bluuebunny/crossref_metadata_2025 huggingface dataset:
BDNF/TrkB Induces pCREBS133 Phosphorylation and Activates Mitochondrial Biogenesis in Rat Phrenic Motor Neurons
Authors: Davis Anne , Dasgupta Debanjali , Fogarty Matthew , Mantilla Carlos B. , Sieck Gary
Published: May 2024

Neural control of the diaphragm muscle (DIAm) is essential to sustain breathing. During breathing, fatigue resistant DIAm motor units are recruited comprising smaller phrenic motor neurons (PhMNs) and type I and IIa muscle fibers. Larger PhMNs comprise more fatigable DIAm motor units that are infrequently active during expulsive behaviors. The difference in activation history is reflected in the higher mitochondrial volume density in smaller PhMNs. In PhMNs, brain-derived neurotrophic factor (BDNF) signals through its high-affnity receptor, tropomyosin receptor kinase B (TrkB.FL). Previously, we found that intrathecal BDNF treatment induces CREB phosphorylation at Serine 133 (pCREBs133). We hypothesized that pCREBs133 binds to the PGC1a promoter, transcriptionally targeting PGC1a expression, mediating downstream increased expression of NRF1, NRF2, and TFAM and mitochondrial biogenesis. To test this hypothesis, we used a chemosensitive TrkBF616 rat model with a knock-in allele sensitive to 1NMPP1 in that TrkB kinase is inhibited following 1NMPP1, a small molecule derivative of the protein kinase inhibitor protein phosphatase 1 that plays a significant role inhibiting the TrkB phosphorylation and activation. Systemic treatment of 1NMPP1 in TrkBF616 rats inhibits TrkB kinase activity. A bioinformatic analysis was performed to identify a putative binding site for pCREBs133 in the PGC1a promoter sequence in rats. Using a chromatin immunoprecipitation (ChIP) assay we found that pCREBS133 binds to the PGC1a promoter in rat cervical ventral horn. We showed that intrathecal BDNF treatment increased pCREBs133 phosphorylation, which was blocked by 1NMPP1 treatment in TrkBF616 rats. PGC1a, NRF1/2, and TFAM protein was compared in cervical ventral horn lysates from BDNF- and BDNF+1NMPP1-treated TrkBF616 rats by qRT-PCR and Western Blot. Relative mtDNA copy number (normalized to nuclear DNA) was quantified by qPCR to assess the mitochondrial biogenesis. All results were analyzed by paired t-test. Following 1NMPP1-induced inhibition of TrkB kinase, PGC1a, NRF1/2, and TFAM mRNA and protein expression was reduced. Consistent with these findings, mtDNA copy number was decreased following TrkB kinase inhibition by 1NMPP1. We conclude that inhibition of TrkB kinase by 1NMPP1 reduces PGC1a-mediated mitochondrial biogenesis in rat cervical ventral horn.

Supported by NIH grants AG44615 and HL146114.

This is the full abstract presented at the American Physiology Summit 2024 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.
Metabolomic and transcriptomic correlative analyses in germ free mice link probiotic-associated metabolites to host intestinal fatty acid metabolism and b-oxidation
Authors: Ambat Jayanth , Antonio Jayson , Bala Ajitha , Suntornsaratoon Panan , Flores Juan , Li Wei Vivian , Jones Abby
Published: May 2024

While a transcriptomics-led examination of the effects of probiotics offers valuable insights into their influence on host gene regulation, the dynamic interactions between host metabolism and probiotics require an integrated analysis and multiomics approach to provide deeper insight into the underlying molecular mechanisms governing these associations. Here, we developed a high throughput bioinformatic tool ‘metabolome-transcriptome correlation analysis’ (METRCA, in press) that can determine whether a certain gene-metabolite association is up or downregulated and predict whether a metabolite’s or gene’s tissue location impacts its interaction with one another. To test this, we analyzed by LCMS and bulk-RNA sequencing then assessed linkages between serum as well as fecal metabolites and disease- or metabolism-associated genes in the ileum of gnotobiotic mice fed either a tryptophan (trp)-deficient or -suffcient diet then mono-associated with Lactobacillus rhamnosus GG (LGG) or administered PBS (control). We discovered significant associations, mainly in LGG mice fed trp, between metabolites and ileal genes involved in fatty acid metabolism (FAM) and oxidation (FAO). This association was specific and not observed in GF mice gavaged with PBS, monocolonized with R. gnavus, or fed trp- diets, and in SPF mice. Many fecal trp metabolites like indole-3-acetamide and serum trp metabolites like indole acetonitrile were significantly, positively correlated with important intestinal FAM genes Dgat1 and Mgat2. Likewise, numerous fecal metabolites like 5-hydroytrp and several serum trp metabolites like serotonin emerged as positively correlated metabolites with the rate-limiting FAO genes Acaa2 and Cpt2. Serum indoles were more strongly associated with both FAM and FAO genes than fecal indoles. Few fecal and serum metabolites were found negatively correlated with FAM and FAO genes. Since most are positive correlators, these findings suggest a testable hypothesis that LGG- and trp-dependent metabolites increase the intestinal metabolism of dietary lipids, providing a mechanism to previous findings that LGG supplementation in mice protected against diet-induced obesity and fatty liver disease. Analytical approaches like METRCA can thus be used to extract valuable information from metabolite and RNA-Seq datasets, generating novel hypotheses that may discover new interactions between microbe- or nutrient-associated metabolites and host metabolic or disease-related pathways.

