bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial Biogenesis and Remodeling Protein Response to Bouts of High-load vs High-volume Resistance Training
Authors: Mueller Breanna , Vann Chris , Roberts Michael , Kavazis Andreas
Published: May 2024

Introduction: The effects of higher-load (HL; sets of 5 repetitions at 80% of 1-RM) versus higher-volume (HV; sets of 10 repetitions at 60% of 1-RM) resistance training (RT) on various molecular outcomes has proven to be similar between the two paradigms. However, the mitochondrial response to such training paradigms remains understudied. Therefore, the purpose of this study was to interrogate protein level responses of HL vs HV RT in proteins related to mitochondrial biogenesis and remodeling. We hypothesized that HV RT would have a greater effect on mitochondrial markers of biogenesis and remodeling than HL RT. Methods: Fifteen resistance trained males participated in 6 weeks of unilateral RT (single-leg leg press and single-leg leg extension) where each leg was randomly assigned to undergo either the HL or HV RT. Biopsies of the vastus lateralis were collected from both legs at PRE and POST and western blots analysis was used to assess mitochondrial proteins related to biogenesis and remodeling. The data collected were analyzed using two-way within-subject repeated measures ANOVAs. Results: Our results show no statistically significant effect of RT condition on markers of mitochondrial biogenesis and remodeling. However, there was a main effect of time in markers of both mitochondrial biogenesis and remodeling with MFN1 decreasing (p = 0.007, ƞ2 = 0.234) and PINK1 increasing (p = 0.009, ƞ2 = 0.222) from PRE to POST. Conclusion: HL and HV RT paradigms do not differentially affect the mitochondrial markers assessed, and our hypothesis that HV RT would elicit a greater response in markers of mitochondrial biogenesis and remodeling was not supported. However, our data supports the idea that mitochondrial adaptations can be stimulated regardless of the RT paradigm. This discovery could be advantageous for individuals selecting a mode of RT training that is most likely to result in the greatest exercise adherence as well as provide multiple health benefits. Furthermore, research into RT induced mitochondrial adaptations between sexes remains understudied, thus future investigations into these bioenergetic responses is warranted.

Funding for assays and participant compensation was provided through discretionary laboratory funds from MR. Additionally, a portion of CG Vann’s effort was funded through the National Institutes of Health (R01AG054840).

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.
Liver-specific Bmal1 deletion in mice leads to increased aortic stiffness and altered vascular remodeling
Authors: Pati Paramita , Combs Robert , Srivastava Hemant , Colson Jackson , Millender-Swain Telisha , Bailey Shannon , Pollock Jennifer
Published: May 2024

Circadian rhythms have an important role in cardiovascular physiology. Circadian clock genes are critical for vascular homeostasis. Global loss of the circadian clock gene Bmal1 increases aortic stiffness with impaired vascular function and blood pressure rhythm in mice. We previously found that hepatocyte-specific Bmal1 deletion (HBK) in the liver changes perivascular adipose tissue-mediated vascular function in young adult mice, indicating that liver circadian clock disruption distally affects function in another tissue and potential cross-talk between the liver and vasculature. We hypothesized that liver-specific Bmal1 deletion leads to aortic stiffness and pathological vascular remodeling. Studies were performed in 4- to 6- month old male HBK and flox control mice. Aortic stiffness, measured by pulse wave velocity, was significantly elevated in HBK mice compared to flox control mice (Flox: 1.84 ± 0.1 m/s; HBK: 2.9 ± 0.2 m/s; n = 5, p < 0.01). Systolic blood pressure (tail-cuff) in the light phase was similar in both flox control and HBK mice (Flox: 101 ± 1 mm Hg; HBK: 103 ± 2 mm Hg; n = 3-5, p = 0.35). Aortas and plasma were collected at ZT2 for histological analysis and metabolite measurements. MetaMorph software analysis of Masson’s Trichrome-stained aortic sections revealed significantly increased aortic wall fibrosis in HBK mice (% fibrosis per area, Flox 17.0% ± 3.3%, HBK 27.4% ± 2.1%, p=0.03). The medial cell number in H&E stained-aortic sections was increased in HBK mice compared to flox control mice (Flox: 534.6 ± 31.9; HBK: 1069 ± 90.1; p < 0.01). TUNEL stained apoptotic cells were also counted and more apoptotic cells were observed in young HBK mice aortas compared to flox controls (Flox: 224.4 ± 56.1; HBK: 408.4 ± 20.8; p = 0.02). Aortic wall thickness was not different in young adult HBK mice compared to flox control mice. Aortic levels of the lipid peroxidation marker vascular 4-hydroxynonenal (4-HNE), a measure of oxidative stress, were similar between flox control and HBK mice. In plasma, the oxidative stress marker, 8-hydroxy-2’-deoxyguanosine (8-OHdG), a DNA damage product, and plasminogen activator inhibitor-1 (PAI-1) were not different between genotypes. In conclusion, our findings indicate that liver circadian clock disruption increases aortic stiffness and fibrosis with higher cell numbers as well as aortic apoptosis. These data suggest that altered vascular remodeling in HBK mice may contribute to aortic stiffness resulting in cardiovascular disease.

Funded by P01HL136267 to JSP, R21AA026906 to SMB, and AHA 856877 to PP.

