokines such as TNFα mediate the airway hyperreactivity and remodeling characteristic of asthma. In various diseases, inflammation leads to an increase in mitochondrial fission, associated with decreased expression of mitochondrial fusion proteins, such as Mfn2. Mitochondrial fission is generally coupled with mitochondrial biogenesis and cell proliferation. Some studies in other cell types suggest that mitochondrial fission may be a result of the accumulation of unfolded or misfolded proteins i.e., endoplasmic reticulum (ER) stress. ER stress activates three intracellular pathways to induce a homeostatic response to restore normal ER function. The IRE1α pathway is activated by autophosphorylation and splices XBP1 mRNA. Previous studies have shown that the XBP1 has downstream effects on mitochondrial proteins. In this study we hypothesized that TNFα induces activation of the IRE1α pathway which leads to a decrease in Mfn2 expression. hASM cells were isolated from lung specimen incidental to patient surgery. The patients were normal as evaluated by the clinical pathologist (no history of asthma or COPD). hASM cells were stimulated with TNFα (20 ng/ml) for 1, 3, 6, 12 or 24 h. Expression of total and phosphorylated IRE1α, Mfn2 and cleaved and uncleaved XBP1 were evaluated by Western blot. RNAscope® was used to measure Mfn2 mRNA expression in hASM cells transfected with non‐phosphorylatable IREα ( Delta P‐IRE1α; GFP tagged) compared to non‐transfected cells. Results were analyzed by 2‐way ANOVA with Bonferroni analysis when justified. We found that exposure of hASM cells to TNFα activated the IRE1α/XBP1 pathway and decreased Mfn2 expression in a time dependent fashion. In addition we found that TNFα exposure in cells expressing the inactive Delta P‐IRE1α did not lead to a significant decrease in Mfn2 expression. Our results suggest that the IRE1α/XBP1 pathway may be involved in the regulation of mitochondrial fission, mitochondrial biogenesis and cell proliferation. Support or Funding Information Supported by NIH grant HL126451 (GCS) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Ischemia/reperfusion (I/R) injury occurs during acute myocardial infarction and is a frequent cause of contractile failure, morbidity and mortality. There is evidence that reduced cardiac contractility following I/R injury is associated with disruption of excitation‐contraction (E‐C) coupling. We hypothesized that I/R injury alters E‐C coupling in cardiomyocytes by reducing the Ca 2+ sensitivity of contraction. Cardiomyocytes were isolated from adult rat (Sprague Dawley) hearts using a modified Langendorff system. Cardiomyocytes were then loaded with fura‐2 AM (Ca 2+ fluorescent indicator; 1 μM) and electrically stimulated every 2 s (0.5 Hz). Cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) and sarcomere length shortening responses to stimulation were simultaneously measured using an IonOptix System equipped with a hypoxia‐regulated incubator system (Live Cell Instrument). To model I/R, cardiomyocytes were initially exposed to normoxia (21% O 2 ) for 30 min followed by 30 min exposure to 2.5% O 2 and finally 30 min exposure to normoxia. Throughout the I/R protocol, Ca 2+ sensitivity of cardiomyocytes was assessed using a phase loop plot of concurrently measured [Ca 2+ ] cyt and sarcomere length shortening. I/R reduced the extent of sarcomere length shortening, but it had no effect on the amplitude of evoked [Ca 2+ ] cyt transients. Furthermore, I/R reduced Ca 2+ sensitivity reflected by a rightward shift in the phase loop plots. These results support the hypothesis that I/R alters E‐C coupling in cardiomyocytes by reducing the Ca 2+ sensitivity of contraction. Support or Funding Information Supported by Mayo Foundation This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Mitochondrial dysfunction and fragmentation precede neuronal death in many neurodegenerative disorders, including those involving motor neurons. In other cell types, pro‐inflammatory agents such as TNF‐α cause marked mitochondrial fragmentation. These inflammatory insults may underlie the vulnerability of specific neurons during age‐associated conditions, such