NIHR01-AT010243 (NG,RF), R01DK119198 (NG), NSF 1754783 (RF)).

This is the full abstract presented at the American Physiology Summit 2024 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.
A conserved interaction between the GRIP and finger domains in ENaC subunit is critical for proteolytic activation
Authors: Wang Xue-Ping , Humeda Sami , Kashlan Ossama
Published: May 2024

The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the lung and the aldosterone-sensitive distal nephron. ENaC is a heterotrimer comprising ⍺βγ or δβγ subunits. Though sharing similar structural folds as other ENaC/Degenerin family members, ENaC subunits have a unique extracellular GRIP (Gating Release of Inhibition by Proteolysis) domain, and the a and g subunits have polybasic sites that can be cleaved by proteases. Aldosterone promotes double cleavage of the α and γ subunits at these sites, leading to release of the first two b-strands of the GRIP domain (P1 and P2) and channel activation. Structural and sequence conservation suggest that the interaction between P1 and the channel are mediated by a Pro-Leu motif in P1 and a conserved Trp residue in helix α2 of the finger domain. We hypothesized that this interaction is critical for the inhibitory properties of the imbedded tract, defined by P1, and thus channel activation upon release. To test our hypothesis, we substituted the conserved Trp with Ala or Phe in each of the human ENaC subunits and performed two-electrode voltage clamp experiments in Xenopus oocytes to measure the effects on function. Compared to wild type channels, we found that ⍺W251F, βW218A, βW218F, γW229F, and δW396F had no effect on amiloride-sensitive currents. Interestingly, δW396A decreased amiloride-sensitive current by 20% (p=0.01). However, amiloride-sensitive currents were greatly enhanced for Ala mutations in subunits subject to cleavage: ⍺W251A (p=0.02) and γW229A (p=0.01). In line with channel activation, ⍺W251A weakened sensitivity to extracellular Na+ (Na+ self-inhibition), and γW229A completely lost its sensitivity to both extracellular Na+ and chymotrypsin. We also tested the effect of inhibitory peptides (α8 and γ11) corresponding to the imbedded inhibitory tracts (i.e., P1 of the GRIP domain) of the ⍺ and γ subunits. ⍺W251A was insensitive to 10 μM ⍺8 compared to wild type ENaC (85% inhibition), and γW229A was insensitive to 100 μM γ11 compared to wild type ENaC (40% inhibition). Collectively, our results provide evidence for the idea that the interaction between the Pro-Leu motif in GRIP domain’s P1 β-strand and the conserved Trp in the finger domain’s α2 helix is critical for the autoinhibitory properties of P1, and therefore activation upon P1 release after proteolysis of the ⍺ and γ subunits.

R01 DK125439 Kashlan (PI) ENaC Regulation by Biliary Factors.

This is the full abstract presented at the American Physiology Summit 2024 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.
Resting Beat-to-Beat Blood Pressure Variability in Young, Healthy, Non-Hispanic Black Females
Authors: Taherzadeh Ziba , Young Benjamin , Skow Rachel , Stephens Brandi , Fadel Paul
Published: May 2024

Background: Non-Hispanic Black (BL) individuals are at greater risk for cardiovascular disease and hypertension compared to other racial groups. Beat-to-beat blood pressure variability (BPV) is a known predictor of adverse cardiovascular outcomes. Our laboratory recently reported an augmented resting beat-to-beat BPV in non-Hispanic BL males compared to their White (WH) male counterparts. However, racial differences in beat-to-beat BPV between non-Hispanic BL and non-Hispanic WH females are unknown. Therefore, we tested the hypothesis that young, healthy, non-Hispanic BL females have elevated resting beat-to-beat BPV compared to healthy age and body mass index matched WH females. Methods: We studied 12 young healthy non-Hispanic BL females (20 ± 2 years, 25.2 ± 4 kg/m2, mean ± standard deviation) and 12 young healthy non-Hispanic WH females (22 ± 3 years, 22.7 ± 3 kg/m2; P = 0.13 and P = 0.10, respectively). Beat-to-beat arterial blood pressure (finger photoplethysmography) was continuously recorded during ten minutes of supine rest. Mean, standard deviation (SD), range, coeffcient of variation, and average real variability (ARV) were calculated for systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP). Results: Resting absolute SBP (BL: 113 ± 12 mmHg; WH: 112 ± 7 mmHg; P = 0.98), DBP (BL: 69 ± 7 mmHg; WH: 66 ± 5 mmHg; P = 0.22), and MAP (BL: 83 ± 7 mmHg; WH: 81 ± 4 mmHg; P = 0.48) were not different between groups. Likewise, beat-to-beat SBP SD (BL: 3.5 ± 0.78 mmHg; WH: 4.0 ± 1.16 mmHg; P = 0.21), range (BL: 21 ± 5 mmHg; WH: 24 ± 6 mmHg; P = 0.26), coeffcient of variation (BL: 3.01 ± 0.82%; WH: 3.56 ± 0.96%; P = 0.20), and ARV (BL: 2.02 ± 0.46; WH: 2.26 ± 0.6; P = 0.27) were not different between BL and WH females. There were also no differences in any of the beat-to-beat BPV measures for DBP or MAP between BL and WH females (all P > 0.05). Conclusion: In contrast to our hypothesis and previous work in non-Hispanic BL males, these preliminary data suggest that beat-to-beat BPV is not different between non-Hispanic BL and WH females.