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.
In Cardiac Myocytes the SGLT2 Inhibitor Ertugliflozin Attenuates Cytosolic Ca2+ Peaks After Adrenergic Stimulation
Authors: Dixit Mihir , Alam Sifat , Zhao Tengteng , Nguyen Quoc , Gierden Kyle , Yuan Jason , Vallon Volker
Published: May 2024

A novel class of antidiabetic drugs, called gliflozins, inhibit sodium-glucose cotransporter 2 (SGLT2i) to lower blood glucose levels in patients with Type 2 diabetes (T2DM). Gliflozins reduce mortality and heart failure (HF) hospitalizations in HF patients with or without T2DM, but the molecular mechanisms are unclear. We hypothesize that modulating effects on cardiac calcium homeostasis are involved in SGLT2i-induced benefits during HF, and that the SGLT2i ertugliflozin (ERTU) will alter cytosolic calcium concentration ([Ca2+]cyt) in contracting cardiomyocytes after adrenergic stimulation. Cardiomyocytes were isolated from 0–2-day old C57BL/6J mice, treated for 72 hours with either ERTU (100 nM, and 1 μM), the NHE-1 inhibitor cariporide (CARI, 10 μM), ERTU and CARI, or vehicle (Ctrl). After 72 hours, cells were either A) loaded with Fura-2, baseline cytosolic calcium levels ([Ca2+]cyt) recorded, cells stimulated with phenylephrine (PE, 100 μM), and [Ca2+]cyt recorded for additional 30 minutes, B) lysed and protein isolated for analysis by immunoblotting, or C) fixed for immunohistochemical assessment. Results: 100 nM and 1 uM ERTU reduced [Ca2+]cyt peaks after PE, but 100 nM ERTU to a lesser degree. In contrast, 72-hour-long NHE1 inhibition with CARI increased [Ca2+]cyt at baseline when compared to vehicle and ERTU, and further enhanced [Ca2+]cyt peaks after PE. Immunoblotting revealed increased protein levels of S16 phosphorylated phospholamban, decreased total phospholamban, and upregulated sarcomeric α-actinin post-ERTU. Immunohistochemical assessment of sarcomeric α-actinin localization revealed improved sarcomeric Z-line organization after ERTU administration. Sustained exposure to the SGLT2i ERTU reduced, but the NHE-1 inhibitor CARI increased [Ca2+]cyt in contracting cardiac myocytes. These data indicate that ERTU’s beneficial effect on [Ca2+]cyt homeostasis is independent of NHE1 inhibition. Since disturbed calcium handling and calcium overload are hallmarks of HF, reduced [Ca2+]cyt levels after sympathetic stimulation may contribute to the cardioprotective effect of SGLT2i. Further studies are needed to unravel the molecular mechanisms behind ERTU’s influence on cardiac calcium homeostasis.

This study was funded by Merck & Co., Inc. (Kenilworth, NJ).

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.
Chaos in the Colon: Unlocking the Puzzle of DRA (SLC26A3) Downregulation with IL-1α and p38 MAPK Amidst Brachyspira Turmoil
Authors: Challa Nitin , Enns Cole , Keith Brandon , Harding John , Loewen Matthew
Published: May 2024

This study examines the impact of Brachyspira hyodysenteriae and Brachyspira hampsonii infection on chloride (36Cl−) transport within the porcine colon. Utilizing 3H-mannitol and 36Cl−, we evaluated the bidirectional flux across colonic tissues from both healthy and infected pigs. We observed no change in 3H-mannitol flux, indicating unaffected paracellular movement. However, we observed a significant decrease in mucosal-serosal 36Cl− flux (Jm-s) in the apical segment of the spiral colon of infected pigs, suggesting a compromised chloride absorption due to the downregulation of DRA (SLC26A3), confirmed by reduced mRNA and protein levels. To dissect the molecular dynamics in vitro, we exposed polarized Caco-2 cells to B. hampsonii lysate. This model recapitulated the in vivo responses, including the downregulation of DRA and the concomitant IL-1α upregulation. This in vitro model demonstrated that B. hampsonii lysate upregulates IL-1α expression through p38 MAPK phosphorylation. More importantly, in vitro studies with B. hampsonii lysate, recombinant IL-1α, SB 203580 (p38 MAPK inhibitor), and blocking IL-1α signalling with IL-1 receptor antagonist (IL-1RA) revealed that IL-1α has a dose-dependent negative effect on DRA expression and a positive effect on IL-1α expression. Furthermore, inhibiting IL-1α expression with SB 203580 and blocking IL-1α signalling with IL-1RA not only prevented this downregulation of DRA but also promoted its upregulation, highlighting the regulatory role of IL-1α in chloride absorption. In summary, this study presents an in-depth understanding by which Brachyspira infections can impede chloride absorption in the colon and offers potential intervention targets for conditions involving impaired DRA-mediated Cl− transport.

This research was supported by Alberta Livestock Meat Association (ALMA 2013R054R) and Natural Sciences and Engineering Research Council of Canada Discovery Grant 02743-2021 (to M. E. Loewen).

