bluuebunny/crossref_metadata_2025 huggingface dataset:
Immune Metabolic Reprogramming by Excess Dietary Salt in Salt Sensitive Hypertension via CXCL8
Authors: Jamison Sydney , Pitzer Mutchler Ashley , Kirabo Annet
Published: May 2024

Salt sensitivity of blood pressure (SSBP) is when blood pressure changes parallel to salt loading/ depletion and is an independent risk factor for cardiovascular disease. Despite its importance, there is no feasible diagnosis, which limits investigational studies to identify therapeutic targets. We have found that immune cells mediate SSBP via Epithelial Sodium Channel (ENaC). CXCL8/(IL-8), a known chemoattractant plays a well-known role in glycolysis. We hypothesize that CXCL8 mediates immune metabolic reprogramming of immune cells to glycolysis via ENaC. To test this hypothesis, we performed bulk and single cell RNA sequencing in human monocytes from individuals from a modified Weinberger inpatient salt loading/ depletion protocol. The participants were instructed to refrain from taking their blood pressure medication for 2 weeks prior to the salt loading/depletion protocol. Blood and urine samples were collected, as well as ambulatory blood pressure recording. We found that changes in immune cell activation via isolevuglandin (IsoLG) protein-adducts mirror changes in salt balance and blood pressure in salt sensitive but not salt resistant individuals. We confirmed that these in vivo changes can be recapitulated in vitro. In addition, we found that salt-loading both in vivo and in vitro increases antigen presenting cell production of IL-8, which was reversed after salt depletion in salt sensitive, but not salt resistant people. ENaC inhibition by amiloride prevented the high salt-induced production of IL-8. Moreover, high salt induced blood pressure changes of expression of CXCL8 (p = 0.01553, r = 0.8067) were significant along with varying expression of glycolytic genes, including hexokinase 1(HK1)(p = 0.04190, r = -0.7249), phosphofructokinase (PFKP)(p = 0.05932, r = 0.6879) and galactose mutarotase (GALM) (p = 0.04176, r = 0.5138). Seahorse analysis revealed increased extracellular acidification rate (ECAR) in high salt conditioned monocytes, which was associated with mitochondrial dysfunction. Our results suggest that high salt intake reprograms antigen presenting cell metabolism to glycolysis via IL-8 signaling. Furthermore, targeting IL-8 and mitochondrial switching to glycolysis may not only provide novel therapeutic approaches, but also important biomarkers for diagnosis of salt sensitivity of blood pressure.

1R03HL155041-01 (Kirabo, PI), 1R01HL144941-01A1 (Kirabo, PI), 5R01HL147818-22 (Kleyman & Kirabo), 1R01HL157584-01 (Shibao, Kirabo — MPI), R01DK135764 (Kon, Shelton, Kirabo — MPI), R21TW012635 (Kirabo, Masenga — MPI), Meharry Medical College: School of Graduate Studies T32AI007281, T32GM14492, andT32HL00773.

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.
Determination of surfactant secretion in precision-cut lung slices
Authors: Khade Bipin , Islam Mohammad , Bhattacharya Sunita , Bhattacharya Jahar
Published: May 2024

Surfactant, secreted from type 2 (AT2) cells of the alveolar epithelium (AE), maintains alveolar stability. However, the variability of surfactant secretion, a possible cause of regional lung disease, remains unquantified. To address this, we cultured precision-cut lung slices (PCLS) of mouse lung explants, using low-melting point agarose as per reported protocols. For real-time confocal imaging (RCM), we loaded day-old PCLS with the cytosolic marker calcein green (CG) and the AT2 cell marker, lysotracker red (LTR). RCM revealed alveoli as CG-stained AE that circumscribed the non-fluorescent alveolar lumen. Each slice (n=3) contained >300 alveoli, each with an LTR+ AT2 cell. Exposing the slice to ATPgamma-s (4 microM) induced progressive loss of LTR fluorescence, denoting induction of surfactant secretion. In 60% of AT2 cells, the fluorescence decreased to 40±2% of initial in 50 min (p<0.01). In 11%, the decrease was to 59±1%. In 29%, there was no decrease. We conclude: (i) PCLS provide a robust platform for quantifying AT2 secretion; (ii) surfactant secretion rates vary considerably amongst AT2 cells. Mechanistic basis of the variation, hence its impact on lung function must be understood.

HL36024.

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.
Potential contribution of liver sinusoidal endothelial cell expressing EphrinB1 in fibrogenesis during metabolic dysfunction-associated steatohepatitis in mice
Authors: Yoshigi Masaaki , Miles Veronica C. , Pellizzari Sarah , Kamdem Severin Donald , Kameni Earnest , Helms My N , Mimche Patrice
Published: May 2024

