bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Role of Endothelial CD47 in the Regulation of Pathogenesis of Coronary Artery Disease
Authors: Chen Hong
Published: May 2024

Background: The anti-phagocytic molecule CD47 is upregulated in atherosclerotic plaques of patients with CVD. However, the role of endothelial cell-specific expression of CD47 in the pathogenesis and progression of atherosclerosis has not yet been fully defined. Kojima et.al. described atheroprotective effect of anti-CD47 antibody by enhancing efferocytosis in atherosclerosis. In this study, we determined whether endothelial cell-specific CD47 regulates efferocytosis in atherosclerosis. Methods: Endothelial cell-specific, tamoxifen-inducible CD47knockout (CD47iECKO) mice were injected with a single dose of AAV-mutant PCSK9 via the tail vein. Susceptibility to atherosclerosis was evaluated in cross sections of the aortic sinus in mice fed Western Diet for 12 weeks. Using single-cell RNA sequencing and in vitro efferocytosis assays, we investigated the role of CD47 in regulating efferocytosis in an atherosclerotic mouse model. Results: Endothelial cell-specific deletion of CD47 led to a significant plaque reduction in the aortic sinus of athero-CD47iECKO mice. Cluster analysis and gene expression profiles of 20,000 aortic endothelial cells from athero-control and athero-CD47iECKO mice identified 10 major cell types from the integrated data: endothelial cells, vascular smooth muscle cells, fibroblasts, immune cells (B cells, T cells, macrophage), erythrocytes, oligodendrocytes cells, Schwann cells, and pericytes. After Western Diet intake, in the CD47iECKO group, a subpopulation of endothelial cells as well as fibroblasts, T-cells, B-cells, resident aortic macrophages, erythrocytes, oligodendrocytes, and pericytes were increased, while vascular smooth muscle cell numbers decreased. Using an in vitro efferocytosis assay on isolated primary endothelial cells from control and CD47iECKO mice, we observed CD47 deletion induced engulfment of apoptotic Jurkat cells, which had been exposed to BAY11-7082 (NF-κB inhibitor) to stimulate the apoptotic attributes. Quantitative PCR analyses showed that CD47 deletion increases the expression of efferocytosis receptor FasL, CX3CL1, Gpr132, MerTK, Scarb1, Sirpa, TSP1, and HMGB1. Conclusion: Our study indicates that genetic deletion of CD47 in endothelial cells attenuates atheroma progression by enhancing efferocytosis. This study also reveals phenotypic heterogenicity of endothelial cells at the single cell level during atherogenesis and provides a deeper understanding of endothelial cell biology in atherosclerosis that may facilitate the development of novel therapeutic interventions for this cardiovascular disease.

NIH, AHA.

This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Induction of Alcoholic Ketoacidosis in Mice
Authors: Ryu Clara , Rivadeneira Ana , Narayanan Ashwini , Kim Gou Young , Choi Inyeong
Published: May 2024