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.
Metabolic effects of Type 2 Diabetes mellitus might be mitigated by grape pomace flour
Authors: Monteiro Rodrigues Luis , Nicolai Marisa , Piccoli Raphaela , Pereira Paula , Spanevello Roselia , Stefanello Francieli , Palma Maria Lidia
Published: May 2024

Type 2 diabetes mellitus (T2DM) is a multifaceted metabolic disorder characterized by insulin resistance and pancreatic β-cell dysfunction, leading to varying metabolic insults. Grape pomace, a byproduct of the winemaking industry, is under investigation as a potential therapy for T2DM due to its abundant content of bioactive polyphenols and dietary fiber. Within the current backdrop of environmental concerns, the conversion of grape pomace into a safe and applicable form, with the aim of promoting sustainable management of environmental waste, acts as a catalyst for the emergence of viable alternatives. Grape pomace flour (GPF) represents an economical and feasible solution. We hypothesized that dietary supplementation of GPF would improve the glycemic control and the associated risk factors in T2DM-induced animals. In this sense, the present study, approved by CEUA UFPel No 033578/2022-14, aimed to evaluate the effect of the GPF from ‘Arinto’ and ‘Touriga Nacional’ grape varieties on biochemical and histopathological parameters in an animal model of T2DM. To induce T2DM, rats were given a high-fat diet and a Streptozotocin (STZ) injection (35 mg/kg). The rats were divided into five groups: Control (CT), T2DM, T2DM+Metformin (Met) (250 mg/kg), T2DM+'Arinto' GPF 10% (WGPF), and T2DM+'Touriga Nacional' GPF 10% (RGPF). Results showed that Met, WGPF, and RGPF groups had significantly lower blood glucose ( p<0.05, P<0.001, and p<0.01, respectively) and triglyceride levels ( p<0.001) compared to the T2DM group. Also, we observed a significant increase in serum cholesterol levels in the T2DM when compared to the CT ( p<0.01), that was reversed in the treated groups. Additionally, the T2DM+RGPF group had higher levels of high-density lipoprotein (HDL) compared to the control group ( p<0.05). Histopathological analysis revealed structural changes in the pancreas in the T2DM animals, while tissue damage was not observed in the Met and GPF supplementation groups. In conclusion, both 'Arinto' and 'Touriga Nacional' GPF demonstrated protective effects against increased blood glucose, triglycerides, and cholesterol levels in a T2DM animal model. 'Touriga Nacional' GPF also increased HDL levels and showed protective effects on pancreatic cell structure. This suggests that GPF could be a promising dietary option for T2DM and its complications.

FCT-Foundation for Science and Technology (UIDB/04567/2020 CBIOS, and UIDP/04567/2020 CBIOS), Pelotas Federal University (UFPel), and Brazil's Federal Government (CNPq).

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.
Angiotensin-(1-7) Mas Receptors are Highly Colocalized to GABAergic Proopiomelanocortin Neurons in the Arcuate Nucleus of the Hypothalamus
Authors: Vernail Victoria , Bingaman Sarah , Silberman Yuval , Arnold Amy
Published: May 2024

Previous studies from our laboratory have shown that circulating levels of angiotensin (Ang)-(1-7), a protective hormone of the renin-angiotensin system, are greatly reduced in obese hypertensive mice. Additionally, restoration of this hormone reduces cardiovascular sympathetic tone and lowers blood pressure in this model suggesting a central mechanism of action. In support of this, our recent data show that the sympatho-inhibitory and blood pressure lowering effects of Ang-(1-7) involve activation of mas receptors (masR) within the arcuate nucleus of the hypothalamus (ARC). More specifically, we have found that masR are highly colocalized to proopiomelanocortin (POMC) neurons in the ARC. Due to the heterogeneous nature of POMC neuron subtypes, we wanted to explore the neurotransmitter phenotype of these POMCmasR neurons. We hypothesized that due to the blood pressure lowering effects of Ang-(1-7), POMCmasR are likely colocalized to inhibitory GABAergic neurons. To test this, we performed RNAscope in-situ hybridization in male C57Bl/6J mice after twelve weeks of either a 60% high fat diet (HFD) or matched control diet (n=12-18/group). We used RNAscope to colocalize expression of POMC, masR, and the vesicular GABA transporter, Vgat, mRNA transcripts within the ARC. Colocalization of POMCmasR and Vgat was quantified and percent-colocalization compared between control and HFD groups. We found that POMCmasR are highly colocalized with the GABAergic marker, Vgat, to a similar extent in control and HFD groups (84±4 vs. 90±2% of neurons, respectively; p=0.294 unpaired t-test). The population of POMC neurons that did not express masR also did not colocalize with Vgat. As an initial step to determine if masR modulates GABAergic signaling in the ARC, we assessed gene expression of glutamate decarboxylase 2 (Gad2; a GABA synthetic enzyme) in the ARC following chronic six-week treatment with systemic Ang-(1-7) versus saline infusion in male mice fed a control diet versus HFD (n=9-13/group). We found that Ang-(1-7) increases ARC Gad2 mRNA in HFD mice (1.03±0.09 control diet vs. 0.93±0.07 HFD vs. 1.23±0.07 HFD plus Ang-(1-7); p=0.023 one-way ANOVA). Supporting specificity of these actions, Ang-(1-7) did not alter POMC mRNA in the ARC. These overall data suggest that masR primarily localize to GABA-expressing POMC neurons in the ARC, and Ang-(1-7) may directly modulate GABA synthesis pathways in this brain region. Further studies are needed to elucidate the functional importance of these ARC POMC GABAergic neurocircuits to the depressor effects of Ang-(1-7) in obesity.

Funding: NIH R01 HL156986.