Study Objective and Hypothesis: The pathogenesis of fibrosis during metabolic dysfunction-associated steatohepatitis (MASH) is not fully understood. Our previous work indicate that the Eph/Ephrin signaling could be a major cell-cell signaling hub regulating fibrogenesis in MASH. While cell-cell interaction between HSC and liver sinusoidal endothelial cells (LSEC) has long been proposed as a driver for liver fibrosis, molecular mechanisms of their interaction have not been elaborated. In this study, we tested the hypothesis that EphrinB ligands expressed in LSEC plays a role in the pathogenesis of MASH fibrosis. Methods: Single nuclear RNA-sequencing of liver from human and mouse MASH were analyzed for Efnb1 and Efnb2 expression. Human liver tissues obtained from patients with MASH were assessed for pEphrinB expression. Plasma level of EphrinB2 was measured in MASH and healthy individuals. EphrinB1 deletion in LSEC were generated by crossing Efnb1fl/fl mice with CDH5(PAC)-CreERT2 mice. Experimental MASH was developed in LSEC-specific Efnb1 knockout and wild type mice by feeding them with a choline-deficient amino-acid defined (CDAA) high fat diet for 8 weeks. Histology, flow cytometry, qPCR, biochemical assays and immunohistochemistry were used to dissect the contribution of LSEC- Efnb1 function in MASH. Statistical analysis was performed using ANOVA and Mann Whitney U test and a p-value < 0.05 was considered significant. Data: Single nuclear RNA sequencing showed that Efnb1 and Efnb2 are upregulated in LSEC of mice with MASH. Plasma level of Ephrin-B2 is significantly elevated in MASH patients compared to healthy individuals. Deletion of Efnb1 was achieved after tamoxifen administration, as depicted by reduced expression of Efnb1 mRNA level and EphrinB1 protein in the liver of LSEC Efnb1−/− mice compared to littermate ( Efnb1fl/fl) fed the CDAA-HFD. However, we noted an increased expression of Ephb receptors mRNA level in the liver of LSEC Efnb1−/− mice compared to littermate fed the CDAA-HFD. In addition, we noted an increased CDAA-HFD-mediated inflammation and fibrosis after deletion of Efnb1 in LSEC compared to littermates mice fed CDAA-HFD. Conclusions: Although EphrinB1 is upregulated in LSEC during advanced MASH, its deletion does not protect from MASH fibrosis. Given the functional redundancy between Ephrin-B ligands, deletion of one member of this family might not be suffcient to abolished EphB-EphrinB bidirectional signaling.

NIDDK, NIAMS.

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.
Glucocorticoid receptors regulate hypothalamic CRH neuron activity in primary hypertension
Authors: Zhang Hua , Shi Yao , Li De-Pei
Published: May 2024

The hypothalamic-pituitary-adrenal axis is important in maintaining homeostasis in response to physical and psychological stressors. The corticotropin-releasing hormone (CRH) is a neuropeptide synthesized by CRH neurons in brain regions including paraventricular nucleus (PVN) of the hypothalamus. The glucocorticoids synthesized and released in adrenal gland act on hypothalamic CRH-expressing neurons inhibit the CRH production and maintain the level of glucocorticoids. The glucocorticoid receptor (GR) is critical for this negative feedback to regulate CRH synthesis and release. Hypertension is associated with high stress response and CRH mRNA levels. The number of CRH-expression neurons are significantly increased in PVN of the hypothalamus in primary hypertension patients. this study determined if the HPA axis negative feedback is altered and contribute to maintaining hypertension. We measured mRNA expression levels of GRs and MR in PVN tissue obtained from adult Wistar-Kyoto normotensive rats (WKYs) and spontaneously hypertensive rats (SHRs) and found that the GR mRNA expression level was significantly lower in SHRs that in WKY rats, while the MR gene expression did not differ between WKY rats and SHRs. Furthermore, we found that acute perfusion of dexamethasone (DEX), a synthetic glucocorticoid, significantly decreased frequency of spontaneous excitatory postsynaptic synaptic currents (EPSCs) in identified PVN-CRH expressing neurons in brain slices prepared from WKY and SHRs. DEX caused a greater reduction of frequency of EPSCs in SHRs was than in WKY rats. Acute perfusion of DEX significantly increased the firing rate of PVN-CRH neurons in WKY rats but did not change the firing rate in SHRs. In addition, incubation of brain slices with DEX for 1 hr significantly inhibited the firing activity of PVN-CRH neurons in WKY rats but did not change the firing activity of PVN CRH neurons in SHRs. These data suggested that the glucocorticoid excites PVN-CRH neurons in acute phase while inhibits these neurons in chronic phase in normotensive rat. In hypertension, downregulated GR may be responsible for impaired the HPA axis feedback and contribute to hypertension.

HL139523, HL142133, and HL159157.

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.
Nogo-B modulates physiological and pathological angiogenesis during development and disease
Authors: Shan Dan , Zhang Cheng , Baldini Margaret , Palos-Jasso Andrea , Wang Rui , Chu Shuhan , Zhang Xiaoying
Published: May 2024

