bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Impact of Diaphragm Muscle Sarcopenia in Fischer 344 Rats
Authors: Sarrafian Tiffany L. , Khurram Obaid U. , Fogarty Matthew J. , Bhatt Arjun , Mantilla Carlos B. , Sieck Gary C. 
Published: June 2021

The diaphragm muscle (DIAm) is the primary inspiratory muscle responsible for force generation across a range of ventilatory and higher force airway clearance behaviors throughout life. Our laboratory has previously reported DIAm sarcopenia, the age‐related atrophy and weakening of skeletal muscle, in multiple rodent models. Interestingly, in Fischer 344 rats, there is an increase in body mass from 6 to 24 months of age, which is greater in females (~140%) than in males (~110%), suggesting potential sex‐related differences in the manifestation of sarcopenia. Transdiaphragmatic pressure (Pdi) measurements provide information about force generation in vivo across a range of motor behaviors. The purpose of this study was to evaluate if there are sex‐related differences in DIAm sarcopenia, and if such differences translate to a functional impact on Pdi generation. We hypothesized that because of differences in weight gain, DIAm sarcopenia is more pronounced in male vs. female Fischer 344 rats, and that there is a greater impact of sarcopenia on maximum Pdi generated by bilateral phrenic nerve stimulation (Pdimax). Since Pdi generated across a range of ventilatory motor behaviors only requires <50% Pdimax, we also hypothesize that sarcopenia does not impair Pdi generation during ventilatory behaviors. Compared to 6‐month old rats, DIAm sarcopenia was apparent in ‐24‐month old rats of both sexes with a 30% reduction in maximum specific force. There was also a ~20% decrease in the cross‐sectional area of type IIx and/or IIb fibers with age, and this reduction was more pronounced in male rats. Thus, these mixed results do not entirely support our hypothesis of more pronounced DIAm sarcopenia in male rats. However, DIAm sarcopenia was associated with a ~30% decrease in Pdimax that was comparable between male and female rats. Importantly, the Pdi generated during ventilatory behaviors was unaffected by DIAm sarcopenia, even during more forceful ventilatory efforts induced by complete oropharyngeal airway occlusion. Although ventilatory behaviors are preserved with aging, a reduction in Pdimax indicates an impaired ability of the DIAm to generate higher force expulsive airway clearance behaviors necessary for maintaining airway patency.Support or Funding InformationNIH R01‐AG044615 and T32‐HL105355This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Respiratory EMG responsiveness to hypercapnic hypoxia in mdx mice
Authors: Murphy Kevin Humphrey , Burns David Patrick , Lucking Eric , O'Halloran Ken 
Published: June 2021

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease characterised by the absence of the structural protein dystrophin. Respiratory failure is the leading cause of premature death in DMD. Although respiratory insufficiency is recognized as a hallmark of DMD, respiratory control is relatively understudied. We hypothesized that enhanced drive in accessory respiratory motor pathways preserves ventilatory capacity compensating for severe respiratory muscle weakness.We utilised the C57BL/10ScSn‐Dmdmdx/J (mdx) mouse model. Respiratory flow recordings were performed in freely‐behaving wild‐type (WT, n=9) and mdx (n=7) mice at ~8 weeks of age using whole‐body plethysmography to determine ventilation (VE), oxygen consumption (VO2) and carbon dioxide production (VCO2). Baseline recordings were performed in normoxia, followed by chemostimulation using hypercapnic hypoxic gas challenge (FiO2=0.10; FiCO2=6; balance N2). In urethane (1.6 g/kg) anaesthetised WT (n=13) and mdx (n=14) mice, diaphragm, intercostal and genioglossus EMG and motor unit recordings were performed during baseline conditions and in response to chemostimulation.Plethysmographic recordings revealed that during chemostimulation, minute ventilation (VE) is equivalent in WT and mdx mice, highlighting a considerable ventilatory reserve in young mdx despite substantial diaphragm muscle weakness. Moreover, diaphragm, intercostal and genioglossus EMG responsiveness to hypercapnic hypoxia was equivalent in WT and mdx mice.Despite substantial diaphragm weakness and evidential muscle fibre damage and remodelling, freely behaving mdx mice can achieve enhanced ventilation during chemostimulation to levels equivalent to that of WT animals. We found no evidence of enhanced activity in respiratory muscle EMGs. Assessment of the motor unit population will allow for the comparison of the phenomena of rate coding and recruitment during increased respiratory muscle activation. Our study suggests that respiratory muscle weakness in young mdx does not compromise ventilator behaviour but is likely to have considerable impact on airway protective behaviours.Support or Funding InformationDepartment of Physiology, University College Cork, Cork, IrelandThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Intralingual AAVrh10‐miRSOD1 Injection on Respiratory Function in SOD1G93A Mice
Authors: Lind Lori A. , Johnson Katherine A. , McCall Angela L. , Mueller Christian , Brown Robert H. , Lever Teresa E. , Elmallah Mai K. , Nichols Nicole L. 
Published: June 2021