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.
Renin-Angiotensin System Signaling in Parietal Epithelial Cells
Authors: Fedoriuk Mykhailo , Stefanenko Mariia , Van Beusecum Justin , Palygin Oleg
Published: May 2024

INTRODUCTION. Bowman's capsule surrounds the kidney glomerular tuft and consists of a monolayer of parietal epithelial cells (PECs) creating a physiologically functioning barrier between the urinary space and the renal interstitium. Pathophysiological PEC activation describes the phenotypic switch from their normal quiescent state to one of proliferation and migration to the glomerular tuft, leading to glomerular injury. The development of several proteinuric kidney diseases is closely associated with PEC activation, however, the mechanisms of functional receptor signaling in these cells are not well described. This study investigates the functional presence of renin-angiotensin system (RAS) components and corresponding intracellular mechanisms in PECs. Methods. The vibrodissociation method was utilized to obtain freshly isolated Bowman’s capsules from discarded adult male human transplant tissues and 12-week old Wistar male and female rats. Confocal fluorescent microscopy of freshly isolated Bowman’s capsule PECs was performed using Fluo-8 or DAF-FM fluorescent dyes to detect changes in intracellular Ca2+ concentrations and nitric oxide (NO) response to the variety of RAS peptides. RESULTS. In separate experiments on freshly isolated rodent and human Bowman’s capsule PECs, we demonstrate the changes in redox response to the stimulation of RAS components. In particular, the acute application of Ang II peptide caused a rapid increase in NO production. Interestingly, we did not indicate significant changes in intracellular Ca2+ influx. To find out which receptor is responsible for the NO release we preincubated PECs with AT1R and AT2R blockers (Losartan and PD123319 respectively). Our data indicate that RAS mediates NO production through the AT2R activation. There is no sex difference was observed in these experiments. Further experiments with the variety of nitric oxide synthase (NOS) pharmacology reveal the functional distribution of NOS in rodent PECs. CONCLUSION. Our study provides an overview of the RAS signaling within PECs. For the first time, we present evidence demonstrating a direct connection between Ang II and redox homeostasis in PECs. Further investigation using the developed approach will be essential for understanding the role of Bowman's capsule cells in the glomerulus and the development of potential treatment against the hyperplasia of activated PECs and corresponding chronic kidney pathologies.

Funding Sources: R01 DK129227, R01 DK126720, VA CDA-2 1IK2BX005605-01.

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.
Chronic Intermittent Hypoxia Alters GABAergic Inhibition in Median Preoptic Nucleus (MnPO) Neurons of Rats
Authors: Farmer George , Little Joel , Cunningham J Thomas
Published: May 2024

Chronic intermittent hypoxia (CIH) is an animal model simulating the hypoxemia associated with obstructive sleep apnea (OSA) in humans. Male rats exposed to CIH exhibit increased sympathetic tone and persistent elevations in blood pressure similar to that observed in patients with. In MnPO neurons, CIH reduces GABAa inhibition and, in some neurons, GABAa activation produces excitation. Here, loose patch and perforated patch recordings were conducted in MnPO neurons in response to optogenetic stimulation of GABAergic SFO (efferent) and MnPO (local) neurons to assess the role of GABAergic signaling in these nuclei on MnPO excitability. Our working hypothesis was that CIH would diminish the inhibitory effects of optogenetic stimulation of GABA neurons. This hypothesis was tested using an AAV vector that selectively expresses channel rhodopsin in GABA neurons Using isoflurane (2-3%) anesthesia, male Sprague-Dawley rats (250-350g) received infusions (0.2 μL) of pAAV-mDlx-ChR2-mCherry-Fishell-3 in the SFO or MnPO. After recovery, rats were subjected to 7 days of CIH (0800-1600 hr) or normoxia (Norm). CIH consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) on 7 consecutive days. For in vitro recordings, rats were anesthetized with isoflurane (2-3%), and sagittal or coronal slices (300 μm) containing MnPO were cut using standard slice procedures. Voltage-clamp loose patch or perforated patch recordings using gramicidin (100 ug/ml in 140 mM K-gluconate) were collected in MnPO neurons in response to optogenetic stimulation of GABAergic neurons (2 ms pulse, 20 Hz, 1 sec). MnPO neurons from CIH-treated rats showed differing responses to GABAergic stimulation while MnPO neurons from Norm-treated rats predominantly exhibited GABAergic inhibition. MnPO neurons from Norm rats exhibited mIPSCs and outward currents in response to muscimol. MnPO neurons from CIH-treated rats show variable mIPSC frequencies and heterogeneous responses to GABAergic neuronal activation in the SFO and MnPO. The results demonstrate that CIH alters the Cl− homeostasis in some MnPO neurons altering their response to GABAa stimulation. These changes may contribute to the increased sympathetic tone and reactivity, as well as the persistent hypertension associated with CIH.

Supported by R01 HL155977.