Angiogenesis, i.e., the formation of new blood vessels, is essential for normal development and functional vessel formation in ischemic diseases. During angiogenesis, endothelial cells (ECs) coordinate migration, proliferation, and metabolic fluxes to adapt to branching vessels' increasing energy demands. Nogo-B is a ubiquitously expressed reticulon family protein mainly localized to the endoplasmic reticulum (ER) membrane. Our previous studies have shown that the genetic loss of Nogo-B results in aberrant vascular remodeling and dysregulated endothelial barrier function. The current study aims to examine the role of endothelial Nogo-B in regulating developmental angiogenesis and pathological angiogenesis in retinopathy of prematurity (ROP). We hypothesized that endothelial Nogo-B is required for physiological and pathological angiogenesis, and Nogo-B depletion attenuates pathological angiogenesis. Using tamoxifen-inducible, endothelial cell-specific Nogo-B−/− (Nogo-BiECKO) mice, Nogo-B global knockout mice, and C57Bl/6 (WT) mice, our results show that loss of Nogo-B in ECs significantly delayed Isolectin B4 (IB4)-labeled retinal blood vessel sprouting. Impaired EC proliferation was observed by the staining of and incorporation of 5-ethynyl-2′-deoxyuridine (EdU). Furthermore, using the Oxygen Induced Retinopathy (OIR) model to mimic ROP, we could demonstrate that loss of Nogo-B in EC resulted in reduced neovascularization compared to WT. Mechanistically, using Percoll gradients, ultra-resolution microscopy, proximity ligation assay, and electron-microscopy, we show that Nogo-B is localized in the mitochondria-associated ER membrane (MAM) and regulated MAM remodeling. Using seahorse assay, quantitative RT-PCR, and metabolomics analysis, we determined that loss of Nogo-B altered VEGF-mediated EC glycolysis, which is needed for the energy demand during angiogenesis. These data suggest that Nogo-B regulates glucose metabolism in EC and plays a pivotal role in physiological and pathological angiogenesis. Targeting endothelial Nogo-B may provide a plausible approach to controlling the formation of pathological neovascularization in retinal diseases, such as ROP, diabetic retinopathy, and wet forms of age-related macular degeneration.

This work was supported by the National Institutes of Health (R01HL153599) and awards from the American Heart Association award to JY.

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.
Engaging Alumni as Peer Mentors: Fostering Belonging and Forging Paths to Success
Authors: Sarathy Jayashree , Perez Morales Tiara , Narayanan Madhavan
Published: May 2024

Peer mentoring positively impacts students’ learning experience. It not only improves their content knowledge in courses but also reduces stress and anxiety levels, while increasing participation and engagement within the academic community. The MS in Integrative Physiology (MSIP) - graduate peer mentoring (GPM) program at Benedictine University, launched in 2020 during the COVID19 pandemic, aids in student and mentor success. The MSIP program, started in 2017 with 18 students, now has a total of 187, with 30 students enrolled in 2023. The cohort-based learning model, small class sizes, personalized advising, and a well-designed curriculum benefit diverse students. The shift to online learning during the pandemic challenged connections crucial for student success. An alum of the program was hired as a peer mentor to assist with classroom management, facilitate breakout room discussions, and provide insights into professional school applications. The students and the GPM establish an environment conducive to learning through in-person and virtual interactions. The MSIP-GPM program equips students with crucial learning skills that are needed for future success. It is a win-win situation for mentors and mentees alike. Mentors develop communication, interpersonal, and leadership skills along with confidence and motivation. They also deepen their understanding and exposure to content knowledge during their glide year. Mentees receive practical advice, academic strategies, career insights, and new friendships. Intentional selection of GPMs from underrepresented communities and first-generation students fosters inclusivity, support and a sense of belonging — all essential for their success. Positive feedback after the first semester led to program continuation, with GPMs providing support and mentorship. Since 2020, 13 students served as GPMs. 54% are in professional programs, and 23% are in the interview process for admittance. Based on the success of GPMs in Physiology, peer mentors were introduced in Immunology and Medical Biochemistry in Fall 2023. Results from student surveys, interviews, performance-metrics in tests and GPM feedback demonstrated the program’s effectiveness and peer mentor success. Peer mentoring aids active learning, boosts confidence and success, particularly for first-generation and underrepresented students.

JS, TPM and MN are supported by BenU COSH and NSSRP Funds, Benedictine University, Lisle, IL.

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.
Phenotyping adult mice with inducible depletion of sequestosome-1 (p62) in endothelial cells
Authors: Mookherjee Sohom , Gardner Colton G , Gapelu Lynasi , Judge Michael , Fatahian Nima , Boudina Sihem , Symons J David
Published: May 2024

Macroautophagy (autophagy) maintains intracellular homeostasis by targeting damaged / dysregulated organelles for their transport to and degradation by the lysosome. Autophagy regulated gene 3 ( Atg3) enables autophagosome maturation which is required for the process of autophagy to occur. We reported that inducible depletion of Atg3 specifically in endothelial cells (ECs) impairs arterial function in the absence of metabolic disruptions (Cho et al., Cardiovasc Res, 2023). Sequestosome-1 (p62/SQSTM1) tethers proteins into the autophagic pathway for subsequent lysosomal degradation. The contribution from EC p62 to arterial and metabolic function is unknown and we addressed this. Adult male and female C57BL/6 mice with tamoxifen inducible depletion of p62 specifically in ECs ( p62EC−/−) were compared to littermate controls ( p62EC+/+). p62/18S mRNA expression was lower (p<0.05) in ECs but not vascular smooth muscle cells (VSM) from p62EC−/− [n=6] vs. p62EC+/+ [n=9] mice, whereas Beclin-1/18S was similar in ECs and VSM from both groups. Neither body composition, glucose disposal nor insulin tolerance were impacted by EC specific p62 depletion. Vascular contractile responses to: (i) phenylephrine mediated α1-activation and (ii) potassium chloride evoked L-type Ca2+ channel opening; and relaxation responses to (iii) acetylcholine (endothelium-dependent) and (iv) sodium nitroprusside (endothelium-independent); were similar between groups in mesenteric resistance arteries (isometric myography) and middle cerebral arteries (isobaric myography). Further, intrinsic arterial stiffness was not different between p62EC−/− [n=6] and p62EC+/+ [n=9] mice i.e., neither the elastic modulus nor stress vs. strain curves generated using segments of thoracic aorta were altered. These findings indicate EC specific p62 depletion does not impact arterial function or the indexes of systemic metabolism we measured. Because p62 is upregulated during cellular stress, it is possible that a challenge (e.g., hypoxia, ischemia) might be required to unmask a protective role for intact EC p62 and studies are ongoing to test this.