This work was supported by the College of Nursing and Health Innovation.

This is the full abstract presented at the American Physiology Summit 2024 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.
Examining The Effects Of Gallein On Opioid Induced GIRK Currents
Authors: Keldsen Charles , Beeman Carley , Bartol Kai , Levitt Erica , Smrcka Alan
Published: May 2024

Opioids are powerful analgesics, but carry a risk of overdose that has become a pressing issue to public health in the United States. Overdose deaths are caused by the opioid-induced shutdown of neuronal circuits that operate unconscious breathing cycles, leading to respiratory depression. Respiratory depression, alongside antinociception, is mediated by the activation of μ-opioid receptors, which are coupled to inhibitory heterotrimeric G proteins. Recent studies have shown that gallein, a Gβγ subunit inhibitor, amplifies the antinociceptive effects of opioids through Gq-phospholipase C (PLC) mediated mechanisms, while either attenuating or having no effect on respiratory depression. The molecular mechanisms of gallein effects on respiratory depression are unclear. Based on the binding interface, gallein is predicted to inhibit Gβγ binding to phospholipase C, but not G protein inwardly rectifying potassium channels (GIRK). The goal of this study is to examine the effects of gallein on opioid-induced GIRK currents, with or without Gq subunit activation, in mouse locus coeruleus (LC) neurons. The LC is an area of particular interest due its involvement in respiratory circuits, and activation of Gq-coupled pathways decreases the amplitude of opioid-mediated GIRK currents in LC neurons. We hypothesized that gallein would have no effect on opioid mediated GIRK currents without Gq activation, but would prevent Gq-mediated inhibition of GIRK. We tested this hypothesis using whole-cell voltage-clamp recordings of LC neurons contained in brain slices from adult male and female mice. The endogenous opioid agonist Met-enkephalin induced outward GIRK currents that were not significantly altered by gallein. The Gq-coupled receptor agonist oxotremorine-M reduced the amplitude of Met-enkephalin mediated GIRK currents, as expected. Preliminary data suggests no effect of gallein on GIRK currents in the presence of Gq activation, contrary to our hypothesis. These results will increase our understanding of the role of PLC in Gq-mediated inhibition of GIRK currents at the cellular level and provide insight regarding the potential respiratory related impacts associated with using gallein derived compounds in combination with opioids to increase opioid antinociception and reduce the doses of opioids needed to treat pain.

This research is supported by NIH Grant R01DA047978-05 to ESL.

This is the full abstract presented at the American Physiology Summit 2024 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.
An Ex Vivo Approach to Studying Renin Dynamics
Authors: Vanden Avond Mark , Grzybowski Michael , Greene Andrew , Liang Mingyu , Cowley, Jr Allen , Geurts Aron
Published: May 2024

Hypertension is the leading risk factor for cardiovascular disease and impacts one in three adults globally. One non-coding mechanism that may affect blood pressure regulation and alter hypertension risk in the general population is the disruption of chromatin conformation. CCCTCF-binding factor (CTCF) has many important functions including regulating the conformation of chromatin by binding to specific DNA sequences. Renin has multiple CTCF motifs surrounding the gene and cellular expression of CTCF is critical for proper Renin expression. Using CRISPR-Cas9 in the Dahl salt-sensitive (SS) rat, we have developed three mutant rat models with one or more mutations to the CTCF motif surrounding the Renin gene. CTCF binding was confirmed to be disrupted in all three models at the CRISPR-targeted locations. Plasma renin activity (PRA) was measured in wild-type (WT) and mutant rats fed a 0.1%, 0.4% or 4.0% NaCl diet for four days. PRA in WT males responded as expected to dietary NaCl (17.52±0.81, 12.87±1.00, and 7.13±0.19 ng/mL; p<0.05 for 0.1% and 4.0% vs. 0.4%). Interestingly, two of our male mutant models failed to increase their PRA on the 0.1% NaCl diet (CTCF1=13.87±0.86; CTCF2=13.95±0.59; p<0.05 compared to WT 0.1% NaCl). Despite this, no differences in Renin mRNA were detected. We hypothesize Renin transcriptional response to salt depletion is delayed in the mutant models compared to WT. To test this, we developed an ex vivo approach to culture kidney slices and evaluate Renin expression to a variety of stimuli. Kidneys from SS rats fed low salt (LS; 0.4% NaCl) or high salt (HS) diet (4.0% NaCl) for 24 hours were harvested, sliced, and cultured in basal media or media containing increasing salt. Renin expression was examined at 1, 2, 4, 8 and 24 hrs of incubation. Kidney slices from SS rats fed HS that were incubated basal media stimulated Renin expression as soon as 2 hrs and continued to increase until 8 hrs. This was the first time for establishing an ex vivo approach for motoring the dynamic changes in Renin expression in response to a stimulus by culturing kidney slices. This method could be used to evaluate other stimuli, such as therapeutics, and could decrease the number of animals needed for a study.