as sarcopenia. We hypothesize that motor neuron exposure to pro‐inflammatory cytokines induces mitochondrial fragmentation. Experiments were conducted using a mouse motor neuron‐like cell line, NSC‐34. These cells were differentiated by serum depletion until neurite structures (>50 um) were observed, ~24 h. The differentiated NSC‐34 cells were then treated with mouse TNF‐α at varying concentrations; control (0 ng), 10 ng/ml, and 20 ng/ml for 24 h. The cells were then loaded with MitoTracker Green to image mitochondria using a Nikon A1R confocal microscope (60x oil immersion 1.4 NA). Images were analyzed using MatLab macro to calculate form factor, aspect ratio and mitochondrial volume density. TNF‐α exposure caused mitochondrial fragmentation as reflected by decreased form factor (27% reduction) and aspect ratio (12 % reduction). These results are consistent with inflammatory cytokine exposure leading to mitochondrial fragmentation in motor neurons which may increase their vulnerability mitochondrial dysfunction and cell death. Support or Funding Information NIH: RO1AG044615 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The diaphragm muscle (DIAm) comprises four muscle fiber/ motor unit types: Fatigue resistant type S and FR motor units with type I and IIa fibers, respectively that are recruited for ventilatory behaviors; and more fatigable type FInt and FF motor units with type IIx and/or IIb fibers that are only recruited during high force expulsive/straining behaviors of the DIAm. With advanced aging in Fischer 344 rats (F344), type IIx and/or type IIb DIAm fibers are vulnerable to sarcopenia (reductions in specific force and cross‐sectional area, CSA), while type I and IIa fibers are resilient. This is consistent with the fact that ventilatory behaviors are conserved with aging, while airway clearance behaviors are compromised. Throughout life, breathing imposes a high duty cycle (>33 %) of DIAm activity and thus, a high energy demand for type I and IIa fibers that is met by a high oxidative capacity. However, the effect of advanced age on DIAm fiber oxidative capacity has not been examined. Accordingly, we hypothesize that the oxidative capacity in type I and IIa DIAm fibers is not affected by age, whereas concomitant with sarcopenia, the oxidative capacity of type IIx and/or IIb fibers is reduced. To assess DIAm fiber oxidative capacity, we measured succinate dehydrogenase (SDH) activity in type‐identified DIAm fibers from 6, 18 and 24‐month old male and female F344 rats. Following euthanasia, the DIAm was excised and 2–3 mm strips from the mid‐costal region were dissected, stretched to optimal length and rapidly frozen in melting isopentane. Serial transverse sections (6 μm) were cut to assess fiber type (based on myosin heavy chain, MyHC isoform), cross‐sectional area (CSA) and SDH activity. For SDH, sections were incubated at 25°C for 8 min in solutions containing 0 or 80 mM succinate and 1.5 mM nitro blue tetrazolium (reaction indicator), 5 mM EDTA, 0.2 mM PMS and 0.1 mM azide in 0.1 M phosphate buffer. Images were acquired every 5 s across a 15‐min period using a 20x objective on an Olympus IX71 inverted microscope. Linearity of the SDH reaction (change in optical density‐ OD) was confirmed across the 15‐min period for each fiber type. The presence of DIAm sarcopenia was confirmed in 24‐month old rats by a 25 % decrease in specific force and a 20% decrease in type IIx and/or IIb CSA. At all ages, the SDH activity of type I and IIa fibers was higher than type IIx/IIb fibers. No age‐related difference was observed in the SDH activity of type I or IIa fibers, whereas SDH activity of type IIx and/or IIb DIAm fibers was reduced at 24 months compared to younger animals. These results underscore the remarkable vulnerability of IIx and/or IIb fibers to sarcopenia, with profound resilience in I and IIa fibers. We propose that mitochondrial abnormalities may underlie the selective vulnerability of IIx and/or IIb fibers possibly due to changes at the phrenic motor neuron level. Support or Funding Information Supported by NIH grant AG44615 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .