bluuebunny/crossref_metadata_2025 huggingface dataset:
The myocardial inflammasome and heart failure with reduced ejection fraction
Authors: MacLaren Janna , Freeburg Lisa , Samani Stephanie , Churillo Amelia , Barlow Shayne , Jones Traci , Shazly Tarek , Spinale Francis
Published: May 2023

Background: Left ventricular (LV) injury, particularly myocardial infarction (MI), is a leading cause of heart failure with reduced ejection fraction (HFrEF). However, whether and to what degree activation of the innate immune system, such as the NOD-LRR pyrin-containing protein 3 (NLRP3) inflammasome, occurs post-MI and the development of HFrEF remains unclear. This study tested the central hypothesis that LV myocardial induction of NLRP3 occurs and causes downstream activation of proinflammatory cytokines, notably interleukin-18 (IL-18). Methods and Results Pigs (n=6, 25kg) underwent an intracoronary occlusion (90 mins) followed by reperfusion and LV function (echocardiography) and myocardial sampling performed at 28 days post-MI. Age and weight matched pigs (n=6) served as controls. All results are reported as mean ± SEM. LV ejection fraction (EF) decreased (64.2 ± 5.1 vs. 44.6 ± 3.8 %, p<0.05) and end diastolic volume (EDV) increased (49.4 ± 1.2 vs.68.5 ± 11.6 mL, p<0.05) - consistent with the HFrEF phenotype. LV myocardial NLRP3 expression (rtPCR, 1.14 ± 0.28 vs. 61.55 ± 26.55 fold change, p<0.05), protein levels (digital immunoblot, 1271 ± 646 vs. 9177 ± 2491 pixels, p<0.05), and activity (caspase-1;YVAD-FAC substrate; 147 ± 122 vs.398 ± 58 slope of the fluorescence/time relation, p<0.05) were all increased. These results identified that NLRP3 at the mRNA level yielded functional inflammasome activation, and IL-18 mRNA levels (rtPCR) increased concordantly (1.05 ± 0.17 vs. 16.65 ± 7.20 fold change, p<0.05). Conclusions: While exogenous inflammatory pathways have been identified post-MI and with HFrEF, the unique findings of this study were clear demonstration that the innate immune system, the inflammasome, is also induced with a potent downstream induction of IL-18. These findings suggest that the inflammasome, notably NLRP3, as a potential therapeutic target in the context of ischemic HFrEF.

This work was supported in part by National Institutes of Health grant R01HL130972-01A1 (F.G.S.) & R01HL5949 (F.G.S.) as well as a Merit Award from the Veterans Health Administration, BX000168-10A1 (F.G.S.) & BX005320 (F.G.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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.
Acute and reversible modulation of kidney proximal tubule endocytic capacity by fluid shear stress
Authors: Lackner Emily , Shipman Katherine , Long Kimberly , Cowan Isabella , Weisz Ora
Published: May 2023

The proximal tubule (PT) of the kidney is comprised of cells that are uniquely specialized for efficient apical uptake of albumin and other proteins that escape the glomerular filtration barrier. These proteins bind to the multiligand receptors megalin and cubilin and are internalized via clathrin-dependent endocytosis into apical early endosomes that rapidly mature into larger apical vacuoles (AVs). Dissociated ligands are delivered from AVs to lysosomes for degradation, while receptors are collected into dense apical tubules for recycling to the cell surface. When cells are grown under continuous fluid shear stress (FSS), the endocytic uptake is approximately 9-fold greater than static cells. Additionally, cells cultured in this manner rapidly and reversibly adjust their endocytic capacity in response to changes in FSS. We hypothesize flow-dependent modulation of endocytic capacity enables PT cells in vivo to preserve uptake efficiency in response to changes in glomerular filtration. We are combining biochemical, imaging, and mathematical modeling approaches to determine the mechanism(s) by which cells chronically and acutely modulate endocytic uptake. Increased levels of megalin transcripts (~2x) and protein (~4x) is insufficient to account for the large increase in endocytic uptake observed in cells cultured under FSS vs static conditions. We used cell surface biotinylation experiments to compare the fraction of megalin at the surface, the fraction endocytosed, and the half-life of surface-tagged megalin under FSS and static conditions. These data were used to adapt a kinetic model that describes kinetics of megalin biosynthesis, trafficking, and degradation in cells. Overall, the cells grown under FSS have a greater endocytic rate and recycling rate compared with static grown cells. By contrast, megalin degradation is faster than recycling in static-grown cells. Colocalization of megalin with known markers of apical compartments will enable us to extend our model to determine whether changes in endosomal maturation rates contribute to these differences. Additionally, cells cultured under continuous FSS rapidly and reversibly adjust their endocytic capacity in response to changes in shear stress. We hypothesize flow-dependent modulation of endocytic capacity enables PT cells in vivo to preserve uptake efficiency in response to changes in glomerular filtration. We are currently adapting our model to determine how acute changes in shear stress trigger alterations in endocytic uptake.