Funded by NIH 1HL149620.

This is the full abstract presented at the American Physiology Summit 2024 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 32 d of spaceflight on tendon collagen fibril morphology and nanomechanics
Authors: Bojorge Guillermo , Ortiz-Delatorre Julissa , Bialy Patrick , Ichimura Andrew , Lorenz Kent , Scott Ryan , Bagley James R.
Published: May 2024

INTRODUCTION: Collagen is an essential component of connective tissue and plays a crucial role in elasticity, stability, and force distribution. While the adaptations of tendon to internal stimuli have been extensively researched, the impact of environmental factors (such as microgravity) on its structure and function at the nanoscopic scale (i.e., fibril level) remains largely unexplored. The purpose of this study was to determine the effects of 32 days (d) aboard the International Space Station (ISS) on murine tendon collagen fibril morphology and nanomechanics (Young’s Modulus, YM). We hypothesized there would be a decrease in fibril size and YM after spaceflight. Methods: Quadriceps tendon samples from female C57BL/6J mice from NASA Rodent Research-1 underwent chemical processing and mechanical isolation. Subsequently, collagen fibrils (n=56) were studied via atomic force microscopy (AFM; JPK NanoWizard 4a) to compare the morphology (diameter, height) and nanomechanics (average YM) between spaceflight (SF) and ground control (GC) specimens. The qp-BioAC-CB1 cantilever (spring constant: 0.3 N/m, tip radius: 10μm) was used under quantitative imaging mode (QI) for all force and morphological measurements. The data from 56 micrographs (SF n=30; GC n=26) was processed via the JPK Data Processing Software (Version 6.4.5.), and finally analyzed with Gwyddion (Version 2.62). A 2-way MANOVA compared all measurements. Results: More ‘small fibrils’ (height <50nm) were identified in the SF vs GC samples. SF fibrils averaged 20.3% smaller (width) compared to GC, but this difference was not significant (p>0.05). Additionally, while there was a trend for a lower YM in the SF samples, YM for SF specimens was not statistically different between groups (GC=3.26±1.94 MPa; SF= 2.76±1.44 MPa) (p>0.05). CONCLUSION: To our knowledge, this is the first investigation to use AFM QI-mode on tendon collagen fibrils after spaceflight. While there were no statistically significant changes in mean fibril size and YM between SF and GC, these results suggest that collagen fibrils may require more time to adapt to changes in their environment, and/or more rapid changes may take place at different levels of the collagen hierarchy. Our data provides valuable insight into mammalian tissue adaptations during spaceflight. Future research should continue these investigations with longer-duration missions to build a time-course of tendon adaptations to microgravity.

FUNDING: CSUBIOTECH New Investigator Grant (JRB).

This is the full abstract presented at the American Physiology Summit 2024 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.
PTSD-like mice display negative cardiac characteristics post-inescapable foot shock
Authors: Corker Alexa , Troncoso Miguel , Learmonth Maya , Broughton Philip , Sidles Sara , Kelly Ryan , LaRue Amanda
Published: May 2024

Post-traumatic stress disorder (PTSD) is a risk factor for cardiovascular disease, however the mechanism behind this correlation is unknown. We hypothesized that PTSD alters cardiac homeostasis by increasing macrophage numbers and initiating cardiac fibrosis. To induce experimental PTSD, male and female C57BL/6 mice (4-weeks: n=16-17/sex, males=4.7±0.4 months old; females=4.8±0.3 months old; 8-weeks: n=13-16/sex, males=6.0±0.3 months old; females=6.3±0.5 months old) were exposed to 5 separate foot-shock incidences (IFS; 1.0 mA, 1 sec duration) in 6 min. Control mice (n=8-9/sex) were placed in the same chambers but experienced no foot shocks. Behavioral tests (16 parameters based on the DSM-5 clinical standards) were performed to characterize mice that do not demonstrate PTSD-like behavioral characteristics (NR) and PTSD-like mice. Doppler echocardiography measurements were collected serially up to 8-weeks post-IFS. Histological and gene assessments were collected 4- and 8-weeks post-IFS to measure markers of adverse cardiac remodeling that resulted in later functional changes. Over time PTSD-like mice demonstrated a decline in cardiac function becoming significantly impaired by 8-weeks as shown by an increase in E/e’ ratio along with left atrial diameter compared to controls, indicative of impaired LV filling pressure and diastolic dysfunction (p<0.05 for all). Beginning at 4-weeks post-IFS, PTSD-like mice had increased picrosirius red staining (PSR) compared to control mice (p<0.05) that remained elevated at 8-weeks. PTSD-like mice had increased macrophage numbers (Mac3) at 4-weeks post-IFS compared to control mice (p<0.05) and showed an increased trend when compared to controls 8-weeks post-IFS (p=0.09). To determine potential signaling mechanisms, Ccl2, Ccl5, and discoidin domain receptor tyrosine kinase 2 (DDR2) mRNA levels were assessed. Gene expression of Ccl2 demonstrated a decreased trend in PTSD-like mice (p=0.13), while Ccl5 (p=0.08) and DDR2 (p=0.06) mRNA demonstrated an increased trend in PTSD-like mice when compared to controls. Our data indicates that in a mouse model of PTSD, animals that demonstrate PTSD-like behaviors show signs of increased cardiac inflammation stimulating cardiac remodeling and cardiac dysfunction.