Chronic alcohol use depletes glycogen stores in the liver and impairs gluconeogenesis, causing decreased glucose availability and increased reliance on fatty acid breakdown. This also alters counterregulatory hormone balance that further increases free fatty acid breakdown and ketone production, resulting in ketoacidosis. The main features of alcoholic ketoacidosis (AKA) are high ketone bodies, low blood bicarbonate levels, and high anion gaps. This metabolic disorder was first reported in 1940s and pathophysiological data were collected from patients taken several decades ago, which helped to lay the foundation for the current understanding of its pathophysiology, diagnosis, and management. Nonetheless, our understanding of AKA is presently limited and has many challenges. To better understand its biochemical and pathophysiological properties, we induced AKA in mice (8-weeks; male) by modifying the clinically relevant NIH alcohol feeding protocol. Compared to mice pair-fed with control diet, mice fed chronic/binge alcohol under a fasting condition developed low blood HCO3− (20.07 mM for pair-fed vs. 13.04 for EtOH-fed mM; p < 0.05; n = 4-6/group), high β-hydroxybutyrate (0.54 mM for pair-fed vs. 3.62 for EtOH-fed mM; p < 0.05), and high pH anion gap (19.7 for pair-fed vs. 33.3 for EtOH-fed; p < 0.05), all of which are hallmarks for AKA. Blood pH was also low (7.32 for pair-fed vs. 7.14 for EtOH-fed; p<0.05) and base excess was more negative, consistent with metabolic acidosis. Mice subjected to a fasting alone exhibited a mild increase in β-hydroxybutyrate. Furthermore, [HCO3−], anion gap, and blood pH remained unchanged in these fasting-alone mice. In mice with AKA, the NADH/NAD+ ratio in the liver was decreased, consistent with the conversion of acetoacetate to β-hydroxybutyrate. These mice also had decreased glucose and insulin levels, consistent with hormonal changes in response to impaired gluconeogenesis and glycogen depletion. The mice also had increased aspartate transaminase AST levels in the blood. Histopathological analysis of the livers revealed diffuse mixed microvesicular and macrovesicular steatosis and mild lobular inflammation in AKA mice. Few acidophilic bodies (necrosis) and ballooning degeneration were seen. Except for the liver, most organs showed no significant difference between the two groups of mice. In summary, AKA mice showed biochemical parameters that are similar to those in humans diagnosed with AKA. The animal model of AKA will be useful for preclinical studies that search and test prevention strategies to alleviate AKA or evaluate the therapeutic effcacy of drug candidates.

This work was supported by the grant from the National Institute of Alcohol Abuse and Alcoholism (R21AA028606).

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.
Cerebrovascular Conductance Responses to Carbon Dioxide are Attenuated by the Presence of Hypertension in Patients with Atrial Fibrillation
Authors: Walsh Harvey , Junejo Rehan , Lip Gregory , Fisher James
Published: May 2024

The cerebral vasculature is highly sensitive to carbon dioxide (CO2) and cerebrovascular CO2 reactivity (CVRCO2) is widely used to index cerebral vascular health. Some studies have shown that both atrial fibrillation (AF) and hypertension (HTN) are independently associated with reduced CVRCO2. However, it is unknown if these effects on CVRCO2 are synergistic when AF and HTN are present together. In this study we tested the hypothesis that CVRCO2 was lower in patients with both AF and HTN (AF+HTN) compared to normotensive patients with AF. Transcranial Doppler ultrasound was used to measure middle cerebral artery blood velocity (MCA Vm) in AF (n=31) and AF+HTN (n=57) during stepped increases and decreases in end-tidal carbon dioxide (PETCO2). The ratio of MCA Vm and mean arterial pressure (MAP) was defined as cerebrovascular conductance index (CVCi). CVRCO2 was taken as the linear slope for MCA Vm and MCA CVCi versus PETCO2. As expected, baseline MAP was higher in AF+HTN than AF (107±9 vs. 98±9 mmHg, respectively; p<0.001). Baseline MCA Vm was numerically lower in AF+HTN than AF, but this did not reach statistical significance (50 [44-60] vs. 52 [45-63] cm.s−1, respectively; p=0.075), while baseline CVCi was lower in AF+HTN compared to AF (0.46 [0.42-0.57] vs. 0.54 [0.44-0.63] cm.s−1.mmHg−1; p<0.001). MCA Vm CVRCO2 was not different between groups (1.70 [1.47-2.19] and 1.74 [1.54-2.52] cm/s/mmHg−2 for AF+HTN and AF, respectively; p=0.221), whereas MCA CVCi CVRCO2 was 13% lower in AF+HTN compared to AF (0.013 ± 0.004 vs. 0.015 ± 0.005 cm.s.mmHg−2; p=0.047). Stepwise linear regression analysis identified that cardiac rhythm during data acquisition (AF or normal sinus rhythm) was a predictor of baseline MCA CVCi and MCA CVCi CVRCO2. In summary, our results demonstrate impaired MCA CVCi CVRCO2 in AF+HTN and suggest that the presence of HTN worsens cerebrovascular function in AF. Such cerebrovascular alterations may be important for the development of cognitive decline and diminished brain health in AF.