Supported by AHA23PRE1025910 (SM); Rural and Underserved Utah Training Experience T32 (CG), Pacific Islander Research Internship Program (LG), NIHR01HL149870-01A1 (SB) and NIHRO1HL141540 (JDS).

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.
Exosomal non-coding RNA exhibit sex-specific profiles in salt sensitive hypertension
Authors: Tarhriz Vahideh , Xia Huijing , Eivazi Mortaza , Lazartigues Eric
Published: May 2024

Exosomes are secreted by various living cells and can be found in all body fluids, especially in cerebrospinal fluid (CSF) and plasma, thereby providing a source of biomarkers for cardiovascular diseases that can be used for patients’ diagnostic. Using high-throughput sequencing technologies, exosomes have been found to contain different RNA populations particularly non-coding RNA. Recently, many non-coding RNA have been recognized as key regulators in the progression of hypertension. In this study we hypothesized that female and male C57BI/6J mice exhibit different microRNA (miR) profiles after deoxycorticosterone acetate (DOCA)+salt hypertension treatment. CSF (10 μl) and plasma (100 μl) were collected from 3-month-old mice and again after 3 weeks of DOCA-salt treatment before exosomes isolation and processing for high throughput small RNA sequencing (Creative Biolabs). Analysis was performed using R language and RStudio ( https://rstudio.com/ ) as an integrated development environment. Global miRNA analysis of CSF exosomes shows different expression miRNA profiles in DOCA+salt male mice compared with their own baseline values. In addition, we observed a remarkable difference in global miRNA expression profile between DOCA+salt male mice and DOCA+salt females (p-value < 2.2e-16), while no significant difference was detected between DOCA+salt females and their baseline values (p-value = 0.6679). Moreover, we observed that there are three clusters of around 200 miR that have different expression levels in DOCA+salt males. Our analysis identified around 74 upregulated miRNA in DOCA+salt males compared to DOCA+salt females. The maximum level of expression belonged to the muscle-specific miRNA (myomiR) miR-1a-3p, miR-206-3p and miR-26a-5p which were highly accumulated in CSF derived exosomes. These top three upregulated miRNA were associated with 282 validated target genes among which, two pivotal genes, ACE2 and ADAM17, were identified in the PPI network, miRTargetLink, and by KEGG analysis. miR-26a-5p was mostly associated with ACE2 and ADAM17 and the mRNA expression of ADAM17 was associated with reduced miR-206-3p. In conclusion, our bioinformatic analysis identified three miRNA and associated them with two key molecules involved in salt sensitive hypertension. Further analysis is likely to highlight more diagnostic biomarkers and potential candidates for the development of new therapeutic approaches.

This work was supported in part by a research grant from the National Institutes of Health (HL163588).

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 2-Year Calorie Restriction Intervention Improves Diet Quality Without Compromising Nutritional Adequacy: The CALERIE Trial
Authors: Racette Susan , Garr Barry Valene , DeGraff Jasmyn , Gilhooly Cheryl , Das Sai Krupa
Published: May 2024

Background: The Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE) was the first randomized controlled trial of calorie restriction (CR) on biomarkers of aging and cardiometabolic health in humans without obesity. Objective: To determine diet quality and nutritional adequacy during a 2y calorie restriction intervention among healthy adults without obesity. Design: CALERIE phase 2 was a multi-site, randomized controlled trial of 2y of CR vs. ad libitum (AL) control. CR participants received extensive dietary education and support. Food diaries were completed at baseline and months 6, 12, 18, and 24 and analyzed using NDSR. Diet quality was evaluated using the PANDiet diet quality index. Nutritional adequacy was defined using sex- and age-specific Estimated Average Requirement (EAR) or Adequate Intake (AI) criteria for each nutrient. Results: 220 participants completed baseline assessments and were randomized (145 CR, 75 AL; 70% women, age 38±7 y, BMI 25.1±1.7 kg/m2); 86% completed the 2y study. The level of calorie restriction achieved averaged 11.9±0.7% during the 2y intervention. Diet quality improved in the CR group (BL 74.3±5.1%; CR average 76.3±5.3%, p < 0.0001), but not in the AL group ( p < 0.0002 between groups). Nutritional adequacy improved modestly in CR, but not in AL. More than 90% of CR participants achieved 100% of EAR or AI during CR for protein, carbohydrate, 6 of 12 vitamins (A, B1, B2, B3, B6, B12), and 5 of 9 minerals (copper, iron, phosphorus, selenium, sodium). Nutrients for which <90% of participants achieved adequacy included fiber, omega-3 fatty acids, vitamins B5, B9, C, E, and K, and the minerals calcium, magnesium, potassium, and zinc. Conclusion: A 2y CR intervention designed to promote healthy aging and comprised of comprehensive nutrition counseling promoted improvements in diet quality and maintenance or improvements in nutritional adequacy.