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 stiffness is altered by age and fatigue at the cellular level
Authors: Privett Grace , Ricci Austin , Callahan Damien
Published: May 2024

Advanced age is associated with increased skeletal muscle stiffness at the cellular and whole-tissue levels. In older adults, fatigue increases falls risk. Our lab has previously observed fatigue-induced reductions in skeletal muscle cellular stiffness in younger adults, prompting the question of whether the same occurs in older adults. If so, it is possible that fatigue-induced reduction in cellular stiffness of older adults would reduce the rate of tendon loading and/or increase tissue compliance, ultimately contributing to increased falls risk. Therefore, the OBJECTIVE of the present study is to measure cellular passive and active stiffness in younger and older adults across fatigue conditions to test the hypothesis that fatigue induces tissue compliance that predisposes older adults to falls. Methods: 11 untrained males and females (7 younger, 20±1 yrs.; 4 older, 74±4 yrs.) performed unilateral knee extensions to fatigue, followed by bilateral biopsy of the Vastus Lateralis providing fatigued (F) and non-fatigued (NF) samples. Single fibers (146 younger, 130 older) were passively stretched to 156% initial length and passive modulus was calculated as the slope of the resulting stress-strain curve. Separately, Residual Force Enhancement (RFE) was measured in a subset of fibers (9 younger, 40 older) to assess passive contributions (titin) to active (actin-myosin) stretch. Main effects (age/fatigue) on passive modulus and RFE were tested via linear mixed model and t-tests, respectively. Results: Passive modulus was increased with age (p=0.04) and reduced by fatigue in both the younger (p<0.01) and older (p<0.01) participants. In a subset of fibers, RFE was increased in older women compared to younger (p<0.01) and older men (p<0.01). No main effect of fatigue on RFE was detected (p=0.49). However, RFE trended towards reduction with fatigue in a younger male (p=0.13) and was significantly reduced by fatigue in myosin heavy chain (MHC) I fibers from an older male (p=0.02). CONCLUSION: In agreement with existing literature, cellular passive modulus was higher in older versus younger adults. Additionally, fatigue reduced passive modulus in skeletal muscle fibers from both older and younger adults. Though no main effect of fatigue on RFE was detected, these results suggest that cellular RFE measures are age- and fiber-type specific.

This work was supported by funds from the Wu Tsai Human Performance Alliance.

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.
Effect of CaSR Deletion in the Esophagus on Gene Expression and Immune Function
Authors: Abdulnour-Nakhoul Solange , Kolls Jay , Flemington Erik , Ungerleider Nathan , Nakhoul Hani , Song Kejing , Nakhoul Nazih
Published: May 2024

Background: The calcium sensing receptor (CaSR) regulates Ca2+ concentration in plasma by regulating parathyroid hormone secretion. In other tissues, CaSR plays roles in cellular differentiation and migration, and in secretion and absorption. We reported previously that CaSR can be conditionally deleted in the mouse esophagus. This conditional knock-out ( EsoCaSR−/−) model showed significant reduction in levels of adherens and tight junction proteins and had significant build-up of bacteria on the luminal esophageal surface. Objectives: We aimed to further understand the role of CaSR and to examine the modification in gene expression induced by CaSR deletion in the esophagus. Methods: We performed mRNA sequencing and bioinformatic analyses of mice esophageal tissues. Gene expression was characterized using an unbiased pathway approach in EsoCaSR−/− and control mouse esophageal mucosae. Next generation sequencing was used to obtain a profile of the differences in RNA expression between tissues. RNA expression data was used to analyze differences in pathways and functional networks between EsoCaSR−/− and control esophageal tissues. Results: Our data indicated significant upregulation of gene sets involved in DNA replication and cell cycle in EsoCaSR−/−. This is accompanied by marked downregulation of gene sets involved in the innate immune response and in protein homeostasis including peptide elongation and protein traffcking. Ingenuity Pathway Analysis (IPA) demonstrated that these genes are mapped to important biological networks including Calcium and RhoA signaling pathways. Conclusions: The major findings of this study are that CaSR impacts important pathways of cell proliferation, differentiation, cell cycle and innate immune response in esophageal epithelium. The disruption of these pathways also causes significant modifications of the microbiome.

Funding: U54 GM104940 grant from NIH, VA Merit grant, Paul Teschan Research grant, Carol Lavin Bernick grant, Tulane institutional 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.
Comparing Nitric Oxide Generation Pathways using Dietary L-Arginine and Dietary Nitrate: A 15N tracer study in Wistar Rats
Authors: Brown Haleigh , Piknova Barbora , Park Ji Won , Tunau-Spencer Khalid J , Schechter Alan N
Published: May 2024

Mammals generate nitric oxide (NO) through two main pathways. In the direct production pathway, endogenous nitric oxide synthase (NOS) enzymes catalyze the reaction of L-arginine with oxygen, producing nitric oxide and citrulline. When oxygen accessibility is low, the reductive pathway supplies nitric oxide through the nitrate-nitrite-NO reduction cascade. Additional research is needed to understand whether the two NO generation pathways interact and what effects the two pathways have on the body and each other. We investigated how dietary supplementation of 15N-L-arginine (0.5 g/kg), 15N-nitrate (0.013 g/kg), or a combination of L-arginine and nitrate influences nitrate concentrations in plasma, skeletal muscle, and liver tissue from Wistar rats. We administered treatments via oral gavage. Two hours after treatment administration, we euthanized the rats and collected tissue samples. The nitrate concentrations were measured using chemiluminescence, and future mass spectrometry analysis for 15N/14N ratios will be performed. The calculated concentrations were compared between treatment groups using single factor analysis of variance. Firstly, we observed that nitrate administration significantly increased total nitrate levels in plasma, liver, and muscle tissues compared to the control group. Interestingly, L-arginine alone did not result in a significant rise in total nitrate concentrations. Notably, liver tissue from the L-arginine-treated group had slightly lower concentrations of nitrate than the control group. The combination of L-arginine and nitrate showed no statistically significant difference when compared with the nitrate-treated group in all three tissues. These results suggest that a bolus of L-arginine does not have a substantial short-term effect (within 2 hours after ingestion) on total nitrate concentrations in rat tissues. Future mass spectrometry analysis will indicate how the tissues handle acute ingestion of two different NO sources: L-arginine and nitrate. Further research must be performed for a more detailed understanding of the kinetics of arginine and nitrate, as well as for the effects of these dietary supplements on improving NO homeostasis.