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which patients normally succumb to the disease within 5 years of onset. Death is usually a result of impaired respiratory function due to loss of motor neurons that control upper airway muscles and/or the diaphragm. The motor neurons that control upper airway muscles and thereby airway patency are found in the brainstem in a region called the hypoglossal motor nucleus. One cause of ALS is a mutation in the superoxide dismutase‐1 (SOD1) gene that results in the buildup of SOD1 protein in neurons. For this subset of patients, reducing expression of the mutated gene may slow the progression of the disease. The purpose of this study was to determine if delivering a microRNA against SOD1 (miRSOD1) to the hypoglossal motor nucleus of mice that overexpress mutant SOD1 (SOD1G93A) would increase respiratory function. Thus, we hypothesized that intralingual injection of AAV‐miRSOD1 into SOD1G93A mice would knockdown mutant SOD1 in hypoglossal motor neurons and improve respiration. Six‐week‐old male and female mice received intralingual injections of either AAVrh10‐miRSOD1 or vehicle (phosphate buffered saline). Starting at 13 weeks of age, respiratory parameters at baseline and in response to hypoxia (11% O2) + hypercapnia (7% CO2) were studied in miRSOD1‐injected SOD1G93A mice (n=11), vehicle‐injected SOD1G93A mice (n=12), and vehicle injected wild‐type mice (n=17) via whole‐body plethysmography every other week until the SOD1G93A mice reached end‐stage (20% body weight loss). Minute ventilation during hypoxia + hypercapnia and mean inspiratory flow at baseline were significantly reduced (p<0.05) in vehicle‐injected but not miRSOD1‐injected SOD1G93A mice as compared to wild‐type mice. miRSOD1 injections also extended survival in females by ~1 week; although this did not reach statistical significance, we suggest that any increase in survival could be clinically significant. In conclusion, this study indicates that intralingual miRSOD1 treatment restored respiratory function to wild‐type levels (potentially via increasing upper airway patency) and is worthy of further exploration as a possible therapy to preserve respiratory capacity in ALS patients. Future directions include studying the vector genome expression and SOD1 knock down in local and systemic tissues to understand the degree of transduction. In addition, we will measure the impact of intralingual miRSOD1 on swallowing and upper airway function.Support or Funding InformationSupported by NIH R21 NS098131‐02 and K99/R00 HL119606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developmental Nicotine Exposure: Diaphragm Contractility and Neuromuscular Transmission in Neonatal Rats
Authors: DeLucia Claire M. , Whalen Mikayla , Fregosi Ralph F. 
Published: June 2021

Developmental nicotine exposure (DNE) results in chronic desensitization of nicotinic acetylcholine receptors (nAChRs) in the brain. Due to the presence of nAChRs at the neuromuscular junction, we investigated whether DNE alters the fatigability and/or neuromuscular transmission (NT) in neonatal rat diaphragm. Neonatal rat (P1–P12) phrenic nerve‐hemidiaphragm preparations were excised and secured in a dish perfused with warmed (37°C), oxygenated Krebs solution. Force production was measured during both supramaximal muscle or phrenic nerve stimulation. Fatigue was induced using five minutes of repeated stimulation (0.2 ms pulse duration) delivered to the muscle or nerve at 40Hz, in 330ms trains every 2 seconds. To quantify NT, repeated trains were applied to the nerve with superimposed muscle stimulation every 15 seconds for five minutes. When collapsed across all postnatal ages, there were no significant differences in NT. In DNE animals, force fell to 51.26 ±3.26% of baseline (n=13), while in unexposed animals force fell to 53.79 ±4.37% of baseline (n=12; p=0.644). Five minutes of either nerve only or muscle only stimulation also resulted in similar rates of force decline in DNE and unexposed control animals. In addition, no differences in contractile properties evoked with supramaximal muscle stimulation were found between DNE and unexposed animals, including peak force, time to peak, or ½ relaxation time. Taken together, our preliminary results suggest DNE does not alter diaphragm neuromuscular transmission, fatigability, or contractility in neonatal rats studied in the first two weeks of life, though early developmental effects cannot be ruled out.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lung epithelial PDIA3 plays a critical role in influenza infection
Authors: Chamberlain Nicolas , Mihavics Bethany , Nakada Emily , Bruno Sierra , Heppner David , Chapman David , Hoffman Sidra , Siddesha Jalahalli , van der Vliet Albert , Suratt Benjamin , Dienz Oliver , Alcorn John , Anathy Vikas 
Published: June 2021