Funding provided by Bristol-Myers Squibb/Pfizer Alliance Project Grant (CV185-481), British Heart Foundation Project Grant (PG/15/45/31579) and the National Institute of Health Research Clinical Research Network (NIHR CRN).

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.
Trimethylamine-N-oxide aggravated sympathetic activation in D-galactose induced aging rats by down-regulating P2Y12 receptor in microglia
Authors: Wu Yuming , Wang Ping , Mi Yuan , Yu Hao , Teng Xu , Jin Sheng , Xiao Lin
Published: May 2024

This study aimed to determine whether trimethylamine N-oxide (TMAO) was involved in sympathetic activation in aging and the underline mechanisms. Our hypothesis is that TMAO reduces P2Y12 receptor and induces microglia-mediated inflammation in the paraventricular nucleus, then leading to sympathetic activation in aging. Eighteen young adults (20-40 years old) and sixteen old adults (65-90 years old) were involved in the study. Aging rats were established by injecting D-galactose (200 mg/kg/d) subcutaneously for 12 weeks. TMAO (120 mg/kg/d) or 1% 3, 3-dimethyl-l-butanol (DMB, a structural analog of choline, inhibiting TMA formation) was administrated in drinking water for 12 weeks to investigate their effects on neuroinflammation and sympathetic activation in aging rats. Heart rate variability was analyzed by 24 h ambulatory ECG multichannel recording system (CT-083S Holter ECG recorder, BENEWARE, Hangzhou) in young and old adults. The PowerLab 15T data acquisition system (AD Instruments, Australia) was used to record blood pressure and renal sympathetic nerve activity in the rats. Plasma levels of TMAO, noradrenaline, and IL-1β in old adults were higher than those in young group. In addition, standard deviation of all normal to normal intervals and standard deviation of the average of normal to normal intervals were lower in old adults and negative correlated with TMAO, which indicating sympathetic activation in old adults and related to the increased TMAO. Rats treatment with D-gal showed increased senescence-associated protein, microglia-mediated inflammation levels and decreased P2Y12 receptor in the paraventricular nucleus. Plasma levels of TMAO, noradrenaline, and IL-1β were increased, accompanied by enhanced renal sympathetic nerve activity. While TMAO supplementation aggravated the above phenomenons, and DMB abated them. These findings suggested that TMAO might contribute to the sympathetic hyperactivity of aging via downregulating P2Y12 receptor in microglia and increasing inflammation in the paraventricular nucleus. These results may provide a new target for the prevention and treatment of aging and aging-related diseases.

This work was supported by the Program for the National Natural Science Foundationof China (32271155 and 31671185), the Science and Technology Project of the Hebei Education Department (ZD2021076), the Youth Top Talent Foundation of Hebei Province of China and the Program of Clinical Medicine Innovation Research Team of Hebei Medical University (2022LCTD-A1).

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.
SLC26A6 Deficiency Differentially Alters the Expression of MicroRNAs and RNA Binding Proteins in the Intestine
Authors: Natarajan Anbazhagan Arivarasu , Husain Nazim , Kapoor Shaunik , Kulkarni Neelkanth , Kumar Anoop , Jayawardena Dulari , Liqyanah Tala
Published: May 2024

