Sieck, Gary C.

Airway inflammation is a key aspect of asthma and is associated with airway smooth muscle (ASM) hyperreactivity (airway narrowing due to increased Ca 2+ and force responses to agonist stimulation) as well as airway remodeling (ASM cell proliferation and altering extracellular matrix composition). Proinflammatory cytokines, such as TNF‐α mediate this airway inflammatory response. Previously, we found that the TNF‐α induces an increase ASM force, which is associated with an increase in ATP hydrolysis, imposing a metabolic stress on ASM. Mitochondria play an important role in providing ATP to support contraction in ASM. Mitochondria are dynamically remodelling within ASM cells displaying changing filamentous or fragmented tubular networks. Changing expression of Mitofusin 2 (Mfn2), which is associated with mitochondria fusion, or Dynamin Related Protein 1 (Drp1), which is associated with fission mediate this mitochondrial remodelling. The stressors that induce mitochondrial remodelling in ASM are unknown, but mitochondrial fragmentation may be an initial step in mitochondrial biogenesis to meet increasing energy demand. We hypothesized that human ASM (hASM) cells exposed to TNF‐α will display reduced Mfn2 and increased Drp1 expression resulting in mitochondrial fragmentation and mitochondrial biogenesis. To test our hypothesis, serum‐deprived primary hASM cells were exposed to 20 ng/ml TNF‐α for 24 h after which they were loaded with 500 μM MitoTracker Green to visualize mitochondria and imaged using confocal microscopy. Images were analyzed using MATLAB Image J software to quantify mitochondrial morphometry: form factor and aspect ratio. An increase in form factor and aspect ratio indicates greater mitochondrial filamentous networks. Expression of Mfn2 and Drp1 were determined by Western blot analysis. In hASM exposed to TNF‐α, Mfn2 expression decreased by ~40%, while Drp1 expression increased by ~75% compared to controls. These changes in fusion/fission proteins were associated with mitochondrial fragmentation (reduced form factor and aspect ratio) and an increase in mitochondria number. Our results indicate that hASM cells exposed to TNF‐α display mitochondrial fragmentation due to changes in Mfn2 and Drp1 expression. Mitochondrial fragmentation may be involved in mitochondrial biogenesis in response to increased metabolic demands imposed by airway hyperreactivity. Support or Funding Information APS fellowship (EK) and NIH grant HL126451 (GCS)

In excitation‐contraction coupling, a transient elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) triggers a force response. The Ca 2+ sensitivity of muscle reflects the relationship between [Ca 2+ ] cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca 2+ ] cyt at which 50% force is generated. In smooth muscle, Ca 2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca 2+ ] cyt . However, this measurement overlooks the dynamic nature of Ca 2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca 2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca 2+ ] cyt and force and determined their temporal dependence using a phase loop plot. We hypothesized that [Ca 2+ ] cyt /force phase loop plot in ASM would be altered by conditions in which the extent of regulatory myosin light chain (rMLC) phosphorylation was affected. Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips, were loaded with Fura‐2 AM (Ca 2+ fluorescent indicator) and stimulated using 1 μM ACh. Isometric force and [Ca 2+ ] cyt responses were simultaneously measured using a Guth Muscle Research System. In the first set of experiments we examined the effect of Y27623, a Rho‐kinase inhibitor, and calyculin A, a protein phosphatase inhibitor that affected Ca 2+ ‐induced rMLC phosphorylation. In a second set of experiments, we examined the effect of cytochalasin D (Cyto‐D), an inhibitor of actin polymerization, which reduces force independent of Ca 2+ ‐induced rMLC phosphorylation. As expected, Y27623 decreased ACh‐induced isometric force but had no effect on evoked [Ca 2+ ] cyt transients. Cyto‐D also decreased force with no effects on evoked [Ca 2+ ] cyt . By comparison, calyculin A increased force with no effect on [Ca 2+ ] cyt . Although these results suggest changes in Ca 2+ sensitivity, the phase loop plots showed no significant shift indicative of changes in dynamic Ca 2+ sensitivity. These results do not support our hypothesis that dynamic Ca 2+ sensitivity in ASM is modulated by the extent of rMLC phosphorylation. Under these experimental conditions, it appears that isometric force generation in ASM is not entirely dependent on [Ca 2+ ] cyt . Support or Funding Information Supported by NIH grant HL126451 (GCS)