This research was supported by grants U01AG020478, U01AG020480, U01AG020487, U01AG022132, U24AG047121, R33AG070455, and R01AG071717 from the National Institute on Aging.

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.
Phrenic Motor Neuron Fractalkine Signaling to Nearby Microglia Regulates Phrenic Long-Term Facilitation
Authors: Marciante Alexandria , Mitchell Gordon
Published: May 2024

Although microglia are traditionally regarded as innate CNS immune cells, recent studies demonstrate that microglia also play key roles in normal CNS functions. For example, microglia engage in bidirectional communication with other cell types ( e.g., neurons) to regulate diverse processes including neuronal excitability, architecture and synaptic plasticity. Although neuron-microglia interactions regulate hippocampal synaptic plasticity, little is known concerning the role of microglia in regulating respiratory motor plasticity. One well-studied form of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a prolonged increase in phrenic motor output following acute intermittent hypoxia (AIH). Whereas AIH consisting of moderate hypoxic episodes (mAIH) elicits pLTF by a serotonin-dominant, adenosine-constrained mechanism, severe AIH (sAIH) elicits pLTF by an adenosine-dominant, serotonin-constrained mechanism. Neurons interact with microglia via release of fractalkine (CX3CL1), activating responses from nearby microglia, the only CNS cell-type known to express fractalkine receptors (CX3CR1). Cervical spinal fractalkine protein injections elicit phrenic motor facilitation similar to pLTF and trigger glial ATP/adenosine release, consistent with the hypothesis that neuronal fractalkine release during hypoxia elicits extracellular adenosine accumulation, thereby modulating AIH-induced pLTF. Here, we tested the hypothesis that phrenic motor neuron fractalkine protein underlies the adenosine-constraint to mAIH-induced pLTF, and drives sAIH-induced pLTF. In adult male Sprague Dawley rats, siRNA knockdown of phrenic motor neuron fractalkine mRNA and protein was achieved via intrapleural injections of siRNAs targeting fractalkine mRNA for 3 successive days. Fractalkine knock-down: 1) enhanced serotonin-dependent mAIH-induced pLTF (100±7%; n=6; vs controls: 50±5%; n=7; p<0.001), but 2) attenuated adenosine-dependent, sAIH-induced pLTF (23±12%; n=5; vs controls: 88±7%; n=7; p=0.001). These findings suggest that hypoxia triggers dose-dependent phrenic motor neuron fractalkine release, activating CX3CR1 on nearby microglia. Subsequent microglial ATP release and conversion to adenosine activates phrenic motor neuron adenosine 2A receptors which: 1) undermines mAIH-induced (serotonin-dominant) pLTF; or 2) reaches levels suffcient to drive sAIH-induced (adenosine-dominant) pLTF. These findings increase our understanding of how spinal intercellular interactions regulate AIH-induced phrenic motor plasticity.

NIH R01HL148030 (GSM), NIH R01HL149800 (GSM), NIH T32HL134621-5 (ABM).

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.
The Effect of Gestational PFOS Exposure on Metabolism in the F344 Rat
Authors: Pina Doriann , Holl Katie , Wagner Valarie , Kwitek Anne
Published: May 2024

Perfluorooctanesulfonate (or PFOS) is a type of PFOA known as (per-and poly-fluorinated alkylated substance) which is a manufactured compound chemical made of a strong carbon-fluorine and sulfonic acid. Due to its hydrophobic and lipophobic properties, industries use it in food wrappers, fire-fighting foam, sweat-proof clothing, and household cleaning products. PFOS exposure is an endocrine disruptor that may induce Metabolic Syndrome (MetS), an association of disorders that increases the risk of significant health issues like heart disease, diabetes, and stroke. Gestational and lactational exposure to Perfluorooctanesulfonate (PFOS) in F344/Stm rats was hypothesized to induce metabolic dysfunction in both female and male offspring. Pregnant F344 rats were given a control diet or diet containing 15mg/kg or 30mg/kg of PFOS; measures of body weight and body composition through nuclear magnetic resonance (NMR) were performed, and tissue was measured at the euthanasia of the dams. Both female and male offspring exposed during pregnancy and lactation were subject to the same protocol with tissue collection at three ages. On both low and high doses of PFOS, the pregnant females showed a decreased body weight gain compared to the control group; males and females also showed decreased body weight at three weeks of age when weaned from the exposed female. At 16 weeks of age, the same patterns were not revealed. The results from NMR suggested a 21% decrease in fat percentage and a 23% decrease in fat for the high exposure in the dams. The offspring at three weeks old showed a reduction in fat mass, fat mass %, and fat-free % in both high and low-dose groups compared to the control group; on the other hand, at 16 weeks old, this trend is not shown. The results suggest that PFOS had a short-term metabolic effect on the offspring at the younger; however, at the adult age, they seem to recover their initial phenotypes. For future studies, it suggested the focus on underlying mechanisms responsible for the rodent's recovery.