National Institute of Health: 5T32GM133353-03, R01 DK118726, R01 DK125049

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.
Inflammatory response to radiation exposure of immune cells
Authors: Didier Kaylin , Sanchez Sophie , Forsberg Matthew , Shahi Ankita , Wannebo Brett , Capitini Christian , Schrage William
Published: May 2023

Background: The purpose of this study was to determine the effect of radiation on the inflammatory signaling response of immune cells. Radiation exposure can increase the risk of developing cardiovascular disease (CVD), which is likely initiated as an inflammatory process. In turn, inflammation initiates atherosclerosis via endothelial cell damage and subsequent immune cell infiltration. We hypothesized that CD14+ immune cells will exhibit an amplified inflammatory response, as demonstrated by larger increases in inflammatory cytokine expression 24 hours post radiation exposure. Methods: Human venous blood was collected from 6 healthy young adults (28 ± 5 years, 22.4 ± 0.7 BMI, 3 Male) using an institutional review board (IRB)-approved protocol. Women were studied in days 1-5 of menstrual cycle. Peripheral blood mononuclear cells (PBMCs) were harvested by density gradient separation. PBMCs were re-suspended and incubated with anti-human CD14 microbeads for 15 minutes at 4°C. After washing unbound antibody, cell separation was done using an autoMACS Pro Separator. CD14+ immune cells were incubated in plates, exposed to 4 gray (Gy) of x-ray radiation, and then incubated for 24 hours. CD14+ immune cells were exposed to 20ng/ml of TNF-α for 30 mins. The culture media was recovered, centrifuged at 100 × g for 1 minute to remove any floating cell debris and assayed for secreted factors, specifically for IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, INF-γ, and TNF-α. Cytokine responses to radiation were analyzed by paired t-tests. Differences in sex were analyzed by two-way repeated measures ANOVA (Condition x Sex). Results: Radiation exposure (4 Gy) did not change the levels of IL-1β (p=0.15), IL-2 (p=0.13), IL-4 (p=0.16), IL-6 (p=0.18), IL-8 (p=0.35), IL-10 (p=0.18), IL-12p70 (p=0.15), IL-13 (p=0.27), INF-γ (p=0.12), and TNF-α (p=0.14). TNF-α stimulated expression levels of IL-1β (p=0.15), IL-2 (p=0.16), IL-4 (p=0.36), IL-6 (p=0.72), IL-8 (p=0.89), IL-10 (p=0.87), IL-12p70 (p=0.43), IL-13 (p=0.24), and INF-γ (p=0.21) were not different post radiation. There was no sex difference in inflammatory cytokine expression post radiation (p>0.2) or TNF-α stimulation post radiation (p>0.1). Conclusion: These data suggest that expression of inflammatory signals of CD14+ immune cells are not different after exposure to acute radiation (4 Gy x-ray). Further studies are needed to investigate whether increased exposure (duration and/or dose) to radiation could lead to increases in inflammatory cytokines in the adaptive immune response.

Translational Research Institute for Space Health through NASA Cooperative Agreement NNX16AO69A and NIH R01 HL150361

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.
Inflammatory cytokine elaboration following secondhand smoke exposure is mediated in part by RAGE signaling
Authors: Curtis Katrina , Homer Kyle , Wendt Ryan , Chang Ashley , Van Ry Pam , Arroyo Juan , Reynolds Paul
Published: May 2023

The receptor for advanced glycation end products (RAGE) is a key contributor to the immune and inflammatory response in a myriad of diseases. RAGE is a transmembrane pattern recognition receptor with special interest in pulmonary anomalies due to its naturally abundant expression in the lungs. Our previous studies demonstrated a role for RAGE in inflammation following acute exposure to secondhand smoke (SHS). However, chronic inflammatory mechanisms associated with RAGE have yet to be fully elucidated. In this study, we address the impact of long-term SHS exposure on RAGE signaling. RAGE knockout (RKO) and wild type (WT) mice were exposed to SHS five times weekly via a nose-only delivery system (Scireq Scientific, Montreal, Canada) for six months. SHS animals were compared to mice exposed to room air only. Immunoblot and colorimetric high throughput FACE assays (Active Motif) were used to assess phospho-AKT and NF-κB, respectively. A mouse cytokine antibody array (Abcam) was used to screen secreted cytokines in bronchoalveolar lavage fluid (BALF). Phospho-AKT was decreased and NF-κB was elevated in both groups of SHS exposed mice, with RKO+SHS mice demonstrating tempered outcomes for both intermediates compared to WT+SHS exposed mice. BALF contained increased levels of pro-inflammatory cytokines including IFNγ, IL-13, MIP-1γ and Eotaxin1 in exposed groups and diminished secretion was observed in exposed RKO mice. These results validate a role for RAGE in the mediation of chronic pulmonary inflammatory responses and suggest AKT signaling as a viable pathway of RAGE dependent inflammatory responses. Additional characterization of RAGE-mediated pulmonary responses to prolonged exposure will provide valuable insight into cellular mechanisms of lung diseases such as chronic obstructive pulmonary disease.