BACKGROUND: Putative anion transporter 1 (PAT1, SLC26A6), expressed at the luminal membrane of the intestinal epithelial cells, plays a key role in oxalate homeostasis and bicarbonate secretion. Additionally, we have recently shown that PAT1 knockout mice (PKO) exhibit compromised barrier integrity, gut dysbiosis and enhanced susceptibility to colitis. As defective PAT1 function and/or expression alters intestinal vulnerability to inflammation and miRNAs and RNA binding proteins are implicated in pathophysiology of gut inflammation, the focus of the current study was to investigate if loss of PAT1 modulates the expression of these post transcriptional regulators. Methods: microRNA was extracted from the colonic mucosa of age-matched PKO and wild type (WT) littermates (Qiagen miRNeasy mini kit) and subjected to miRNA expression profiling by Nanostring nCounter Mouse miRNA panel. Acquired raw data was further analyzed using the nSolver Analysis Software and ROSALIND online software platform to identify the differential gene expression. RT-PCR and western blotting was used to analyze the mRNA and protein expression of RNA binding proteins. Results: Genetic deficiency of PAT-1 resulted in differential expression of miRNAs compared to WT mice, where 60 miRNAs were notably upregulated, and 5 miRNAs were significantly downregulated in the colon. miR-150, known to be downregulated in ulcerative colitis patients, showed the maximum decrease (~6.9-fold change, P< 0.02) in PKO mice. Of note, miR-767 (known to promote tumor progression) was the top upregulated miRNA (~5.2-fold change, p<0.01). Interestingly, in silico analysis using TargetScan revealed that most of the upregulated miRNAs have been associated with the decrease in gut barrier function as previously shown in PKO mice. Further, a remarkable decrease in the protein expression of RNA binding proteins, HUR (0.82 ± 0.07 WT vs 0.49 + 0.03 PKO, P< 0.5) and CUGBP-1(0.37 ± 0.08 WT vs 0. 0.12 ± 0.03 PKO, P< 0.5) was also noted in PKO compared to WT mice. CONCLUSION: Our data provide evidence that PAT-1 deficiency in mice results in differential expression of several posttranscriptional regulators including miRNAs and RNA binding proteins with gene targets implicated in maintenance of epithelial barrier integrity. We speculate that altered expression of posttranscriptional regulators may potentially contribute to altered gut homeostasis observed in diseases associated with PAT1 deficiency.

Supported by VA/NIH.

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 intermittent hypoxia on glucose tolerance in adults with type 2 diabetes
Authors: Zhao Jiahui , Stray-Gundersen Sten , Lalande Sophie
Published: May 2024

Hypoxia stimulates glucose uptake through an insulin-independent pathway. The purpose of this single-blind randomized study was to determine the acute effect of intermittent hypoxia, consisting of alternating short bouts of breathing hypoxic and normoxic air, on glucose and insulin concentrations during an oral glucose tolerance test in adults with type 2 diabetes. It was hypothesized that intermittent hypoxia would attenuate the increase in glucose and insulin concentrations during an oral glucose tolerance test. Six adults with type 2 diabetes (5 men, age: 51±15 years, HbA1c: 7.3±1.5%) visited the laboratory on two occasions. On both visits, a 2-hour oral glucose tolerance test was performed, with venous blood samples collected 0, 30, 60, 90, and 120 min after ingestion of a high-glucose drink. Following ingestion of the drink, participants were exposed to either an intermittent hypoxia (IH) protocol, consisting of eight 4-min hypoxic cycles at a targeted arterial oxygen saturation of 80% interspersed with breathing room air to resaturation, or an intermittent normoxia (IN) protocol consisting of eight 4-min normoxic cycles interspersed with breathing room air. By design, oxygen saturation was lower during intermittent hypoxia than intermittent normoxia (81±3 vs. 97±1%, p<0.01). Relative changes in plasma glucose concentrations in response to the oral glucose tolerance tests were not different between conditions (IH vs. IN: 30: 42±15 vs. 35±15; 60: 57±26 vs. 65±30; 90: 73±32 vs. 88±30; and 120: 78±26 vs. 83±29 mg/dl, interaction effect: p=0.13). Similarly, the relative changes in insulin concentrations in response to the oral glucose tolerance tests were not different between conditions (IH vs. IN: 30: 14±17 vs. 25±17; 60: 42±36 vs. 48±35; 90: 70±58 vs. 86±60; and 120: 103±72 vs. 125±94 ulU/ml, main effect for condition: p=0.12). While these preliminary results did not reach statistical significance, the observed trends for reduced glucose concentrations in combination with lower insulin concentrations during intermittent hypoxia suggest that short bouts of hypoxia improve glucose tolerance by stimulating glucose uptake independently from the action of insulin. Data from a larger sample size of adults with type 2 diabetes are needed to confirm these preliminary findings.