This work was supported by the National Institutes of Health T32GM123055; the American Heart Association Innovator Project IPA35260039, GM113278; the Biomedical Laboratory Research and Development Service of the Veterans Affairs Offce of Research and Development Award IK2BX003922, I01BX00584; and South Carolina Translational Research Center UL1TR001450.

This is the full abstract presented at the American Physiology Summit 2024 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.
Ineffective matching of oxygen supply to myocardial metabolism in primiparous compared to nulliparous swine
Authors: Tucker Selina , Essajee Salmon , Warne Cooper , Heard Michael , Dick Gregory , Goulopoulou Styliani , Tune Johnathan
Published: May 2024

Maternal mortality rates in the United States are higher than any other developed country in the world. Most postpartum maternal deaths are directly related to adverse cardiovascular events and often occur in mothers who have had a healthy pregnancy. Although pregnancy is recognized as a cardiovascular stress test for the pregnant patient, understanding of how the longitudinal and dynamic cardiovascular adaptations during pregnancy contribute to augmented risk of cardiovascular complications through the postpartum period remains poorly understood. We propose that pregnancy-induced adaptations in the coronary circulation contribute to adverse cardiovascular outcomes, especially during the puerperium period (1-60 days following delivery). Accordingly, this study tested the hypothesis that the balance between coronary blood flow and myocardial metabolism is compromised in the puerperium heart. Experiments were performed in anesthetized, open-chest control nulliparous (control) and primiparous (postpartum) swine during the puerperium period (35-45 days post-delivery). Systemic and coronary hemodynamic responses were assessed under baseline conditions and in response to continuous intravenous infusion of dobutamine (1 – 30 μg/kg/min). Under baseline conditions, blood pressure averaged 107 ± 6 mmHg in both control and postpartum swine and was unaffected by dobutamine administration (P = 0.94). Heart rate increased from 77 ± 6 to 165 ± 9 beats/min in response to dobutamine in control swine vs. 52 ± 3 to 139 ± 13 beats/min in postpartum swine (P = 0.53). Coronary blood flow and myocardial oxygen delivery were significantly diminished by ~60 ± 8% at rest in postpartum swine, which is consistent with reduced myocardial oxygen consumption (MVO2). The relationship between coronary blood flow and heart rate (P < 0.0001) or MVO2 (P = 0.0002) showed a marked parallel downward shift in postpartum swine at rest and in response to dobutamine infusion. This reduction in myocardial perfusion was associated with significant reductions in myocardial lactate uptake at rest in postpartum vs. control (0.34 μmol/min/g vs. 0.08 μmol/min/g, P = 0.02). Dobutamine increased myocardial lactate uptake in control by ~47 ± 3% with little change in the postpartum group. Together, these results suggest an ineffective matching of myocardial oxygen supply to metabolism in postpartum swine. This imbalance is consistent with increased incidence of myocardial ischemia and cardiac deaths in postpartum puerperium period.

This work was supported by AHA.23PRE101281, NIH R01 HL158723.

This is the full abstract presented at the American Physiology Summit 2024 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.
High Potassium Suppresses Glycosuria and Gluconeogenesis in Tubule-specific mTORC2 Knockout Mice
Authors: Demko John , Saha Bidisha , Takagi Enzo , Weber Robert , Pearce David
Published: May 2024

Background: Insulin promotes renal proximal tubule glucose reabsorption and suppresses gluconeogenesis (GNG). The kinase mTORC2 is critical for insulin signaling in multiple cell types. In the kidney tubules, mTORC2 knockout has recently been shown to cause glycosuria via reduced plasma membrane SGLT2 and SGLT1 as well as inappropriately increased renal GNG. Potassium (K+) also plays an important role in systemic glucose homeostasis. However, the overall importance of K+ for the regulation of renal glucose reabsorption and GNG is poorly understood. Methods: Rictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. To examine the role of dietary K+ on renal glucose homeostasis, TRKO mice and wild-type (WT) littermates were fed with either a normal 0.5% K+ diet or high 3% K+ diet while in balance cages. Kidney tissue was harvested after feeding and processed to study plasma membrane glucose transporters, gluconeogenic enzymes, and mTORC2-regulated targets by western blot and qPCR. Results: On a normal K+ diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane fractions showed decreased SGLT2 and SGLT1, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes. On a high K+ diet, glycosuria resolved and plasma membrane SGLT2 and SGLT1 were restored in TRKO mice. In addition, TRKO animals fed with a high K+ diet had suppressed gluconeogenic enzymes compared to WT mice. Regardless of dietary K+ content, TRKO animals had similar reductions in phosphorylation of mTORC2 substrates compared to WT controls. Conclusion: Renal tubule mTORC2 is critical for coordinated glucose reabsorption by SGLT2 and SGLT1 as well as suppression of renal GNG. High dietary K+ promotes sodium-glucose cotransport by restoring plasma membrane SGLT2 and SGLT1 in TRKO mice. High dietary K+ also prevents excessive GNG in TRKO mice. High K+ appears to regulate these processes independently of mTORC2 and its downstream substrates, such as Akt. High K+ could potentially provide an alternative signaling mechanism to regulate renal glucose reabsorption and GNG in states of insulin resistance.