This work was supported by funding from the National Institutes of Health (NIH 1R15-HL152257, PRR and JAA).

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.
Stellate Ganglion Sympathetic postganglionic efferent innervation in the flat-mount of the whole heart of rats: Anterograde Tracing
Authors: Chen Jin , Bendowski Kohlton , Madas Jazune , Zhang Yuanyuan , Cheng Zixi
Published: May 2023

The autonomic nervous system (sympathetic and parasympathetic) plays an essential role in controlling cardiac functions. Previously, we used anterograde tracing to examine the vagal afferent and efferent (preganglionic) innervations of the normal heart, their anatomical remodeling and their functional changes in disease models of rats and mice. In addition, we used vesicular acetylcholine transporter (VAChT, a parasympathetic marker;) and tyrosine hydroxylase (TH, a sympathetic marker,) to determine the parasympathetic and sympathetic postganglionic innervation of the whole heart (atria and ventricles). Immunohistochemical labeling with TH is useful for determining the broad distribution of sympathetic efferent axons in the heart. However, it is difficult to distinguish its extrinsic and intrinsic origins using TH labeling. Furthermore, the topographical organization and morphological terminal structures of cardiac sympathetic postganglionic efferent axons from the stellate ganglia (SG) remains unknown. To address this we anterogradely labeled individual sympathetic postganglionic axons projecting to the heart, using tracer Dextran-Biotin microinjections into the right SG of Sprague-Dawley rats (male, n=6, 3-6 months). Fourteen days after the injection, animals were euthanized, and their hearts were dissected into the left/right atria (LA and RA) and ventricles and prepared as flat-mounts. The tracer-labeled axons were digitized and analyzed using The Neurolucida 3D tracing system and imaged using a Zeiss M2 Imager. We found that stellate sympathetic neurons had projections to the LA and RA and left and right ventricles. Individual sympathetic axons formed complex arbors of varicose neurites in the areas of the sinoatrial node (SAN) and atrioventricular node (AVN) as well as the other areas of the atria and throughout the ventricles. Sympathetic axons also innervated blood vessels. Using the Neurolucida Digitization and Tracer system, we determined the regional density and characterized different types of axons. Some axons formed relatively simple terminal structures with only a few branches, whereas other axons produced very complex terminals with extensive branching. For the first time, we anterogradely labeled the sympathetic postganglionic axons and characterized their terminal architecture in the whole heart. These results will provide an anatomical foundation for future study

This study was supported by NIH HEAL/SPARC U01 NS113867-01 and NIH R15HL137143-01A1.

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.
Chronic binge alcohol alters hepatic expression of metabolic genes in SIV-infected female rhesus macaques
Authors: Gallegos Eden , Molina Patricia , Simon Liz
Published: May 2023

Background: Alcohol-related liver injury includes steatosis, fibrosis, and inflammation, which can culminate in alcoholic hepatitis and cirrhosis. The pre-clinical alterations in gene expression underlying these pathophysiological changes are ill-defined. Chronic binge alcohol (CBA) consumption produces marked alterations in metabolic processes and our studies show this is accentuated in chronic infection such as in HIV. This study tested the hypothesis that CBA disrupts expression of hepatic genes involved in glycolytic, gluconeogenic, and lipid turnover pathways in rhesus macaques infected with simian immunodeficiency virus (SIV). Methods: Female rhesus macaques were administered CBA (13-14g ethanol/kg/week, n=7) or vehicle (VEH, n=8) starting 3 months prior to SIV infection. All animals were treated with anti-retroviral therapy starting at 2.5 months post-SIV infection and sacrificed at 14.5 months and liver samples obtained for histological analysis, determination of gene expression by qPCR, triglyceride content by colorimetric assay, and pyruvate kinase activity by a colorimetric enzymatic assay. Results: Expression of lipid handling genes including perilipin 1, peroxisome proliferator-activated receptor γ (PPARγ), carbohydrate responsive binding protein (CHREBP), and acetyl-CoA carboxylase B were higher in CBA-treated compared to VEH-treated macaques. Expression of glycolytic (phosphofructokinase) and gluconeogenic (pyruvate carboxylase) enzymes including aldolase A was increased in the CBA group compared to VEH. No changes were observed in expression of the β-oxidation regulatory protein PPARα. Triglyceride content and pyruvate kinase activity were not significantly different between the groups. Conclusions: These results show significant CBA-mediated alteration of mRNA expression of lipid handling genes that could reflect increased lipid turnover despite lack of alterations in hepatic triglyceride content. Future studies will confirm the findings with protein expression of the differentially regulated genes and levels of non-esterified fatty acid in the liver.

Supported by T32AA007577 & P60AA009803.