None.

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.
Plasma Branched-Chain Fatty Acids Reflect Dairy-Fat Intake in a Pilot Clinical Trial in Individuals with Prediabetes
Authors: Taormina Victoria , Eisenhardt Simonne , Gilbert Matthew , Poynter Matthew , Kien C. Lawrence , Kraft Jana
Published: May 2024

Epidemiological studies often rely on fatty acid (FA) biomarkers of dairy-fat intake to represent diet history and to establish associations with cardiometabolic risk factors. However, a more robust body of direct research is needed to validate the currently utilized FA biomarkers as accurate representations of dairy-fat intake. Thus, our objectives were to i) compare the plasma FA profile of individuals with prediabetes following the consumption of a diet with full-fat (3.25%) yogurt (FFY diet) or non-fat yogurt (NFY diet) to evaluate their validity of currently utilized biomarkers and ii) identify additional FAs to serve as potential biomarkers. We hypothesized that the proportion of the unique dairy-derived FAs including odd-chain FAs (13:0, 15:0, 17:0, 19:0, 21:0, 23:0), branched-chain FAs ( iso-13:0, anteiso-13:0, iso-14:0, iso-15:0, anteiso-15:0, iso-16:0, iso-17:0, anteiso-17:0, iso-18:0), and the trans-FAs 16:1 trans-9 and 18:1 trans-11 would increase in plasma total lipids following the consumption of three daily servings of full-fat yogurt for three weeks. Our 8-week randomized, double-masked, controlled crossover study included six men and seven women aged 45-75 years old. Each experimental diet period, NFY diet and FFY diet, was three weeks in length. The arithmetic difference in fat (8% energy) between the NFY diet (30% fat) and the FFY diet (38% fat) was solely due to the dairy fat in the full-fat yogurt. Following each experimental diet period, fasting plasma samples were taken and analyzed for FA composition of plasma total lipids using gas-liquid chromatography. Consumption of the FFY diet resulted in no change in the proportion of total odd-chain FAs or the individual FAs 16:1 trans-9 and 18:1 trans-11, but the proportion of total branched-chain FAs was greater in response to the FFY diet ( P < 0.01). For individual odd-chain and branched-chain FAs, the proportions of iso-17:0 and anteiso-17:0 were greater in response to the FFY diet ( P < 0.01 for both); however, there were no differences in anteiso-15:0 or iso-15:0 proportions by diet. Results from this pilot trial suggest that commonly utilized FA biomarkers may not be suffciently robust to represent dairy-fat intake. Branched-chain FAs should be further explored for alternatives.

Funding for this project was provided by National Dairy Council Nutrition Research Funding, a USDA Agriculture and Food Research Initiative National Institute of Food and Agriculture Predoctoral Fellowship, and a fellowship from the University of Vermont’s Food Systems Research Center.

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.
KPs improve renal function and blood vessels function by balance the sFlt-1:PlGF ratio in L-NAME-induced SHRSP preeclampsia rats
Authors: Chen Yu-Hsuan , Chen Chuan-Mu
Published: May 2024