RATIONALEThe influenza A virus (IAV) causes severe respiratory illness in humans. Current treatments are rapidly becoming ineffective due to the emergence of viral resistance to available therapies. Given the impact of IAV on human health and health care costs there is an urgent need to explore novel targets for treatments to control IAV. Previous work has demonstrated infection with IAV induces endoplasmic reticulum (ER) stress, effectuating the unfolded protein response (UPR), a highly conserved cellular stress response that seeks to restore normal ER homeostasis. It has also been shown that interactions with host ER based protein disulfide isomerases (PDIs) are upregulated during the UPR and are required for specific IAV proteins to reach their functional conformations in vitro. The viral proteins hemagglutinin (HA) and neuraminidase (NA) both contain numerous disulfide bonds necessary for their function. Our results demonstrate both HA and NA interact with PDIA3; a chaperone responsible for the catalysis and isomerization of disulfide bonds. However it is unknown whether these host‐viral protein interactions are required during active infection in vivo and whether PDIA3 represents a putative therapeutic target during H1N1 infection.OBJECTIVETo investigate the role of host PDIA3 in the establishment of H1N1 infection and development of subsequent immunopathology.METHODSThe impact of PDIA3 in IAV infection was examined using lung epithelial specific PDIA3 knockout mice and various inhibitors of PDIs in isolated primary mouse tracheal epithelial cells (MTEC).RESULTSWe found that the conditional deletion of PDIA3 in our mouse model resulted in a significant decrease in the number of inflammatory‐immune cells in bronchoalveolar lavage fluid and a marked decrease in IAV‐mediated methacholine induced airway hyperresponsivness, illustrating that PDIA3 plays a significant role in influenza‐induced immunopathology. Interestingly, our data further show that deletion of PDIA3 in vivo affected production of IAV proteins HA and NA. Moreover, We also observed significant alterations in the oxidative folding of HA as well as decreases in IAV induced proinflammatory cytokines in MTECs treated with the PDI inhibitors in vitro.CONCLUSIONSThese data suggest that lung epithelial PDIA3 plays a critical role in the establishment of IAV infection and propagation of the virus, and illustrate the potential of utilizing PDIA3 as a target for future anti‐viral therapies.Support or Funding InformationNIH R01 HL12238 (VA) NIH T32 HL076122 (NC) Department of Pathology and Laboratory Medicine (UVM) Startup Fund (VA)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pannexin 1 and a Venous‐specific Purinergic Cascade Induces Endothelial Leak in Response to TNFα
Authors: Comstra Heather Skye , Begandt Daniela , Molina Samuel , Krüger Nenja , Biwer Lauren , Johnstone Scott R. , Lohman Alexander , Good Miranda E. , DeLalio Leon J. , Bacon Hannah , Isakson Brant E. , Koval Michael 
Published: June 2021