ASPIRE Program (R25HL168782) Medical College of Wisconsin.

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.
Muscle Fiber Heterogeneity of Insulin Sensitivity in Syntaxin-4 Enriched Skeletal Muscle
Authors: Hoolachan Joseph , Irimia-Dominguez Jose , Bloom-Saldana Elizabeth , Merz Karla , Zhou Chunxue , Bhattacharya Supriyo , Wang Jinhui
Published: May 2024

Skeletal muscle insulin resistance (IR) is a primary cause for prediabetes, an intermediary condition of higher-than-normal blood glucose levels that left untreated progresses into type 2 diabetes (T2D). A number of contributing factors associated with skeletal muscle IR include aberrant mitochondrial function, elevated reactive oxygen species, inflammation, and reduction in oxidative slow-twitch type 1 skeletal muscle fibers. One leading candidate for reversing skeletal muscle IR is Syntaxin 4 (STX4), traditionally a cell surface transmembrane SNARE protein for GLUT4 vesicle exocytosis that our lab has recently discovered is also localized in the skeletal muscle outer mitochondrial membrane. Importantly, we previously demonstrated that doxycycline-induced skeletal muscle specific STX4 overexpression in obese, prediabetes male transgenic mice (skmSTX4-iOE) reversed IR and mitochondrial dysfunction associated with diabetes muscle. However, the precise mechanisms of how STX4 restores skeletal muscle insulin sensitivity remains to be determined. To test glucose uptake and insulin sensitivity we used the “gold standard” method of hyper-insulinemic-euglycemic clamp in chow-fed skmSTX4-iOE STX4 enriched mice against non-induced controls, we identified improved glucose clearance was only associated with increased insulin sensitivity in type 1 fiber rich soleus muscle. On the other hand, insulin tolerance test studies in doxycycline-induced skeletal muscle STX4 knockout (skmSTX4-iKO) male mice revealed the opposite phenotype, suggesting that STX4 activity in slow-twitch skeletal muscle may be a contributing factor for the reversal of IR. To determine the heterogeneity of STX4 activity between different skeletal muscle fibers, myogenic precursors and intercalating non-muscular tissue in skeletal muscle STX4 enriched skmSTX4-iOE mice, we aim to use our established single nuclear RNA-Sequencing platform (snRNA-Seq) to identify pathways in distinct fiber types associated with STX4 enrichment. Overall, our work aims to determine the functional requirements of STX4 in slow-and-fast twitch skeletal muscle fiber types and identify novel molecular pathways that may serve as useful targets for the reversal/protection against skeletal muscle IR in prediabetes and T2D.

This study was supported by grants from the National Institutes of Health (DK067912, DK1129712 and DK102233) and City of Hope Shared Resources Pilot Grant.

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.
Aldo-keto reductase family 1 member A1 contributes to increase the glycolytic metabolism and to accelerate cellular senescence of bone derived mesenchymal stem cells
Authors: Liou Ying-Ming , CHIANG CHEN HAO , LIN YI-HUI , KAO YU-CUIEH , Chen Chuan-Mu
Published: May 2024

Skeletal aging characterizes the important event where bone loss is accompanied by accumulation of marrow adipose tissues. This event may be due to dysfunctional differentiation of bone derived mesenchymal stem cells (BMSCs) switched from osteoblasts (OBs) to adipocytes (ADs). Our accomplished work demonstrated that Aldo-keto reductase family 1 member A1 (Akr1A1) might serve as an important control to modify the NAD+-dependent SIRT signaling pathway for regulating the adipo-osteogenic lineage differentiation of BMSCs. In this study, we established a senescence cell model for examining whether increased Akr1A1 expression in BMSCs contributes to preferential lineage differentiation to ADs at the expense of osteogenesis by switching on the glycolytic metabolism in senescent cells with D-galactose induction or natural aging in MSCs. Our result suggests that increased expression of the glycolytic enzyme, Akr1A1 contributes to increase the glycolytic metabolism and to accelerate cellular senescence of MSCs. Such information might be useful to help develop the novel therapeutic strategy for treating age-related bone diseases with “perspective Akr1A1 inhibitors”.

Grants supported by NSTC (112-2314-B-005 -004 -, NSC-111-2314-B-005-003-), iEGG and Animal Biotechnology Center from the Feature Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE-112-S-0023-A) in Taiwan, and partially by the grants of CYCH-NCHULS110003, CYCH-NCHULS111003, CYCH-NCHULS112002, TCVGH-NCHU 112-7616 to Y-ML.