Preeclampsia (PE) is one of the major problems for global healthy burden. It affects 2 to 8% of all pregnancies and causes both maternal and infant morbidity and mortality. Kefir peptides are products from many kinds of prebiotic fermentation in whole milk by kefir grain. In this study, we used the N(ω)-nitro-L-arginine methyl ester (L-NAME) in drinking water to mimic the preeclampsia in spontaneous hypertension rat stroke-prone (SHRSP) pregnant rats, the rats were assigned to five treatment groups: the normal group, untreated group, L-NAME group, L-NAME+KPs (LD and HD dose diets) for 20-day experiment. Data showed that rats only gave the L-NAME group induced severe high blood pressure, urine protein, placenta damage, and embryo reabsorption. After KPs pre-administration significantly reduced urine protein production and generalized vessel endothelial dysfunction, anti-oxidative, and anti-inflammation levels in SHRSP rats placenta. In conclusion, kefir peptides is a multipurpose peptide that can suppress oxidative stress, HIF-1a, anti-TNF-α and IL-6 inflammation levels, reducing urine protein production and generalized vessel endothelial dysfunction and balance the sFlt-1:PlGF ratio through blocking eNOS/NO. KPs might be a promising protective nutraceutical agent for early pregnancies.

The Higher Education Sprout Project by the Ministry of Education (MOE-112-S-0023) in Taiwan.

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.
Renal upregulation of NCC counteracts empagliflozin-mediated NHE3 inhibition in normotensive but not in hypertensive rats
Authors: Coelho Paulo , Santos-Rios Thiago , Martins Flavia , Crajoinas Renato , Caetano Marcos , Luchi Weverton , McCormick James
Published: May 2024

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in hypertensive patients. However, the underlying molecular mechanisms remain largely unknown. Proximal tubule (PT) NHE3-mediated sodium reabsorption is inhibited by SGLT2i in normotensive rodents, yet no BP-lowering effect is observed under this scenario. This study examined the effect of empagliflozin (EMPA) on renal tubular sodium transport in normotensive and spontaneously hypertensive rats (SHRs). It also tested the hypothesis that EMPA-mediated PT NHE3 inhibition in normotensive rats is associated with upregulation of distal nephron apical sodium transporters. Administration of EMPA for 14 days reduced BP in 12-week-old SHRs but not in age-matched Wistar rats. PT NHE3 was inhibited by EMPA treatment in both Wistar and SHRs. EMPA increased NCC expression and phosphorylation levels in Wistar but not SHRs. SHRs exhibited higher cortical and medullary NKCC2 and β-ENaC subunit mRNA and higher medullary α-ENaC expression levels than Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), EMPA combined with HCTZ or vehicle for 14 days. In these rats, BP was reduced in response to EMPA and HCTZ combined but not by EMPA or HCTZ alone. Altogether, these data suggest that upregulation of NCC counteracts EMPA-mediated inhibition of PT NHE3 in male normotensive rats, thereby maintaining their baseline BP. Moreover, reducing NHE3 activity without further upregulation of the main apical sodium transporters beyond the PT may contribute to the BP-lowering effect of SGLT2i in experimental models and patients with hypertension.

FAPESP 2016/22140-7 and 2021/14534-3.

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.
K+ Channel Activation Inhibits Allergen-Stimulated ATP Release by Human Bronchial Epithelial Cells
Authors: Srisomboon Yotesawee , Kita Hirohito , O'Grady Scott
Published: May 2024