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.
Increased asprosin in epididymal adipose tissue as a potential contributor to impaired vascular function in obese mice.
Authors: Shah Prachi , Maddie Nicole , Carrillo-Sepulveda Maria Alicia
Published: May 2023

Vascular complications, such as impaired vasodilation, are a major problem that obese individuals face. In obesity, epididymal white adipose tissue (eWAT) expands, undergoes phenotypic changes, and begins to secrete a variety of adipokines that can metabolically regulate other organs and tissues including the cardiovascular system. Human studies report high levels of asprosin, a newly discovered adipokine secreted by WAT, in obesity. In this study, we hypothesize that obesity increases asprosin expression in eWAT which correlates to vascular complications.Eight-week-old male C57BL/6 mice were randomized into two experimental groups. The control group (n=7) received a standard chow diet (5% fat, 48.7% carbohydrates [3.2% sucrose]) and the Western Diet (WD) group (n=11) received a WD (40% fat, 43% carbohydrates [34% sucrose]) for 39 weeks. Obesity was confirmed by increased body weight (45.39 ± 1.98 g vs. 29.21 ± 0.79 g controls, p< 0.05) and increased BMI (0.45 ± 0.12 kg/m2 vs. 0.32 ± 0.01 kg/m2 controls, p< 0.05). At terminal experiments, eWAT was collected for histological and biochemical analysis. As expected, obese mice exhibited a greater amount of eWAT in comparison to the control group (2.69 ± 0.14 mg/g vs. 0.58 ± 0.06 mg/g controls, p< 0.05). Histological analysis of eWAT stained with H&E revealed that obesity induced a significant increase in adipocyte's size (10543.47 ± 694.06 μm2 vs.1726.64 ± 152.20 μm2 controls, p< 0.05) and infiltration of inflammatory cells surrounding adipocytes. Strikingly, greater asprosin expression was detected in eWAT from WD group in comparison to the control group (1.3 fold of increase, p<0.005). To assess whether asprosin has vascular effects, mesenteric arteries from control C57BL/6 mice were acutely incubated with pharmacological concentration of asprosin followed by vascular studies assessed by wire myograph. Our preliminary results show that asprosin impairs endothelium-dependent vasodilation (Emax:72.95% ± 7.38 vs. 93.33% ± 1.33 controls, n=3).The novelty of our results show that asprosin has a negative effect in vascular function. Our findings imply that eWAT may be a tissue contributing to the high levels of asprosin in obese individuals, further suggesting asprosin as a culprit of obesity-associated vascular dysfunction in obese males.

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.
Hypothalamic PVN astrocytes fail to properly set glutamate tone in obese mice contributing to impaired glucose metabolism in this condition
Authors: Kirchner Matthew , Herrera Moro Chao Daniela , Pham Cuong , Li Dongdong , Martin Claire , Luquet Serge , Stern Javier
Published: May 2023

The hypothalamus is key in the control of energy balance, but strategies targeting hypothalamic neurons have failed to provide viable options to treat most metabolic diseases. Conversely, the role of astrocytes in systemic metabolic control has remained largely unexplored. Here, we show that obesity promotes anatomically restricted exacerbated Ca2+ activity in hypothalamic paraventricular nucleus (PVN) astrocytes. Bidirectional chemogenetic manipulation of astrocyte Ca2+ activity in lean mice stimulated or inhibited the activity of neighboring parvocellular neurons. Importantly, these effects translated into bidirectional regulation of sympathetic activity, glucose metabolism, and energy balance. We found that the astrocyte-mediated effects on PVN neurons were blocked by the glutamate receptor blocker kynurenic acid, and the astrocyte glutamate transport blocker TBOA, suggesting that the astrocyte-neuron signaling and the effects on glucose metabolism involved the astrocytic control of ambient glutamate levels. Notably, we found that TBOA alone stimulated neurons in lean mice but failed to evoke a response in obese mice. This suggests that the astrocyte glutamate transporter activity is impaired in obese mice. Finally, we found that chemogenetic inhibition of astrocyte Ca2+ activity in obese mice improved glucose metabolism in this condition. Together, our results indicate that exacerbated astrocyte Ca2+ activity leads to impaired glutamate transporter activity, standing as a likely mechanism contributing to altered PVN neuronal activity and glucose homeostasis during this condition. Collectively, these findings highlight a yet unappreciated role for astrocytes in the direct control of systemic metabolism and suggest potential targets for anti-obesity strategy.

NHLBI F32 HL158172 (MKK) NHLBI HL090948 (JES) NINDS NS094640 (JES)

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.
The Proliferative and Functional Roles of CEBP/A in Ins-1 832/13 Cells and Primary Rat Islets
Authors: Ellsworth Peter , Herring Jacob , Elison Weston , Leifer Aaron
Published: May 2023

The beta cell transcription factor Nkx6.1, when overexpressed in primary rodent and human islets, is sufficient to enhance beta cell proliferation, increase insulin secretion and enhance cell survival. We have sought to define the transcriptional targets of Nkx6.1 that allow for its ability to increase functional beta cell mass. We have shown that Nkx6.1 induces expression of the transcription factor CEBP/A. CEBP/A overexpression is sufficient to induce proliferation of Ins-1 832/13 cells and primary rat islets. CEBP/A overexpression enhances glucose stimulated insulin secretion from primary rat islets, while decreasing total insulin content. Finally, CEBPa overexpression protects Ins-1 832/13 cells from thapsigargin and glucolipotoxicity induced cell death but fails to protect against etoposide and camptothecin induced cell death. These data suggest that CEBP/A may play a critical role in Nkx6.1 mediated expansion of functional beta cell mass through a endoplasmic reticulum stress-mediated pathway.