This work was supported by intramural NIDDK/NIH grant ZIA DK 025104-15 to Alan N. Schechter.

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 Myocardial Infarction-induced Heart Failure on Diaphragm Arteriolar Vasorelaxation
Authors: Behnke Bradley , Horn Andrew , C Poole David , Musch Timothy
Published: May 2024

INTRODUCTION: Diaphragm atrophy and contractile dysfunction are prevalent in heart failure (HF), diminishing physical capacity and quality of life. The delivery of blood flow and oxygen necessary to support the metabolic demand of contractions is largely governed by the resistance vasculature (i.e., arterioles) with the endothelium playing a central role in arteriolar vasorelaxation. Adequate perfusion and blood flow distribution, and thus resistance vessel vasodilation, is a major determinate of diaphragm contractile function. While the mechanistic bases of HF-induced diaphragm dysfunction continue to be elucidated, the impact of HF on diaphragm vasomotor control has not been investigated. METHODS: Adult (6 mo) Sprague-Dawley rats (n=7) were divided into two groups: 1) Healthy Control (HC; n=4) and 2) MI-induced heart failure (HF; n=3). First-order arterioles (1A) from the medial costal diaphragm were isolated, cannulated, and pressurized at 90 cmH2O and allowed to equilibrate for 60 min to develop spontaneous tone. Thereafter, endothelium-dependent and -independent vasorelaxation responses to cumulative doses of acetylcholine (ACh; 10−9 to 10−4M) and sodium nitroprusside (SNP; 10−9 to 10−4M) were determined. RESULTS: There were no differences in spontaneous development (%) or maximal diameter (μm) between HC (20 ± 4%; 178 ± 11 μm) and HF (18 ± 2%; 184 ± 29 μm) diaphragm 1As ( P > 0.05). Maximal ACh-mediated relaxation (%) was significantly reduced in HF rats compared to HC rats (52 ± 2 versus 68 ± 5%; P < 0.05). Maximal endothelial-independent vasorelaxation with SNP, was not different between HC and HF rats (71 ± 3 versus 73 ± 1%; P > 0.05). CONCLUSION: The preliminary data herein demonstrate that HF impaired endothelial function and vasomotor function in the medial costal diaphragm resistance vasculature. This suggests that, in HF, there may be a redistribution of blood flow to less effcient regions of the diaphragm (e.g., ventral costal) to sustain contractile function and compensate for the diminished medial costal vasomotor function demonstrated herein. Studies including a larger sample size are warranted to investigate the impact of HF on regional blood flow distribution and resistance vessel function in the diaphragm. Such investigations would yield novel mechanistic insights into HF-induced diaphragm dysfunction.

National Institute of Aging 1R15AG078060, Ruth L. Kirschstein National Research Service Awards from the National Heart, Lung, and Blood Institute 1F31HL167618-01, and Sustained Momentum for Investigators with Laboratories Established (SMILE) Grants at Kansas State 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.
Thick Ascending Limbs (TALs)-specific deletion of ALMS1 (Alstrom syndrome 1) increases surface NKCC2 expression and NaCl reabsorption in the TAL
Authors: Maskey Dipak , Ortiz Pablo
Published: May 2024

The regulation of NKCC2 mediated NaCl reabsorption in Thick Ascending Libs (TALs) is essential to salt homeostasis and blood pressure regulation. Previously, our lab showed that NKCC2 activity is regulated by membrane traffcking and inhibiting endocytosis enhanced surface NKCC2 expression and NaCl reabsorption. We also found that ALMS1, a protein mutated in Alström syndrome, is expressed in TALs. Whole animal gene deletion of ALMS1 caused hypertension, and inhibits NKCC2 endocytosis, increasing its surface expression and renal NaCl reabsorption. Little is known about additional functions of ALMS1, however, global gene deletion of ALMS1 induces obesity, and that, in turn may affect TAL and NKCC2 function. We hypothesized that ALMS1 is a part of protein complex that binds apical NKCC2 in TALs and promotes its endocytosis. To dissect the specific role of ALMS1 in TALs, without interference of systemic effects, we studied surface NKCC2 expression and TAL mediated Na transport after inducible deletion of ALMS1 in TALs. To do this, we generated a loxP flanked exon 7 ALMS1 transgenic mouse line, that we crossed with TAL specific inducible Umod-ERT2 Cre mice (Umod-ALMS1 KO). 3 weeks after inducing Cre expression with tamoxifen, ALMS1 expression in TAL suspensions decreased by 85% (p<0.01) (n=5). In Umod-ALMS1 KO TALs the surface to total NKCC2 ratio, was increased by 37±15% compared to control floxed ALMS1 (p<0.01) (n=5). Recombinant ALMS1 (GST-Carboxyl terminus-ALMS1), or immunoprecipitation of ALMS1, pulled down NKCC2 from TAL lysates (n=3), suggesting that the mechanism by which ALMS1 regulates surface NKCC2 involves protein-protein interactions. Three weeks after inducing ALMS1 deletion, we measured bumetanide-induced natriuresis (4h) as an index of NKCC2-mediated NaCl absorption. We found that bumetanide induced UNa excretion was enhanced in Umod-ALMS1 KO, compared to control (control: 110±7 vs Umod-ALMS1 KO: 173±26 μmols Na/4h, p<0.01). We conclude that ALMS1, binds NKCC2 to regulate its surface expression. Depletion of ALMS1, specifically in the TAL enhances surface NKCC2 and TAL NaCl reabsorption. Our data indicate that regulation of the ALMS1-NKCC2 interaction in the TAL is important for renal NaCl reabsorption. Decreased ALMS1 function or expression increased TAL NaCl reabsorption and this may contribute to the hypertension and kidney disease that is associated with SNPs in the ALMS1 gene in the general population, or in patients with Alstrom Syndrome.