Disruption of the endothelial barrier by pro‐inflammatory cytokines is a complication of multiple diseases, including sepsis that can result in tissue edema. To assess the effect of TNFα on native endothelial permeability, we customized an Ussing chamber to record electrophysiological measurements of ex vivo mesenteric mouse venules or arterioles that were treated with either DMSO vehicle control or 50ng/ml TNFα for 30 minutes. Venules showed a TNFα‐dependent decrease in transendothelial electrical resistance (TEER), whereas arterioles did not. An in situ microvessel perfusion system also demonstrated preferential increases in fluorescein permeability in TNFα treated venules versus arterioles. Human umbilical and saphenous vein (HUVEC and HSaVEC) cells cultured on Transwell permeable supports showed a comparable decrease in TEER in response to TNFα; cultured aortic and coronary arterial (HAoEC and HCoEC) endothelial cells did not. At tight junctions, we found that in addition to claudin‐1 and claudin‐5, venous endothelial cells also expressed claudin‐11. Upon addition of TNFα, venous claudin protein expression was significantly reduced. To investigate molecular mechanisms underlying the differential effect of TNFα on venous and arterial cells, we targeted pannexin 1 (PANX1) channel activity using the antagonists mefloquine and spironolactone in vitro and in venules from conditional PANX1 knockout mice and found that in all cases TNFα‐induced permeability was eliminated. Further analysis of purinergic signaling in venous endothelium revealed that the ectonucleotidase CD39 is expressed at higher levels in veins than arteries and that addition of the CD39‐antagonist ARL67156 abolished TNFα‐induced permeability in venules from wildtype mice. Coupled with expression of CD73, this result suggests that ATP released by PANX1 is metabolized to adenosine as an autocrine signal produced in response to TNFα. Consistent with adenosine signaling, TNFα‐induced permeability was impaired in venules isolated from wildtype mice in the presence of A2A and A2 antagonists as well as in venules from A2A knockout mice. Our results demonstrate that targeting the purinergic signaling pathway mitigates TNFα‐induced permeability in venous cells. These findings offer multiple possibilities for pharmacological interventions aimed at preventing and treating increased vascular permeability during inflammation.Support or Funding InformationR01 AA025854This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
β1Pix Stabilizes Nedd4‐2 and Plays a Critical Role in ENaC Regulation by AMPK in Kidney Epithelial Cells
Authors: Ho Pei‐Yin , Li Hui , Pavlov Tengis S. , Staruschenko Alexander , Hallows Kenneth R. 
Published: June 2021

The epithelial Na+ channel (ENaC) is expressed in many salt‐reabsorbing epithelia at the apical membrane, including in the aldosterone‐sensitive distal nephron of the kidney where it is the final regulator of Na+ balance, blood volume, and blood pressure. Our previous work has established that the metabolic sensor AMP‐activated protein kinase (AMPK) inhibits ENaC by promoting the binding of the ubiquitin ligase Nedd4‐2 to ENaC. Here, we have further demonstrated that functional β1Pix, a Rho‐GEF signaling protein, is required for AMPK‐dependent ENaC inhibition in CHO cells. Epithelial volt‐ohmmeter measurements were performed to examine the role of β1Pix in regulating ENaC‐dependent currents in polarized kidney cortical collecting duct (CCD) cells. Lentiviral shRNA‐mediated knockdown of β1Pix expression in mpkCCDcl1 cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β1Pix dimerization‐deficient mutant unable to bind 14‐3‐3 proteins (Δ602‐611) increased ENaC currents in mpkCCDc14 cells, whereas overexpression of wild‐type β1Pix had an opposite effect. Through additional immunoblotting and co‐immunoprecipitation studies, we explored the role of β1Pix in the regulation of ENaC by AMPK and the interplay of β1Pix, 14‐3‐3 proteins and Nedd4‐2. Treatment with AMPK activators promoted the binding of β1Pix to 14‐3‐3 proteins in CCD cells. However, the association between Nedd4‐2 and 14‐3‐3 proteins was not significantly altered by AMPK activation, β1Pix knockdown, or overexpression of wild‐type or β1Pix‐Δ602–611 mutant in cells. Moreover, β1Pix knockdown or overexpression of β1Pix‐Δ602–611 inhibited Nedd4‐2 protein expression in CCD cells. This inhibition was associated with a parallel reduction in Nedd4‐2 phosphorylation at Ser‐328, a site that is phosphorylated by several kinases including AMPK which is critical for cellular Nedd4‐2 protein stability. Decreased Nedd4‐2 protein stability in the setting of β1Pix knockdown was confirmed by cycloheximide chase studies. Together, these results suggest that functional β1Pix is important for Nedd4‐2 Ser‐328 phosphorylation and thus Nedd4‐2 stability. Overall, our findings suggest that AMPK activation promotes, potentially via AMPK‐dependent β1Pix phosphorylation, the association of β1Pix, 14‐3‐3 proteins, and Nedd4‐2 into a complex that inhibits ENaC by enhancing Nedd4‐2 binding to ENaC followed by ENaC ubiquitination and targeting for degradation. This regulation of ENaC and other epithelial transport proteins by AMPK may have a pro‐survival role by inhibiting energy‐consuming ion transport processes during conditions of ischemia and other cellular stresses that occur with acute kidney injury.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Piezo1 Regulation of Lung Endothelial Barrier Function in Left Heart Failure
Authors: Friedrich Emily , Hong Zhigang , Zhong Ming , Rehman Jalees , Komarova Yulia , Malik Asrar B. 
Published: June 2021