In previous studies, we found that prolonged hypothermia (2 h or greater) followed by rewarming induces cardiac contractile dysfunction (rewarming shock) mimicking the effects of accidental hypothermia followed by rapid rewarming in trauma patients. We found that rewarming shock was also presented in isolated cardiomyocytes in which reduced contractility was attributed to PKA‐mediated TnI phosphorylation and reduced Ca 2+ sensitivity. During hypothermia rewarming in cardiomyocytes there is increased production of reactive oxidant species (ROS ‐ superoxide anion), which may trigger the intracellular mechanisms underlying reduced contractility. We hypothesized that in cardiomyocytes, hypothermia/rewarming‐induced ROS production is due to altered mitochondrial respiratory function. To test this hypothesis, isolated cardiomyocytes from 6 rats underwent the following hypothermia/rewarming protocol in which they were electrically stimulated every 2 s: experimental temperature was cooled from 35°C to 15°C during the initial 30 min, then maintained at 15°C for 2 h, followed by rewarming to 35°C in the final 30 min. Time‐matched control cardiomyocytes were also electrically stimulated every 2 s and maintained at 35°C for 3 h. After the 3‐h protocol, cardiomyocytes were placed into separate chambers of an Oroboros Oxygraph (high‐resolution respirometry) to measure O 2 consumption rate (OCR) under varying conditions. Initially, basal OCR was measured for 5 min followed by serial exposure to oligomycin (1 μM for 5 min) to block ATP synthase activity, FCCP (0.5 μM repeated 3 times for 5 min each) an H + ionophore to dissipate mitochondrial membrane potential; and antimycin‐A (0.5 μM for 5 min) to block the electron transport chain. This procedure allowed assessment of the key parameters of mitochondrial respiratory function: basal and maximal respiration (after FCCP exposure), ATP production and proton leak (after oligomycin), and reserve respiratory capacity (calculated after antimycin‐A). We found that compared to control cardiomyocytes, hypothermia/rewarming decreased key parameters of mitochondrial function, which suggests that altered mitochondrial function may have contributed to increased ROS production. In addition, decreased OCR following rewarming may contribute to contractile dysfunction by limiting ATP supply in response to metabolic demand, as well as by contributing to a decline in cell viability (e.g., triggering apoptosis). Support or Funding Information 5T32HL105355 (NSS); Norwegian Air Ambulance Foundation (YSH)