NIH R01, Henry Ford Fund.

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.
Sirtuin 4 regulates nutrient-stimulated insulin secretion by modulating GLP-1 secretion
Authors: Penney Magan C. , Do Evan , Luong Amy , Huynh Frank K.
Published: May 2024

Sirtuin 4 (SIRT4) is a mitochondrial enzyme that can remove several novel post-translational modifications (PTMs) on target proteins and thereby modify their activity. We and others have previously shown that young mice lacking SIRT4 (SIRT4KO mice) have elevated glucose, leucine, and glutamine-stimulated insulin secretion. As these mice aged, they developed insulin resistance and glucose intolerance. We hypothesize that when these mice are young and eat a mixed nutrient diet, they secrete elevated amounts of insulin in response to each meal and this hyperinsulinemia drives the development of insulin resistance in old age. However, while insulin secretion in response to glucose, leucine, and glutamine individually has been reported in these mice, it has not been shown how much insulin these mice secrete in response to combinations of these nutrients, which would more closely resemble a normal meal. Thus, we sought to characterize insulin secretion in SIRT4KO mice in response to mixed nutrient challenges. Indeed, SIRT4KO mice secrete more insulin response to different combinations of glucose, leucine, and glutamine. In most cases, these increases in insulin secretion were synergistic, rather than just an additive effect of each individual nutrient. In particular, when leucine and glucose were administered together in aged SIRT4KO males, plasma insulin levels were synergistically increased more than 3-fold over glucose or leucine alone. Remarkably, treating aged SIRT4KO mice with leucine during a glucose tolerance test resulted in so much insulin secretion that glucose intolerance was normalized. Importantly, despite these extremely high insulin levels, there were no signs of dangerous hypoglycemia. To determine whether these synergistic effects of glucose and leucine on insulin secretion were due to effects of SIRT4 on pancreatic beta-cells, we treated beta-cell specific SIRT4KO mice with glucose and leucine and found no differences in insulin secretion compared to wild-type mice, suggesting that SIRT4 affects insulin secretion by acting indirectly on the beta-cell. To determine whether SIRT4 may affect insulin secretion by altering incretin secretion, we measured GLP-1 secretion in response glucose and leucine in combination and found that GLP-1 secretion was elevated in SIRT4KO mice compared to wild-type controls. Taken together, our data show that SIRT4 may be a key regulator of insulin secretion in response to mixed nutrients via effects on GLP-1 secretion.

NIH Support of Competitive Research (SCORE) Pilot Project Award #SC2GM141997 San Jose State University Department of Biological Sciences Start-up Funds.

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.
Identification of Vasopressin-Regulated Aquaporin-2 Traffcking Mechanism with Proximity-Labeling Proteomics
Authors: Liu Chin-Fu , Lin Yi-Jiun , Yu Ming-Jiun
Published: May 2024

Vasopressin controls collecting duct water permeability in part by regulating traffcking of aquaporin-2 to and from the apical plasma membrane in the principal cells. Evidence have shown that aquaporin-2 resides in Rab11-positive recycling endosomes where its PDZ motif is phosphorylated at serine 269, leading to apical aquaporin-2 localization in response to vasopressin. Here, we employed Rab11-conjugated biotin ligase (TurboID) to biotinylate kinases and traffcking machineries recruited to Rab11-positive endosomes in the mpkCCD cells in response to vasopressin vs. vehicle. The biotin-labeled proteins were purified with streptavidin-affnity chromatography and identified with protein mass spectrometry. A total of 2,948 proteins were quantified in three independent repeats of which 119 proteins showed increases in the Rab11-positive endosomes in response to vasopressin while 104 proteins showed decreases. GO term analysis of the 119 increased proteins showed enriched biological processes including transport, protein transport, and vesicle-mediated transport. Microtubule affnity regulating kinase 2 stood out from our Bayes theorem probability analysis as a candidate kinase for aquaporin-2 serine 269 phosphorylation. Purified microtubule affnity regulating kinase 2 phosphorylated a synthetic aquaporin-2 peptide at serine 269 in vitro. Among the 119 proteins is the PDZ-domain containing Rho GTPase-activating protein 21 involved in F-actin dynamics. Switching between GTP- and GDP-bound state, Rho GTPase-activating protein 21 promotes F-actin polymerization and depolymerization, respectively. Our preliminary observations are indicative of a hypothetical model where vasopressin recruits microtubule affnity regulating kinase 2 to the Rab11-positive endosomes where it phosphorylates aquaporin-2 PDZ motif at serine 269. The phosphorylated PDZ motif recruits and activates Rho GTPase-activating protein 21, resulting in the GDP-bound state that promotes F-actin depolymerization. The depolymerized F-actin would facilitate fusion of the aquaporin-2 vesicles with the apical plasma membrane.