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.
Unveiling the Mechanisms of Fluid Secretion in Parotid Gland Ducts: CFTR Dependency, Cellular Contributors, and Signaling Pathways
Authors: Catalan Marcelo , Tribiños Felipe , Kirby Nicolas
Published: May 2024

Compelling evidence indicates that salivary gland ducts mediate fluid secretion, yet the specific cell types responsible for this process remain unclear. Our study aimed to elucidate the fundamental mechanisms governing fluid secretion within ducts and to determine their significance within the context of parotid gland function.Consistent with previous research in exocrine gland ducts, fluid secretion in parotid gland ducts is enhanced when both calcium and cAMP signaling pathways are simultaneously stimulated. Furthermore, we found that duct fluid secretion depends on the activities of CFTR, NKCC1, and the cAMP-activated kinase PKA.To further investigate the cellular contributors to duct-mediated fluid secretion, we generated a mouse model lacking ionocytes in salivary gland ducts using a diphtheria toxin-based approach. Our findings showed that ionocytes play a key role in the process of duct fluid secretion, as their absence resulted in impaired fluid secretion.Overall, our results underscore the significance of ionocytes in facilitating duct fluid secretion, highlighting the essential interplay between calcium and cAMP signaling pathways to achieve optimal secretion. Moreover, our research identified specific basolateral and apical ion-transporting proteins involved in the fluid secretion process. The pharmacological activation of ionocytes may offer a viable alternative secretory pathway when the function of secretory acinar cells is compromised, as observed in pathological conditions that lead to xerostomia.

Fondecyt 1211838.

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.
Skeletal Muscle Mitochondrial Response to Diet Differs by Genotype and Sex in an Alzheimer’s Disease Mouse Model
Authors: Johnson Chelsea , Lysaker Colton , McCoin Colin , Kemna Riley , John Casey , Csikos Drummond Vivien , Thyfault John
Published: May 2024

Introduction: Skeletal muscle mass is reduced in the early stages of cognitive decline. Apolipoprotein ε4 ( APOE4) is the most prevalent genetic risk factor for Alzheimer’s disease (AD) and is associated with faster motor decline in cognitively healthy individuals. APOE4 also increases the risk of insulin resistance and cardiovascular disease, conditions associated with altered bioenergetic function in muscle. However, no studies have examined the role of APOE4 in modulating muscle physiology and the impact this would have on cognitive function. We sought to determine if APOE4 affects skeletal muscle mitochondrial metabolism in response to a high-fat diet (HFD) and if this is reflected in whole-body metabolism. We also examined the role of sex since APOE4 carriers who are female are more likely to develop AD. Methods: Male and female APOE3 (n=32) and APOE4 (n= 29) targeted-replacement mice on a C57BL/6 background purchased from Taconic were fed a standard chow diet until 4 months old. Mice were then switched to a low-fat diet (LFD, 10% kcal fat) or HFD (45% kcal fat) until sacrifice. Glucose tolerance was tested at 7 months by administering an intraperitoneal glucose injection of 1g/kg lean mass. Blood glucose was measured before and 15, 30, 45, 60, 90 and 120 minutes after glucose injection. Resting respiratory exchange ratio (RER) was determined using the Promethion Indirect Calorimetry system at 8 months to determine the relative contribution of carbohydrates versus lipids to whole body metabolism. Mice were sacrificed at 9 months and carbohydrate-driven oxygen consumption was measured at basal, state 3 (ADP), state 3 + glutamate, state 3S (succinate) and uncoupled respiratory states in mitochondria isolated from quadriceps tissue on the Oroboros Oxygraph-2k system. Results: Mice fed a HFD had greater glucose area under the curve (p<0.0001) and reduced RER (p= <0.001) compared to mice fed a LFD, regardless of sex or genotype. At the level of skeletal muscle, APOE4 female mice had increased state 3 (p=0.027) and state 3 + glutamate oxygen consumption (p=0.037) on a HFD compared to a LFD while no diet effect was observed in APOE4 males or any APOE3 mice. Conclusion: We provide evidence that female APOE4 mice are susceptible to diet-induced increases in carbohydrate-driven mitochondrial oxygen consumption in muscle. Future studies are needed to determine the underlying mechanism and consequences of increased muscle mitochondrial activity, which could help identify treatment targets in female APOE4 carriers.

BHTP T32 AG078114, P30 AG072973 (Chelsea Johnson).

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.
MyoD Over-expression Reprograms Elephant Seal Fibroblasts into Muscle Cells
Authors: Lam Emily , Torres-Velarde Julia Maria , Allen Kaitlin , Vázquez Juan Manuel , Crocker Daniel , Vazquez-Medina Jose Pablo
Published: May 2024

Ex vivo tissue culture allows the study of complex cellular mechanisms that are relevant to physiological responses while overcoming the challenges presented by studying animals that are not tractable. In a primary cell culture system, certain proliferating cells can be functionally reprogrammed into other cell types via overexpression of key genes. Dermal fibroblasts can theoretically be reprogrammed into muscle cells, which are often challenging to obtain from wild organisms but offer a unique system to study metabolic responses, by overexpression of the myogenic transcription factor myoD. We derived primary dermal fibroblasts from Northern elephant seal (NES) skin samples and propagated them in primary culture. NES cells respire, stain positive for fibroblast markers (vimentin and PDGFR), and are amenable to electroporation. We overexpressed GFP-myoD in NES fibroblasts and conducted antibiotic selection and purification by FACS. As expected, expression of myoD was higher in transfected cells than in controls according to qPCR analysis (t-test p< 0.05). Treatment with small molecules (CHIR99021, Forskolin and Repsox) enhanced myoD expression. Furthermore, fibroblasts overexpressing myoD also expressed downstream myogenenic markers (myogenin, myosin heavy chain 1 and myosin heavy chain 8). MyoD-overexpressing cells differentiate into myotubes, become multinucleated and stain positively for myosin heavy chain 1. RNAseq analysis confirmed a transcriptional profile of myoD expressing cells, which is unique and different from the naive dermal elephant seal fibroblasts. Establishing differentiated muscle fibers from other mature cell types could provide a unique platform to conduct mechanistic studies in species where muscle tissue samples cannot be obtained from live animals.