Aging‐related changes in lung mechanics of human subjects are characterized by a decrease in resistance and an increase in static compliance (decrease in elastic recoil). Together, these changes result in an increase in residual volume in the absence of an increase in total lung capacity. In C57BL/6x129 mice, it was reported that lung compliance increases with aging while elastic recoil pressure does not change. However, in these studies, “old” mice at 24 months of age were compared to “young” mice at 2 months of age, but at this younger age, alveologenesis is incomplete (completion does not occur until ~6 months of age). Accordingly, it remains to be established if the same aging‐related changes in lung mechanics are observed using fully developed adult C57BL/6x129 mice for comparison. To this end, we compared lung mechanics in C57BL/6x129 mice at 6 months (100% survival, n = 6), 18 months (90% survival, n = 6), and 24 months (75% survival, n = 6) of age. Respiratory system static mechanics (Resistance, R rs ; Compliance, C rs ; Elastance, E rs ) were assessed following tracheal intubation using the FlexiVent. There was a progressive aging‐related decrease in R rs (6 month, 0.47 ± 0.02; 18 month, 0.41 ± 0.02; 24 month, 0.35 ± 0.02 cmH 2 O.s/ml), an increase in C rs (6 month, 0.046 ± 0.002; 18 month, 0.052 ± 0.002; 24 month, 0.062 ± 0.002 ml/cmH 2 O), and a decrease in E rs (6 month, 21.73 ± 1.19; 18 month, 19.30 ± 0.87; 24 month, 16.62 ± 1.25 cmH 2 O/ml). Additionally, lungs were fixed following tracheal instillation of 4% paraformaldehyde at 25 cmH 2 O. Thereafter, lungs were dehydrated, paraffin embedded, sectioned (6 μm thick), and stained (hematoxylin and eosin). Using standard stereological approaches, lung sections were analyzed in a uniform systematic but random fashion to assess alveolar morphology (measurement of average distance between alveolar walls based on mean linear intercept, L m ). There was a progressive aging‐related increase in L m (6 month, 34 ± 2; 18 month, 40 ± 2; 24 month, 45 ± 2 μm) consistent with an aging‐related emphysematous‐like change in lung morphometry. Taken together, these results in appropriately aged C57BL/6x129 mice demonstrate an aging‐related change in lung mechanics and morphometry consistent with what is observed in aging humans. Therefore, the results of the present study confirm the use of rodent models for studying aging‐related changes in lung mechanics and morphometry. Support or Funding Information National Institute of Health grants R01‐AG044615 (CBM & GCS) and T32‐HL105355 (JEE).

Neurotrophic signaling is essential to the maintenance of diaphragm muscle function across the lifespan. With aging, there is an age‐related loss of neurotrophic signaling and a resultant increase in diaphragm muscle denervation and impairment in neuromuscular transmission. We hypothesized that enhancing neurotrophic signaling via the tropomyosin‐related kinase receptor subtype B (TrkB) using the small molecule TrkB agonist 7,8‐dihydroxyflavone (7,8‐DHF) would mitigate age‐related changes in diaphragm neuromuscular transmission and possibly sarcopenia (muscle atrophy and force loss). Adult male TrkB F616A mice (n=32) were randomized to receive 6‐month oral treatment with vehicle (control), 7,8‐DHF, or co‐treatment with 7,8‐DHF and 1NMPP1(a specific inhibitor of TrkB kinase activity in TrkB F616A mice) beginning at 18 and ending at 24 months of age. Mice were analyzed for neuromuscular transmission failure using diaphragm muscle‐phrenic nerve preparations, diaphragm muscle force, and morphologic characteristics of sarcopenia. Chronic 7,8‐DHF treatment did not improve diaphragm neuromuscular function or force (P≥0.25). Co‐treatment with 7,8‐DHF and 1NMPP1 decreased the ratio of the initial nerve stimulation‐derived to initial muscle stimulation‐derived force (~22%) compared to vehicle (p=0.01), consistent with a deleterious effect of inhibiting TrkB kinase activity on aging diaphragm muscle. With age, there was fiber type specific atrophy of type IIa and type IIx and/or IIb diaphragm muscle fibers. There was no evidence of a protective effect of 7,8‐DHF on muscle fiber cross‐sectional area. Taken together, these results support a role for BDNF/TrkB signaling in old age but do not support 7,8‐DHF as a therapeutic agent to mitigate age‐related effects on diaphragm muscle function. Support or Funding Information Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.