National Science and Technology Council, TAIWAN, MOST 111-2320-B-002-084-MY3.

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.
Sesamin Enhances Apoptosis of Activated T cells by physically Interacting with MCL-1 and Shows Therapeutic Effect on Allergic Dermatitis
Authors: Park Hee-Suk , Lee Hyun-Su
Published: May 2024

Sesamin is lignans mainly found in sesame seeds and oil. Previous studies have demonstrated that sesamin has potent pharmacological activities including anti-inflammatory, antioxidative, and cholesterol-and lipid-lowering. Detailed studies performed with sesamin revealed that it possesses anti-proliferative and pro-apoptotic properties that could potentially be used to kill tumors. Nevertheless T cell-mediated disorders usually occur when over-activated T cells are proliferative and less apoptotic, however, it is unknown whether sesamin suppresses T cell activation with underlying mechanism related to proliferation and apoptosis. In the present study, we evaluate whether sesamin upregulates apoptosis on activated T cells and improves T cell-mediated disease. First of all, we confirmed that sesamin suppresses IL-2 production and CD69 expression from activated T cells by quantitative PCR, ELISA and flow cytometry. In silico analysis showed that Myeloid cell leukemia 1 (MCL-1) is predicted to physically interact with sesamin in T cells. According to the in silico prediction, pulldown assay using sepharose4B beads and western blot results showed that treatment with sesamin physically binds to MCL-1 in T cells and regulates S56 phosphorylation level of MCL-1 which represents activity of MCL-1 in activated T cells. Moreover, inhibition of MCL-1 activity by sesamin blocked heterodimer interaction between MCL-1 and BAK in activated T cells by co-immunoprecipitation assay. These results led sesamin to selectively induce cell death pathway only in activated T cells. Oral administration of sesamin alleviated the symptoms of dinitrochlorobenzene (DNCB)/mite extract-induced AD, including ear thickness, mRNA levels of pro-atopic cytokines on the ear lesions of sesamin-treated AD mice. Therefore, these results suggest that sesamin has a therapeutic potential for treating T-cell mediated disease through modulating MCL-1 activity on activated T cells.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2021R1I1A1A01059119).

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.
Mitochondria control IP3-evoked Ca2+ release triggered by voltage-dependent Ca2+ entry in intact resistance arteries
Authors: Zhang Xun , Lee Matthew , Buckley Charlotte , Wilson Calum , McCarron John
Published: May 2024

The contractility of vascular smooth muscle cells (VSMCs) is a major contributor of vascular tone and blood pressure. In VSMCs, intracellular Ca2+ level determines the contractile activity that generates the force to contract arteries. A major source of Ca2+ is via influx through voltage dependent Ca2+ channels (VDCCs). While mitochondria are now recognised as key regulators of intracellular Ca2+ homeostasis in several cell types, their role in modulating Ca2+ signalling mediated by VDCC in VSMCs is unresolved. Given the potential physiological significance, and unclarified interaction between mitochondria and VDCCs in VSMCs, we hypothesize that mitochondria directly regulate Ca2+ signalling mediated by VDCCs. The interplay between mitochondria and VDCCs was investigated by imaging and analysing intracellular Ca2+ signals in smooth muscle cells in intact arteries from rat mesentery. Depolarization of the plasma membrane potential, by high potassium (20 mM) physiological saline solution, triggered Ca2+ influx through VDCCs and a sustained increase in intracellular Ca2+ on which repetitive Ca2+ oscillations occurred. All Ca2+ signals were abolished by removal of external Ca2+ and by dihydropyridine inhibitors of VDCCs. Significantly, the repetitive Ca2+ oscillations, but not the sustained Ca2+ signals, were blocked by the IP3 receptor inhibitor 2-APB and SERCA inhibitor cyclopiazonic acid. Neither the repetitive Ca2+ oscillations or the sustained Ca2+ signals were altered by the ryanodine receptor inhibitors ryanodine and dantrolene. These results suggest that Ca2+ influx via VDCC triggers Ca2+-induced Ca2+ release at IP3 receptors in intact mesenteric arteries. Depolarization of the mitochondrial membrane potential (ψm), with the uncoupler CCCP or complex I inhibitor rotenone, but not ATP deprivation with the ATP synthesis blocker oligomycin, inhibited VDCC evoked IP3 mediated Ca2+ oscillations but not the sustained Ca2+ signals. Furthermore, in intact arteries, depolarization of ψm directly suppressed inositol triphosphate receptors (IP3Rs) mediated Ca2+ release from the internal Ca2+ store evoked by photolysis of caged IP3. These results suggest that mitochondria regulate Ca2+ release via IP3R triggered by Ca2+ entry via VDCC. In return, Ca2+ entry via VDCCs did not alter ψm, but upregulated ROS production. Together, these results suggest that mitochondria regulate Ca2+-induced Ca2+ release at IP3 receptors triggered by Ca2+ influx via VDCCs but do not directly regulate VDCC activity.

British Heart Foundation (RG/F/20/110007; PG/20/9/34859).

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.