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.
The impact of aging on pulmonary microvascular endothelial cell barrier function
Authors: Manji Aminmohamed , Mehta Sanjay , Wang Lefeng , Pape Cynthia , Gill Sean
Published: May 2023

Elderly individuals have substantially elevated morbidity and mortality during conditions of lung injury. However, the precise underlying mechanisms leading to this elevated mortality are ill-defined. Lung injury is associated with damage to pulmonary microvascular endothelial cells (PMVEC) leading to a compromised vascular barrier, fluid and protein leakage into the tissue, and subsequent respiratory dysfunction. Cell-cell junctions, including adherens junctions and tight junctions, are crucial for the maintenance of the endothelial barrier and have been shown to be dysregulated in various disease models. Pilot data from our lab demonstrated increased pulmonary vascular leak in aged mice during lung injury compared to young mice. We hypothesized that aging contributes to PMVEC barrier dysfunction due to impaired cell-cell junction integrity.To address our hypothesis, PMVEC isolated from young and aged mice were cultured in vitro until a confluent monolayer was formed. Endothelial barrier integrity was assessed by Evans blue-labelled albumin flux across the monolayers, as well as immunofluorescence staining of the adherens junction protein, vascular endothelial (VE)-cadherin, and tight junction protein, claudin-5. Localization of leak in relation to junctional proteins was performed using the fluorescently labelled macromolecule, NeutrAvidin. Proteomics analysis was conducted to identify differentially enriched pathways in young and aged PMVEC.Endothelial monolayers from aged mice exhibited a significant increase in albumin leak associated with augmented VE-cadherin and claudin-5 disruption vs. endothelial monolayers form young mice. Furthermore, NeutrAvidin staining localized to paracellular regions with VE-cadherin discontinuity was increased in PMVEC monolayers from aged mice vs. young mice, suggesting that the junctional disruption in aged PMVEC was directly associated with leak. Proteomics analysis revealed pathways known to be implicated in endothelial barrier dysfunction, including regulation of oxidoreduction and protein processing and trafficking, that were altered in PMVEC from aged mice.In summary, PMVEC from aged mice exhibit compromised barrier function, which is directly associated with impaired cell-cell junction integrity. We will next validate and target protein pathways identified in our proteomics analysis that were dysregulated in aged PMVEC to potentially rescue age-induced barrier dysfunction. These findings may highlight molecular pathways involved in predisposing aged individuals to worsened outcomes during lung injury, which can help in the development of therapeutics.

Funded by Lawson Health Research Institute, CIHR, and Western University

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.
Chronic Pelvic Pain Develops in Sensory Neuron Conditional Asic3 Null Mice Subjected to Chemical Injury
Authors: Montalbetti Nicolas , Dalghi Marianela , Apodaca Gerard , Carattino Marcelo
Published: May 2023

Sensitization of primary afferents is an important underlying mechanism for visceral hypersensitivity and pain. We investigated the contribution of acid-sensing ion channels (ASICs) in sensory neurons to the development of pain in a model of chemical-induced cystitis. ASICs are assembled as heterotrimers in bladder sensory neurons; however, the deletion of the ASIC3 subunit results in a loss-of-function phenotype with sensory neurons unable to discharge action potentials in response to acidification. In this study, conditional sensory neuron Asic3 knockout (KO) mice ( Asic3fl/fl;Avil-Cre+/- ) and control littermates ( Asic3fl/fl;Avil-Cre-/- ) received cyclophosphamide (CYP) (IP 80 mg/kg), or saline, every other day for five days. Experimental observations were made after one day (acute) or 14 days (chronic) after the last dose of CYP. In the acute setting, both control and conditional Asic3 KO mice treated with CYP exhibited an irritated bladder phenotype with a high number of voiding events of small volume. However, voiding activity normalized in both groups within two weeks of receiving CYP. In contrast, conditional Asic3 KO mice treated with CYP developed pelvic allodynia that persisted for at least two weeks, whereas control mice had no pain phenotype. In the chronic setting, no apparent edema or inflammatory cells were observed in bladders of control or conditional Asic3 KO mice, indicating that the pelvic allodynia seen in conditional Asic3 KO mice treated with CYP is likely driven by abnormal functioning of the nervous system and not by inflammation. To assess whether, in the chronic setting, the referred pelvic allodynia seen in conditional Asic3 KO mice treated with CYP is caused by sensitized primary afferents, we examined the firing evoked by sustained suprathreshold electrical stimulation of acutely isolated bladder sensory neurons. Sensory neurons were classified based of the sensitivity of the action potential to tetrodotoxin (TTX), as TTX-resistant (TTX-R) or TTX-sensitive (TTX-S). Strikingly, ~ 40% (11/17) of the bladder sensory neurons with TTX-R action potentials from conditional Asic3 KO mice treated with CYP exhibited aberrant firing (i.e., sensitization) in response to suprathreshold stimulation, compared to 3% (1/33) in control mice. These findings indicate that the pelvic allodynia seen in conditional Asic3 KO mice is driven in part by sensitized bladder afferents. Taken together, our studies support the notion that ASICs operate at the nerve terminals to modulate nociceptor excitability and sensitization.