At present, there are limited delivery systems to target neurons in the central nervous system. Neurological diseases are commonly hindered by treatments lacking drug selectivity to the neurons of interest. In particular, motoneuron diseases will likely require selective targeting of therapeutic interventions, which can be delivered via the peripheral nervous system. Nanoparticles are novel drug delivery systems with exceptional therapeutic potential. Specifically, mesoporous silica nanoparticles coated with a lipid bilayer (protocells) are a suitable treatment vehicle to target the peripheral and central nervous system because they offer advantageous fusion characteristics including 1) increased surface area with larger loading capacity, 2) stability and 3) improved biocompatibility. A number of natural toxins exist that already target the nervous system and could be used as a targeting strategy. Cholera toxin produced by the bacterium Vibrio cholerae has an atoxic subunit (CTB) that binds to gangliosides present on neuronal membranes and is a reliable motoneuron tracer. We hypothesized that CTB‐coated protocells specifically target motoneurons. In culture, CTB uptake is evident in motoneuron‐like NSC‐34 cells but not in L6 muscle cells, consistent with the suitability of CTB as a motoneuron targeting strategy. Fluorescently labeled protocells (130 nm in diameter) were bound to CTB using biotin‐neutravidin. CTB‐protocells showed significantly greater motoneuron uptake compared to non‐CTB coated protocells when quantified by measurements of intracellular protocell fluorescence intensity. Confocal images were used for co‐localization of fluorescently labeled protocells and lysosomes. Protocells effectively escape endo‐lysosomal degradation delivering their cargo intracellularly. Biocompatibility was evidenced by lack of cytotoxicity in cultured motoneurons and unimpaired cellular proliferation. To evaluate the targeting efficacy of CTB‐protocells in tissue, we utilized isolated diaphragm muscle‐phrenic nerve preparations. Evidence of protocell uptake at diaphragm neuromuscular junctions measured by protocell fluorescence intensity supports uptake into phrenic motoneuron axon terminals. In conclusion, CTB‐protocells are a promising, customizable approach for targeting and delivering drugs to motoneurons in vivo.

The risk for respiratory diseases is significantly increased in the elderly population (>65 years of age), which may be partially due to an inability to adequately clear the airways resulting from a progressive aging‐related atrophy and weakening (i.e., sarcopenia) of the diaphragm muscle (DIAm). In humans, reduced maximum inspiratory and transdiaphragmatic pressures suggest DIAm weakening, but muscle fiber atrophy has not been examined. In a National Institute on Aging (NIA) supported mouse model of aging (C57BL/6J strain), we recently showed that DIAm sarcopenia exists and affects non‐ventilatory functions (Greising et al., Exp Gerontol 48:881–887, 2013; Greising et al., Am J Physiol Lung Cell Mol Physiol 309:46–52, 2015). Importantly, rodent models such as this provide a valuable tool to explore the mechanisms underlying DIAm sarcopenia. Currently, the NIA also provides two inbred rat models for aging‐related research: the Fischer 344 (F344) and Brown Norway (BN) rat strains. The purpose of this study was to comprehensively examine whether sarcopenia also exists in the DIAm of F344 and BN rat strains provided by the NIA. Young (6 month, 100% survival) and old (24 month, 50% survival) F344 and young (6 month, 100% survival) and old (32 month, 50% survival) BN male, pathogen free, rats were obtained from the NIA rodent colony. In DIAm strips from the right midcostal region, measurements of contractility, contractile protein concentration, relative myosin heavy chain (MyHC) isoform expression, fiber type distribution and fiber cross‐sectional area (CSA) were obtained. Peak twitch specific force (P t ) was reduced by ~17% and ~14% in the aged F344 and BN rats, respectively. Additionally, maximal specific force (P o ) was reduced by ~24% and ~12% in the aged F344 and BN rats, respectively. Thus, although there was significant aging‐related DIAm weakness in both rat strains, reductions in both P t and P o were more pronounced in old F344 compared to BN rats. In F344 rats, aging also affected DIAm fiber type proportions with a significant increase in the proportion of type I fibers in old (45 ± 3%), compared to young (35 ± 3%) rats, with a concomitant decrease in the relative proportions of both type IIa fibres and type IIx and/or IIb fibers. In BN rats, DIAm fiber type proportions were not affected by aging. In both F344 and BN rats, the CSA of type I and IIa fibers were unchanged, whereas the CSA of type IIx and/or IIb DIAm fibers were reduced by ~20% in F344 rats and by ~15% is BN rats. Similar to DIAm weakness, the selective aging‐related atrophy of type IIx and/or IIb DIAm fibers was more pronounced in F344 rats. In conclusion, DIAm sarcopenia is clearly present in both F344 and BN rat strains, but more pronounced in the F344 strain. These results indicate that in addition to the C57BL/6J mouse model, the F344 rat strain is an appropriate model for research pertaining to DIAm sarcopenia. Support or Funding Information Support: National Institute of Health grants R01‐AG044615 (CBM & GCS) and T32‐HL105355 (JEE).