Pulmonary edema is a potentially life‐threatening condition in which interstitial and alveolar spaces fill with fluid. This can be the result of left heart failure and a rise in lung capillary pressure. Piezo1 is a mechanosensitive ion channel expressed in endothelium that is activated by changes in plasma membrane curvature due to high pressure. Here, we address the role of Piezo1 in sensing increased lung capillary pressure that leads to endothelial barrier disruption through disassembly of Vascular Endothelial (VE)‐cadherin (VE‐cad) adhesion at adherens junctions (AJ). We showed that pulmonary capillary pressure rise of 16 cmH2O elicited increased permeability to liquid and albumin in lungs of Piezo1+/+ mice and these responses were prevented in mice deleted of Piezo1 gene in endothelial cells (Piezo1iΔEC). Piezo1iΔEC mice demonstrated no significant change in lung vascular albumin permeability at 24h after aorta constriction, which causes capillary pressure and left heart failure, in contrast to Piezo1+/+ mice, which demonstrated 4‐fold increase in permeability. Consistent with increased lung capillary permeability, activation of Piezo1‐mediated signaling increased VE‐cadherin phosphorylation at both Y685 and Y731 that coincided with VE‐cad internalization and AJ disassembly. Thus, Piezo1‐activated signaling in endothelial cells causes endothelial barrier disruption. These results uncover endothelial cell expressed Piezo1 as a new drug target for treatment pulmonary edema associated with left heart failure.Support or Funding InformationThis work was supported by National Institutes of Health Grants T32 grant and HL45638This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hydrogen Sulfide inhibits store‐operated calcium influx by selectively targeting STIM1‐Orai3 interactions.
Authors: Fresquez Adriana Maria , White Carl 
Published: June 2021

Hydrogen sulfide (H2S) is an endogenous signaling molecule that posttranslationally modifies cysteine residues on target proteins involved in regulating the physiology and pathophysiology of neurodegeneration, blood pressure, metabolism, and inflammation. We recently determined that H2S impinges on Ca2+ signaling‐regulated processes by inhibiting store operated Ca2+ entry (SOCE). The SOCE machinery consists of the plasma membrane‐localized Orai channels and ER‐localized STIM proteins. Interestingly, H2S inhibits SOCE mediated by STIM1‐Orai3 and not STIM1‐Orai1 or Orai2 interactions. The current study tested the hypothesis that H2S achieves selectively and disrupts STIM1‐Orai3 interactions by targeting cysteines unique to Orai3. The approach was to transiently transfect either CFP‐ or mCherry‐tagged Orai3 into HEK293 cells stably expressing STIM1‐YFP and measure SOCE by Ca2+ imaging. Two cysteines (C226 and C232) are present in Orai3 that are absent in the Orai1 and Orai2 sequences. Mutation of either of these cysteines to serine, alone or in combination, abolished SOCE inhibition by the H2S donor GYY4137. Residues C226 and C232 in Orai3 are predicted to form an extracellular intermolecular disulfide. To determine the requirement for disulfide formation, cells were pretreated with DTT. When performed prior to GYY4137 treatment, disulfide reduction also abolished inhibition of SOCE by GYY4137. SOCE is activated upon ER Ca2+ store depletion when STIM interacts with and gates open Orai channels. We postulated that H2S inhibited SOCE by inhibiting STIM1‐Orai3 interactions. To test this, cells were treated with GYY4137 both before and after store depletion. GYY4137 treatment decreased SOCE only when cells were pretreated with GYY4137. These data suggest that H2S modification of Orai3 inhibits the initiation of STIM1‐Orai3 interactions but cannot disrupt them once formed. We next visualized the interaction between STIM1‐YFP and Orai3‐mCherry using confocal microscopy and measured the rate of YFP‐mCherry puncta formation upon store depletion. Pre‐treatment with GYY4137 caused a decrease in puncta formation rate. Taken together, these data are consistent with a model in which H2S impairs SOCE by modifying a disulfide bridge on Orai3 resulting in decreased STIM1‐Orai3 interactions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.