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.
Role of glycocalyx in breast cancer cell adhesion and transmigration across an in vitro blood-brain barrier (BBB)
Authors: Fu Bingmei , Li Yunfei
Published: May 2023

On the surface of every mammalian cell, there is a matrix-like glycocalyx (GCX) consisting of proteoglycans and glycosaminoglycans (GAGs). Disruption of endothelial cell (EC) GCX by vascular endothelial growth factor (VEGF, VEGF-A165), a tumor secretion, was found to be an early event in tumor cell (TC) metastasis across vascular barriers. But how the TC secretion VEGF affects its own GCX to affect its adhesion and transmigration across EC barriers is unknown. Our recent study by employing Super-resolution Optical Microscopy (STORM) showed that while 1 nM VEGF greatly reduces the length and coverage of heparan sulfate (HS) of GCX at an in vitro BBB formed by human cerebral microvascular endothelial cells (hCMEC), it significantly enhances the coverage of HS on a malignant breast cancer cell, MDA-MB-231 (MB231). In the current study, we want to test the hypothesis that the differential effects of VEGF on the GCX of MB231 and that of the BBB favor MB231 adhesion and transmigration across the BBB for the metastasis. To test this hypothesis, we first generated an in vitro BBB by culturing hCMECs on a Transwell filter with 8 um pores. For adhesion, we added MB231 cells to the BBB under 1) no treatment; 2) after VEGF treatment for MB231 for 2h; 2) after VEGF treatment for the BBB for 2h; and 4) after VEGF treatment for both for 2h. After 1h adhesion, the number of adherent MB231 cells increased by 1.7-fold,1.5-fold and 2.4-fold, respectively for 2), 3) and 4) VEGF treatments, compared to no treatment. For transmigration, we add MB231 cells to the BBB under the same conditions as for adhesion. After 6 h, the number of transmigrated MB231 cells increased by 1.3-fold, 1.4-fold, and 1.6-fold, respectively, for 2), 3) and 4) VEGF treatments, compared to no treatment. Our results conform to our hypothesis that the reduced BBB GCX and enhanced MB231 GCX by VEGF favor MB231 adhesion to and transmigration across the BBB. Our findings also suggest a therapeutic intervention by targeting the glycocalyx-mediated breast cancer brain metastasis.

Partially supported by NIH RO1NS101362 and 1UG3UH3TR002151.

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.
Effects of CD8 T-cell transfer on post-myocardial infarction scar formation
Authors: Learmonth Maya , Corker Alexa , Troncoso Miguel , Broughton Philip , Baicu Catalin , Bradshaw Amy , Zile Michael , DeLeon-Pennell Kristine
Published: May 2023

Post-myocardial infarction (MI) left ventricular scar formation and composition is one critical determinant of myocardial remodeling. CD8+ T-cells are adverse regulators of the post-MI remodeling process, leading to decreased cardiac function and survival. Based on our previous studies, we hypothesize that CD8+ T-cells impair cardiac function by altering scar composition, leading to poor tissue biomechanical properties.MI was induced by permanently ligating the left anterior descending coronary artery on C57BL6/J wildtype (WT; 3-7 months of age, n≥4/sex/Day post-MI) and CD8atm1mak (CD8-/-; 3-7 months of age, n≥4/sex/Day post-MI) mice. To test potential effects on the scar, CD8-/- mice were injected with either vehicle or naïve splenic CD8+ T-cells via tail vein, 4 hours post-coronary artery ligation. At day 7 post-MI, infarct tissue was collected and underwent passive stretch biomechanical analysis using an Aurora Scientific tension-length dual mode lever servosystem. To examine a possible mechanism of scar alteration, we tested the effects of recombinant granzymes (Gzm) B and K, which are highly expressed in CD8+ T-cells, on collagen cleavage using a fluorogenic collagen cleavage assay.Scar biomechanics of CD8-/- mice demonstrated increased regional stiffness compared to WT mice. Re-supplementation with splenic CD8+ T-cells decreased scar tissue stiffness by more than 2-fold compared to vehicle treated CD8-/- mice, mimicking the biomechanics of WT mice. Ex-vivo GzmK demonstrated the ability to cleave collagen in a concentration and temporal-dependent manner. Cleavage of the collagen substrate was detected within a 6-hour period at GzmK concentrations as low as 50 nanomolar (nM), and as early as 2-hours at 100 nM. GzmK at a concentration of 100 nM demonstrated a 10-fold greater collagen cleavage capacity compared to GzmB at the same concentration suggesting that GzmK actions may be distinctly separate from that of GzmB in the post-MI setting.In conclusion, our data demonstrates that CD8+ T-cells regulate cardiac fibrosis likely through release of granzymes, leading to alterations in the biomechanical capability of the left ventricle.