Hypothermia/Rewarming (H/R) induces myocardial dysfunction although the underlying intracellular mechanisms remain elusive. We hypothesized that in cardiomyocytes, H/R disrupts intracellular Ca 2+ ([Ca 2+ ] cyt ) homeostasis via an effect on both release and clearance of cytosolic Ca 2+ leading to a prolongation of evoked [Ca 2+ ] cyt transients associated with activation with downstream impact on contractility. To test this hypothesis, isolated rat cardiomyocytes (13–15 cells from 6 rats per group) were loaded with the Ca 2+ indicator, Fura‐2AM (0.5 μM), and exposed to H/R with or without electrical pacing at 0.5 Hz. In both groups, evoked [Ca 2+ ] cyt and contractile (sarcomere length (SL) shortening) responses were simultaneously measured at 35°C before and after hypothermia using an IonOptix system. H/R experiments were conducted as follows: experimental temperature was cooled from 35°C to 15°C in 30 min, maintained at 15°C for 2 h, and then rewarmed back to 35°C in 30 min. Time‐matched controls were implemented for 3 h at 35°C. With electrical pacing throughout hypothermia, basal [Ca 2+ ] cyt increased and the kinetics of the evoked [Ca 2+ ] cyt responses were prolonged. In addition, the extent and velocity of SL shortening was reduced in cardiomyocytes that were paced during hypothermia. In contrast, in cardiomyocytes that were not electrically paced during hypothermia, basal [Ca 2+ ] cyt was maintained and the kinetics of evoked [Ca 2+ ] cyt transients were unaffected. In addition, in the absence of pacing during hypothermia, contractility of cardiomyocytes was unaffected. These results suggest that H/R affects the release and clearance of evoked [Ca 2+ ] cyt transients (e.g., RyR mediated Ca 2+ release and SERCA mediated clearance) such that basal [Ca 2+ ] cyt increases. This effect of H/R on [Ca 2+ ] cyt homeostasis is associated with reduced cardiac contractility.

Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) endoplasmic reticulum (ER) stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyric acid (4-PBA) may reduce ER stress activation. In the present study, we hypothesized that chemical chaperone 4-PBA mitigates TNFα-induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from five patients with no history of smoking or respiratory diseases. The hASM cells’ phenotype was confirmed via the expression of alpha-smooth muscle actin and elongated morphology. hASM cells from the same patient sample were then separated into three 12 h treatment groups: (1) TNFα (20 ng/mL), (2) TNFα + 4-PBA (1 μM, 30 min pretreatment), and (3) untreated control. The expressions of total IRE1α and phosphorylated IRE1α (pIRE1αS724) were determined through Western blotting. The splicing of XBP1 mRNA was analyzed using RT-PCR. We found that TNFα induced an increase in pIRE1αS724 phosphorylation, which was mitigated by treatment with chemical chaperone 4-PBA. We also found that TNFα induced an increase in XBP1s mRNA, which was also mitigated by treatment with chemical chaperone 4-PBA. These results support our hypothesis and indicate that chemical chaperone 4-PBA treatment mitigates TNFα-induced ER stress in hASM cells.

We characterized three phases of neuromotor control—motor unit recruitment, sustained activity, and derecruitment—based on statistical assessments of stationarity of the diaphragm muscle (DIAm) EMG activity in awake rats. Our findings may allow indirect estimates of the contribution of motor unit recruitment and frequency coding toward generating force and provide novel insights about the temporal aspects of DIAm neuromotor control and descending respiratory drive in unanesthetized animals.