Grants from the NIH (T32, R01) and The James Hilton Manning and Emma Austin Manning Foundation.

This is the full abstract presented at the American Physiology Summit 2024 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.
Fibulin-5 Is Required for Small Artery Development and Function
Authors: Halabi Carmen , Lin Michelle , Roth Robyn , Yanagisawa Hiromi
Published: May 2024

Introduction: Fibulins are a family of eight extracellular matrix proteins with diverse functions. Fibulin-4 and -5 (FBLN-4 and -5), in particular, have been shown to be important for elastic fiber assembly. Mutations in each of the genes lead to cutis laxa, a syndrome characterized by loose skin with variable organ involvement, particularly in the vasculature, lungs and skeleton. We recently characterized a mouse model carrying a disease-causing mutation in Fbln4 (E57K) and, contrary to conduit arteries that had elastic fiber fragmentation and stiffness, Fbln4E57K resistance arteries were structurally intact. Functionally however, Fbln4E57K mesenteric arteries exhibited endothelial dysfunction secondary to increased shear stress from large artery stiffness. Loss of FBLN5 has been shown to lead to elastic fiber fragmentation in conduit arteries, but the consequences of its loss on resistance arteries are unknown. Therefore, the objectives of this study were to determine the structural and functional consequences of FBLN5 loss on resistance arteries. Methods: We utilized adult Fbln5 knock-out (KO) mice and examined the ultrastructure of renal and mesenteric arteries via transmission electron microscopy. Functionally, using pressure myography, we characterized the myogenic tone and reactivity of Fbln5 KO and littermate control third-order mesenteric arteries to various vasoactive substances including acetylcholine, papaverine, angiotensin II, phenylephrine and potassium chloride. Results: While the internal elastic lamina (IEL) of Fbln5KO mice resistance arteries was largely intact, the external elastic lamina (EEL) was nearly absent. Similar to Fbln4E57K mice, Fbln5KO mesenteric arteries exhibited impaired endothelial-dependent vasorelaxation, likely secondary to proximal large artery stiffness. Unlike Fbln4E57K mesenteric arteries however, Fbln5 KO mesenteric arteries exhibited increased myogenic tone as well as a hypercontractile response to angiotensin II and potassium chloride, but not to phenylephrine. Conclusions: In addition to highlighting differences in the requirements for elastic fiber assembly along the arterial tree, these data emphasize the differential contribution of FBLN4 and FBLN5 to IEL vs. EEL in resistance arteries and suggest pathway-specific changes in smooth muscle cell contractility secondary to FBLN5 deficiency.

This work was supported by the Washington University Pediatric Nephrology Research Core Pilot Grant Program to CMH. Funds were also provided by The Department of Pediatrics at Washington University in St. Louis School of Medicine to CMH.

This is the full abstract presented at the American Physiology Summit 2024 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.
Cardiac Remodeling in Aged Rats after Early-life Chronic Stress: Sex-specific Outcomes and Associations with Metabolic Parameters
Authors: Sanford Ella , Dearing Carley , Olmstead Nicolette , Morano Rachel , Wulsin Lawson , Myers Brent
Published: May 2024

Cardiovascular disease is the leading cause of death worldwide. Additionally, the incidence of cardiovascular disease varies both across the lifespan and between sexes. While multiple factors influence the development and progression of cardiovascular pathology, prolonged stress is a prominent risk factor for adverse cardiac events. However, the interactions between stress, sex, and aging that impact cardiac susceptibility remain to be determined. To this end, we examined hypertrophy of the aged myocardium in a sex- and stress-specific manner. We hypothesized that sex and stress history would interact to predict cardiac outcomes and that susceptibility/resilience would associate with behavioral, endocrine, and metabolic parameters assessed across the lifespan. In this study, rats were either exposed to chronic variable stress (CVS) in late adolescence (n = 36/sex) or remained as unstressed controls (n = 24/sex). Rats were subsequently challenged with a forced swim test (FST) and glucose tolerance test (GTT) before ageing to 15 months. The FST and GTT were then repeated prior to tissue collection. Cardiac tissue was processed for histological analysis and quantification of ventricular morphology. The data indicated that early-life chronic stress had sex-specific consequences on the aged left ventricle as CVS females had greater concentric hypertrophy. Based on the level of inward remodeling, rats were then classified as susceptible or resilient. Multiple behavioral and endocrine factors in young and aged animals significantly interacted with hypertrophy, including coping behaviors and glucocorticoid responses to hyperglycemia. However, correlations with hypertrophy were only present in males. Male rats that were not exposed to CVS had a positive association between baseline corticosterone and hypertrophy. Alternatively, CVS males had a positive correlation between visceral adiposity and hypertrophy. Thus, sex-specific cardiac susceptibility/resilience relates to endocrine and metabolic measures depending on stress history. This outcome suggests that adverse experiences can have lasting impacts on homeostatic adaptation. Ultimately, these results highlight the importance of sex and prior stress exposure for disparities in cardiovascular risk.