The Department of Defense National Defense Science and Engineering Graduate (NDSEG) Fellowship.

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.
Dietary nitrate flows and accumulation in pig organs:15N-tracer study
Authors: Piknova Barbora , Park Ji Won , Tunau-Spencer Khalid J , Jenkins Audrey , Hellinga David G , Walter Peter J , Cai Hongyi
Published: May 2024

Nitric oxide (NO) is a free radical with multiple functions in virtually all cells. In mammals, NO is generated by nitric oxide synthase from arginine and by reduction of nitrate to nitrite by tissue xanthine oxidoreductase (XOR) and microbiome. Nitrite is then available for reduction to NO by XOR or several heme proteins. Previously we reported that 1) skeletal muscle acts as a body nitrate reservoir, and 2) stored nitrate is systemically, as well as locally used to generate nitrite and NO. Recently, we identified two additional nitrate storage organs - skeleton and skin. At baseline conditions, nitrate concentration in skeleton was 96±63 nmol/g, skeletal muscle 57±39 nmol/g and skin 56±22 nmol/g. For comparison, plasma and liver contained 34±19 nmol/g and 15±5 nmol/g of nitrate, respectively. We used 15N-labeled dose of dietary nitrate to trace its distribution among organs in time. Three hours after nitrate bolus ingestion, total nitrate levels significantly increased in all organs, reaching 221±91 nmol/g in plasma, 126±55 nmol/g in skin, 119±38 nmol/g in skeleton, 100±79 mmol/g in skeletal muscle, and 21±5 nmol/g in liver. As shown by MS, 15N-labeled nitrate distributes in non-uniform way, accumulating largely in skin. As expected, nitrate reductase activity is highest in liver, but also substantial in skin and skeletal muscle, as seen by presence of 15N-labeled nitrite. With consideration of skeletal muscle, skin and skeleton as three largest organs in mammals, these results underline their major role in maintaining homeostasis of the nitrate-nitrite-nitric oxide pathway. Also, important from therapeutic point of view, all three organs are well responding to dietary nitrate interventions by increasing their respective nitrate levels, which leads to general increase of systemic and local NO and perhaps could be used as one of the ways to increase NO availability.

Work supported by NIH internal grant to ANS.

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.
Proteomic Changes during Human Heat Stress and Heat Acclimation
Authors: Barry Hadiatou , Gagnon Daniel , Barhdadi Amina , Oussaid Essaid , Mongrain Ian , Lemieux Perreault Louis-Philippe , Dubé Marie-Pierre
Published: May 2024

INTRODUCTION. To thrive in a warmer climate, humans will need to adapt physiologically. Improved thermoregulatory and cardiovascular responses during heat acclimation have been widely described. However, the cellular and molecular mechanisms mediating these improvements remain understudied in humans. The aim of this study was to perform a broad-spectrum, non-targeted proteomic analysis to determine how plasma protein levels change in response to moderate heat stress and heat acclimation. METHODS. Ten healthy adults (4F/6H, age: 25 ± 3 years) attended two laboratory visits performed before and after a 7-day heat acclimation protocol (hot water immersion, 60 min at a rectal temperature ≥38.5°C). During the visits, blood samples were drawn before and during passive heat stress (~1.5°C increase in esophageal temperature, water-perfused suit). Proteomic analyses were performed on 2938 plasma proteins using the Olink Explore 3072 assay. We quantified the number of proteins that changed by more than one log2(fold change) in concentration. RESULTS. Prior to acclimation, ~1% of the proteins (n=29) changed in concentration during heat stress. The change in concentration for 9 of these proteins reached the p value threshold. After acclimation, 54 proteins changed in concentration under a normothermic state. During subsequent heat stress, 175 proteins (~6%) increased in concentration and 19 (~0.6%) decreased. For 180 of these proteins, the change in concentration during heat stress was unique to the post-acclimation visit. The genes for proteins that changed in concentration during heat stress prior to acclimation are more highly expressed in brain (FGFBP3), arterial (TIMP4), skin (OBP2B), lung/salivary gland/esophageal (CXCL17) tissue. The genes for proteins that changed concentration in response to heat acclimation are highly expressed in skin (MMP7, CA6, KLK14) and brain tissue (DNER). The genes for proteins that only changed in concentration during heat stress post-acclimation are more highly expressed in brain (MOG, SMOC1, PTPRN2), and arterial (TNC, C1QTNF1, MATN2, NTF3, SMOC1) tissue. CONCLUSION. This untargeted and large-scale proteomic analysis identified novel plasma proteins that change in concentration during human heat stress and subsequent heat acclimation. The individual protein results can be utilized as external validation for other studies, as the basis of further data analysis explorations, and/or to guide targeted investigations of the biological responses and adaptations to heat exposure.

Natural Sciences and Engineering Research Council of Canada.

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.