This project was funded by the National Institutes of Health T32GM123055, GM113278; the American Heart Association Innovator Project IPA35260039; the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Award IK2BX003922; and South Carolina Translational Research Center UL1TR001450.

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.
Impact of increasing insulin resistance on cerebral perfusion in adolescents
Authors: Wannebo Brett , Ward Aaron , Bolin Shawn , Eldridge Marlowe , Wieben Oliver , Carrel Aaron , Hermann Bruce , Jones Jana , Schrage William
Published: May 2023

Background: Adolescence is a key growth period that heavily influences future brain health. Approximately 1 in 5 adolescents are obese in the US, and up to half of them exhibit some form of insulin resistance (IR). Notably, insulin acts as a metabolic hormone, a neurotrophic growth factor, and a vasodilator. Reduced vasodilation may lead to reduced cerebral perfusion, and lower perfusion in older adults is associated with reduced cognition and increased risk of stroke and neurologic disease. Given this background, IR may negatively impact adolescent brain health in part due to lower brain perfusion. Therefore, we hypothesized cerebral perfusion would decrease as the level of insulin resistance increased. Method s: Adolescents (n=15, 4 females, age 15±2 yr) were studied. IR was estimated by calculating HOMA-IR (using fasting glucose and insulin), which spanned a spectrum of IR up to but excluding diabetes. Subjects underwent a magnetic resonance imaging (MRI) study visit to assess cerebral perfusion using arterial spin labeling (ASL). The CAT12 toolbox extension for SPM12 was used to process MRI data. Heart rate (HR), mean arterial pressure (MAP), and end tidal CO2 (ETCO2) were assessed. Significance was determined using linear regression and set at p≤0.05. Results: Results are mean±SD. HR (72±10 bpm, p=0.98), MAP (73±6 mmHg, p=0.67), and ETCO2 (37±2 mmHg, p=0.91) were similar across HOMA-IR (0.5 to 3.8). Perfusion (ml/100g/min) at the amygdala (r2=0.33, p=0.02) and parahippocampal gyrus (r2=0.28, p=0.04) decreased as HOMA-IR increased. Borderline significant decreases were found for total gray matter (r2=0.26, p=0.051), anterior cingulate cortex (r2=0.26, p=0.052), and hippocampus (r2=0.23, p=0.07) perfusion as HOMA-IR increased. No associations were found between HOMA-IR and the following: total white matter (r2=0.18, p=0.11), precuneus (r2=0.20, p=0.10), thalamus proper (r2=0.14, p=0.17), and entorhinal cortex (r2=0.16, p=0.14) perfusion. Total gray matter (p=0.49) and white matter (p=0.73) volume had no significant relationship with HOMA-IR. HOMA-IR was not associated with any differences in regional volumes. Conclusions: Our results suggest with increasing HOMA-IR there is a significant reduction in gray matter perfusion in key brain regions associated with memory in adolescents. In contrast, structural changes were not detected, indicating functional decrements occur prior to structural deficits. These findings support our hypothesis that cerebral perfusion is associated with insulin resistance in adolescents, which may negatively impact long-term brain health.

NIH R21 HD097510

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.
Inflammation modulates the activation of autophagy in C2C12 myotubes
Authors: Rogers Robert , Howe Parker , Hayes Annemarie , Yeung Justin , Staudinger Jeffrey
Published: May 2023

Autophagy is a cellular housekeeping process by which proteins and damaged organelles are targeted for engulfment by autophagosomes ultimately culminating in degradation within the lysosomes. Chronic inflammation has been shown to disrupt normal physiological homeostasis leading to increased risk of cardiovascular disease, type 2 diabetes mellitus and muscle atrophy. The purpose of this study was to investigate whether inflammation disrupts the activation of autophagy in C2C12 myotubes. Myoblasts were grown in 10% fetal bovine serum supplemented DMEM until confluent and then differentiated for 5 d in 2% FBS-DMEM. Autophagy was monitored by Western blotting for changes in p62/sequestosome 1 (p62/sqstm1) and in the lipidation status of microtubule-associated protein light chain 3 (LC3) where the protein separates into LC3-II and LC3-I by electrophoresis. Changes in the ratio of LC3-II to LC3-I represent a method to monitor initiation of autophagy. Rapamycin (25-50 nM) for 48 hr successfully initiated autophagy as observed by a decrease in p62/sqstm1 and increase in LC3-II/LC3-I. Incubation of cells with lipopolysaccharide (LPS) to activate the inflammatory process for 48 hr reduced cell diameter and this decrease was partially attenuated by rapamycin (50 nM). LPS disrupted the activation of autophagy by rapamycin. Exposure to LPS for 48 hr increased p62/sqstm1 levels, but this was abolished with co-incubation of LPS and rapamycin; however, LC3-II/I was enhanced by co-incubation with LPS and rapamycin. To further investigate the components of the inflammatory process, cells were exposed to interleukin-6 (IL-6). Acute IL-6 exposure for 3 hr at 10 ng/mL or 50 ng/mL modulated basal autophagy as observed by an increase in the ratio LC3-II/LC3-I. In addition, chronic IL-6 exposure disrupted the initiation of autophagy by rapamycin. B-cell lymphoma 2 (Bcl-2) associated athanogene 3 (Bag3) is a molecular chaperone that plays a role in shuttling proteins to the proteasome but also in targeting proteins for degradation through autophagy. We observed that in our experiments Bag3 levels decreased concurrently with rapamycin treatment, but frequently decreased as p62 increased when exposed to LPS, and vice versa. Furthermore, Bag3 co-localizes with LC3 in primary hepatocytes further indicating a role for Bag3 in the targeted autophagy process. In conclusion, autophagy is disrupted by components of the inflammatory process in C2C12 myotubes. Future investigation may include the protective influence of autophagy in the process of atrophy.