NIH R01 HL 150559 (B Myers) NIHH F30 OD032120 (C Dearing) Pilot Translation Research Program grant from the University of Cincinnati (B Myers).

This is the full abstract presented at the American Physiology Summit 2024 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.
Gut microbiome alterations in Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) suggests variation in the gut-brain axis and gut metabolic potential
Authors: Schiro Gabriele , Shaik Shabana , Laubitz Daniel , Daines Michael , Rice Sydney , Ghishan Fayez , Kiela Pawel
Published: May 2024

PANDAS, or Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections is a form of Children’s Post infectious Autoimmune Encephalopathy (CPAE), a neuropsychiatric condition characterized by the abrupt onset of diverse neuropsychiatric manifestations following streptococcal infection. It is an increasingly recognized neuropsychiatric disorder that affects a subset of children whose underlying etiology remains unclear. Gut microbiome plays a crucial role in its host brain development and is implicated throughout the life in neurobehavior and neurological disorders. Emerging evidence suggest gut dysbiosis resulting in altered intestinal flora can impair gut barrier and increase leakiness in the blood-brain barrier leading to inflammation. We aimed to investigate if the microbiome is altered in pediatric patients with PANDAS. Stool (n=19), oral (n=20) and nasal swabs (n=18) were collected from patients diagnosed with PANDAS or healthy controls (HC) and used for 16S analysis. 13 stool samples (7 HC and 6 PANDAS) samples were further used for shogun metagenomics and 17 for untargeted metabolomics (8 HC and 9 PANDAS). 16S rRNA gene sequencing identified significant difference in microbial α diversity, with lower Shannon H’ in PANDAS compared to HC (Welch’s t-test t(16.95)=-2.6, p=0.02), and in β-diversity (PERMANOVA R2=0.08 p=0.04) between PANDAS and HC fecal but not throat and nasal samples. Shotgun metagenomics identified a significant reduction in gene diversity (Welch’s t-test t(9.9)=-3.06, p=0.01) and changes in various KEGG ortholog modules involved in gut metabolism and gut-brain axis. Significant differences were also detected in metabolite composition (PERMANOVA R2=0.10, p=0.02). We also identified a set of microbial groups and metabolites enriched or depleted in CPAE patients, involved in different aspects of host physiology and gut metabolism. Our results show that PANDAS patients have an altered gut microbial community, which may exacerbate their behavioral symptoms and it should be taken into consideration in unveiling the complex pathogenesis and selection of treatment options.

Arizona Department of Health Services RFGA2022-010-23 (PK) and ADHS17-00007403 (SR, MD); The Alex Manfull Fund (PK).

This is the full abstract presented at the American Physiology Summit 2024 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.
Conditional Knockout of Glycosaminoglycan Synthesis in Bladder Epithelium Has No Effect on Urinary Voiding in Mice
Authors: Hill Warren , de Oliveira Mariana , Zeidel Mark , MacIver Bryce
Published: May 2024

Glycosaminoglycans (GAGs) on the luminal surface of the bladder epithelium have been suggested to play an important role in barrier function and in protection from infection. There is also a substantial literature that posits a breakdown in the GAG layer may underlie, or be associated with the development of painful bladder syndrome (PBS, aka interstitial cystitis) and indeed, GAG mimetics such as pentosan polysulphate provide symptomatic relief for some patients. However, opinions on the presence and role of GAGs vary, and incontrovertible evidence is lacking. We have taken a unique approach to answering this question by engineering a highly cell-specific genetic mouse model that lacks the obligatory initiating enzyme for GAG synthesis in urothelium. Uroplakin 2-Cre (UP2-Cre) mice were crossed with xylosyltransferase 2-floxed mice to knockout the xylt2 gene in urothelium. RT-PCR confirmed the tissue specificity of the knockout in urothelium but not in bladder smooth muscle. Further, lacZ reporter staining in cryosections demonstrated highly specific expression of the Cre construct in urothelium. Mammals express two xylosyltransferases, XylT1 and XylT2 in different proportions in different tissues. While functionally similar they appear to differ in their substrate specificity and kinetics. qPCR in dissected urothelium demonstrated that XylT2 was the dominant isoform, being expressed at approximately 80-fold higher level than XylT1. Void spot assays (VSA) on filter paper (4 hours) have proven to be a reliable indicator of voiding dysfunction in mice. We conducted VSAs on female Up2 cre/xylt2 fl/fl (n=12) and xylt2 fl/fl (n=11) littermate controls aged 8 – 16 weeks. There was no significant difference in void volumes, number of primary voids, volume/void or number of microvoids (<20 μl) between controls and knockouts, whereas transurethral instillation of protamine sulfate, which injures the apical cell layer, induced pain and a significantly greater number of microvoids, with reduced primary void volumes. We conclude preliminarily, that loss of GAGs in urothelium does not affect bladder function, suggesting that GAGs do not play a role in protecting the suburothelium and adjacent sensory afferents from noxious urine components. Future work will characterize and quantitate the distribution of GAGs within the urothelium to determine whether GAGs are deployed on the luminal surface and, whether they play a role in urothelial repair from injury.

NIH grant P20DK097818.

This is the full abstract presented at the American Physiology Summit 2024 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.
Autophagy Is Required for Cell Growth and Protein Anabolism in Lung Cancer
Authors: Ryan Patrick , Fluckey James D.
Published: May 2024

Background: Rampant and unregulated cell growth is a key hallmark of cancer pathology; however, much remains to be discovered about the process that allow for pathological growth in cancerous cells. Recently, we demonstrated that the autophagy-related cysteine protease ATG4B is required for protein anabolism in skeletal muscle, indicating a previously undescribed mechanism by which autophagy directly acts as a recycling/remodeling system for cellular anabolism. Purpose: To investigate if ATG4B is responsible for growth in cancer, and if inhibition of ATG4B is a successful strategy to target cancer growth. Hypothesis: We hypothesized that ATG4B would be higher in cancerous tissues versus healthy ones, and that inhibition of ATG4B by NSC185058 would inhibit cell growth and reduce protein anabolism. Methods: We used the UALCAN web tool to analyze the TCGA data set for differences in ATG4B gene expression in lung adenocarcinoma, comparing healthy to cancerous tissues as well as across cancer stages. Separately, we used the A549 lung cancer cell line as a model to study the effects of ATG4B inhibition, treating cells with 100 μM NSC185058 or equimolar DMSO as a vehicle control, and analyzing treated cells for proliferation and protein synthesis rates. Welch’s t-test was used to calculate differences in TCGA data, while a two-tailed student’s t-test was used to determine differences in proliferation at indicated time points, with an α-level of 0.05. Results: ATG4B expression is 22% higher in cancerous tissues vs healthy controls (p<0.05), and also higher in stages 1-4 of lung adenocarcinoma (Healthy vs Stage 1: +25%, Healthy vs Stage 2: +23%, Healthy vs Stage 3: +13%, Healthy vs Stage 4: +30%, p<0.05). Treatment with NSC185058 suppresses cell growth across 72 hours (24 hour VC vs NSC: -59%, 48 hour VC vs NSC: -83%, 72 Hour VC vs NSC: -90%), and reduces 24-hour rates of protein synthesis by 56% (p<0.05) in cancer cells. Conclusions: ATG4B expression is increased in lung adenocarcinoma, including in early-stage cancer, and inhibition by NSC185058 drastically reduces anabolism in cancerous cells. We posit that autophagy, mediated by ATG4B is required for protein anabolism in lung cancer, and that targeting this process represents a novel strategy to combat cancer pathology.

This work was generously funded by the Huffnes Institute and School of Education and Human Development at Texas A&M University.

This is the full abstract presented at the American Physiology Summit 2024 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.
TAK1 Signaling in Cardiac Fibroblast Contributes to Inflammation Resolution Post Myocardial Infarction
Authors: Nguyen Daniel , Stephan Jonah , Boyd Nolan , Brainard Robert , Hellmann Jason , Wysoczynski Marcin , Hill Bradford
Published: May 2024

Introduction: Resident cardiac fibroblasts are often defined by their extraordinary capacity to deposit extracellular matrix after cardiac injury; however, fibroblasts also react avidly to pro-inflammatory stimuli, suggesting that they may also be mediators of inflammation. Initially identified as a TGFβ responsive enzyme, TGF-β activated kinase 1 (TAK1) has been shown to be significant in regulating pro-inflammatory signaling in several disease models. The goal of this study was to understand how fibroblasts integrate pro-inflammatory signals to regulate acute inflammation following myocardial infarction (MI). We hypothesize that cardiac fibroblasts utilize a TAK1-dependent mechanism to integrate pro-inflammatory signals and orchestrate the cellular immune response following injury. Methods and Results: To create a fibroblast-specific model for TAK1 knockdown, TAK1 fl/fl mice were bred to Col1a2-CreERT mice. Following 2 weeks of tamoxifen chow feeding, both Cre− and Cre+ TAK1 fl/fl mice underwent permanent coronary ligation to induce MI. Three days following MI, immune cell populations from the heart, spleen, and peripheral blood were evaluated through flow cytometry. Deletion of TAK1 in fibroblasts in vivo diminished neutrophils and Ly6C low patrolling monocytes populations in the heart and decreased the number of CCR2neg resident macrophages after MI (n=10/group; p<0.05). Interestingly, circulating levels of neutrophils in the peripheral blood as well as those in the spleen were not significantly different between the groups. To determine how TAK1 may regulate pro-inflammatory signaling, isolated cardiac fibroblasts from TAK1 fl/fl mice were treated with Ad-CMV-iCre and were subsequently treated with IL1β. Notably, TAK1 knockdown suppressed IL1β-dependent p-JNK and NFκB signaling as well as MMP3 production. Further LC-MS/MS evaluation of lipid mediators in conditioned medium showed that deletion of TAK1 significantly increased fibroblast-derived resolvins (RvE2, RvD1, RvD4, RvE4) and lipoxins (LXA4, 15( R)-LXA4) (n= 3-5; p<0.05). Conclusion: Resident cardiac fibroblasts regulate the acute immune response following MI via a TAK1-dependent mechanism. Secreted factors derived from cardiac fibroblasts likely play a pivotal role in shaping the inflammatory and immune response after MI.

NIH and AHA.

This is the full abstract presented at the American Physiology Summit 2024 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.