Intramural grant award from Kansas City University.

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.
Dietary intake of macro and micronutrients correspond with sleep behaviors in collegiate female soccer players
Authors: Rentz Lauren , Leary Miriam , Bryner Randy
Published: May 2023

Both macro and micronutrient intakes have an impact on sleep behaviors in athletes, notably sleep onset latency and sleep efficiency. Proper nutrient intake supports daily muscle repair and synthesis of sleep regulating compounds (e.g., serotonin and melatonin). The objective of this study was to evaluate the relationship between sleep behaviors and dietary intake in elite college female athletes. It was hypothesized that dietary carbohydrates and vitamin intake would be associated with both sleep timing and consistency. Twenty-three female Division I collegiate soccer players were evaluated for their sleep and nutritional patterns. Athletes wore an OURA ring (OURA Health, Oulu, Finland) each night for 31 days during the competitive season. During the final three days, athletes recorded their dietary intake using the Automated Self-Administered 24-hour Dietary Assessment Tool (ASA24). All athletes were exposed to the same travel schedule throughout the evaluation period, which did not differ by more than one time zone. Pearson’s R with Bonferonni Correction was used to assess the relationship between the average daily intake of various nutrients with average nightly sleep behaviors. Total Vitamin B12 ( r=-0.63), added Vitamin B12 ( r=-0.62), and Vitamin C ( r=-0.53) were negatively correlated with average bedtimes (P<0.01) while added Vitamin B12 ( r=-0.61) and Vitamin C ( r=-0.50) were also negatively associated with wake times (P<0.01). Greater dietary intake of kcals from carbohydrates, with respect to recommended consumption, was associated with earlier bedtimes ( r=0.41, P<0.05). In conclusion, greater intake of carbohydrates and vitamins B12 and C are associated with ideal sleep behaviors. A potential mechanism accounting for this relationship could be the enhanced contribution of antioxidants and glycemic load on the synthesis of vital hormones for sleep regulation, including serotonin and melatonin.

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.
Acute fatigue induces paradoxical increases in velocity and power in a sex and myosin isoform dependent manner
Authors: Ricci Austin , Privett Grace , Needham Karen , Callahan Damien
Published: May 2023

OBJECTIVE or our study was to identify effects of acute fatigue on myofibrillar components and their function that persist beyond modifications to intracellular metabolic perturbations. Our HYPOTHESIS was that acute fatigue would induce differential phosphorylation in regulatory proteins in the sarcomere that would reduce tension and increase velocity and power. We used the following METHODS to test that hypothesis. Seven healthy, competitive athletes were recruited to participate (3 male, 4 female), entailing one session of unilateral fatiguing knee extension exercise to the point of task failure. Immediately after, bilateral percutaneous needle muscle biopsy was performed on the vastus lateralis muscle, providing a “fatigued” and “not fatigued” sample. Tissue was separated for phosphoproteomic analysis (western blot and isoelectric separation) and single fiber contractile mechanics (isotonic load clamps; isometric tension, velocity and power). Following mechanical assessment, myosin heavy chain (MHC) isoform was determined for each fiber via SDS-PAGE. Our RESULTS found no effect of fatigue on isometric tension across all fiber types. However, maximum shortening velocity was increased along with contractile power, consistent with our hypotheses. Surprisingly, our results varied by sex and fiber type, such that velocity was increased primarily in MHC IIA/X fibers in males, whereas females demonstrated a blunted response to fatigue. While baseline variation in regulatory protein phosphorylation (Myosin Binding Protein C; MyBP-C) were significantly correlated with power output in contractile power in fast isoforms (MHC IIA, MHC IIA/X) we did not detect significant alterations in phosphorylation of this protein with fatigue. We CONCLUDE that fatigue causes paradoxical increased in velocity and power that are particularly evident in males. While our measurement of MyBP-C phosphorylation did not detect effects of fatigue, other potential sarcomeric proteins may explain this paradoxical response to acute fatigue.

Wu Tsai Human Performance Alliance

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.