Allergens derived from the fungus Alternaria alternata stimulate the release of multiple alarmins including ATP, IL-33 and DNA fragments from human bronchial epithelial cells (hBECs). Oxidative stress induced by Alternaria extract activates ATP release by opening voltage-dependent anion channels (VDAC-1) in the apical membrane and by stimulating exocytosis of ATP containing membrane vesicles. The present study shows that pretreatment of hBECs with K2P channel inhibitors prior to Alternaria exposure significantly increases the initial rate of ATP release and inhibition of ATP loading into membrane vesicles with bafilomycin A1 blocks the response. Furthermore, pretreatment with the K+ channel openers, EBIO (KCa3.1 channels), NS11021 (KCa1.1 channels) and pinacidil (KATP channels) each inhibited the bafilomycin A1-dependent component of ATP release, reducing the initial rate of ATP secretion by ~50%. K2P channel blockers caused depolarization whereas K+ channel openers produced hyperpolarization of the cell membrane. Additionally, K2P channel inhibitors also increased Alternaria-stimulated Ca2+ uptake into hBECs while K+ channel openers reduced the Alternaria response by ~50%. Imaging experiments involving visualization of fluorescently labeled MARCKS proteins in the apical membrane showed that treatment with Alternaria caused a significant decrease in fluorescence intensity, however pretreatment with K+ channel openers, but not K2P channel blockers, inhibited the effect. These results are consistent with recent findings showing that MARCKS proteins bind to the plasma membrane through an electrostatic association with PIP2 and that phosphorylation by PKC or membrane hyperpolarization following K+ channel activation disrupts this interaction resulting in MARCKS detachment from the membrane. MARCKS detachment uncouples the membrane from the actin cytoskeleton, facilitating vesicle docking, fusion and release of ATP.

This study was supported by NIH grants (AI128729 and AI169530) to HK and SMO and by USDA-AES 0016134 to SMO.

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.
Gender Specific Effects of Nr4a1 in Obese Mice
Authors: Herring Jacob , Tessem Jeffery
Published: May 2024

A central aspect of type 2 diabetes disease progression is impaired functional β-cell mass. The hyperglycemic and hyperlipidemic environment present in type 2 diabetes corresponds with impaired beta cell function. The orphan nuclear receptor Nr4a1 is critical for fuel utilization in various tissues, however little is known regarding its function in the β-cell. Nr4a1 expression is decreased in the β-cell of rodent models of type 2 diabetes, as well as in primary human islets from type 2 diabetics. Here we demonstrate sex specific effects of β-cell specific Nr4a1 deletion under high fat chow feeding. While female and male βNr4a1−/− mice were no different than wild type controls when fed a standard chow diet, a clear sex dependent difference is observed when fed a high fat diet. While high fat fed male βNr4a1−/− have improved fasting and non-fasting blood glucose levels and no change in glucose tolerance, high fat fed female βNr4a1−/− mice have impaired glucose tolerance as early as one month after beginning high fat feeding. We also found that βNr4a1−/− female mice have decreased β-cell mass and decreased expression in known targets of Nr4a1 regulation including Eno1 and SDHβ. Our data suggest that Nr4a1 is critical for maintenance of beta cell function, and that loss of Nr4a1 under conditions of obesity and overnutrition in females results in impaired beta cell compensation and function.

NIH R15DK124835-01A1.

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 Long-term Facilitation Following Severe Acute Intermittent Hypoxia Is Preserved in Geriatric Male Rats
Authors: A. Burrowes Kayla , B. Marciante Alexandria , R. Perim Raphael , El-Chami Mohamed , S. Mitchell Gordon
Published: May 2024

Acute intermittent hypoxia (AIH) elicits a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF). Both moderate and severe AIH elicit phenotypically similar pLTF but do so via completely distinct cellular mechanisms. With moderate AIH (mAIH), pLTF arises from a serotonin-dominant, adenosine-constrained mechanism. In aged rats, mAIH-induced pLTF is impaired due to increased basal spinal adenosine levels, which constrain serotonin-dependent pLTF. With severe AIH (sAIH), pLTF arises from an adenosine-dominant, serotonin-constrained mechanism. Since spinal adenosine levels are elevated in aged male rats, we hypothesized that age would actually enhance sAIH-induced- pLTF. Young (~3.5 month) and aged (~20 month) male Sprague Dawley rats were urethane anesthetized, artificially ventilated, vagotomized, paralyzed, and exposed to sAIH (3, 5-minute episodes; arterial Po2 = 25-30 mmHg). Integrated phrenic nerve burst activity was measured before (baseline), during hypoxic episodes and 60 minutes after sAIH. Neither baseline phrenic burst amplitude, the short-term hypoxic phrenic response nor pLTF magnitude (assessed as % change in phrenic burst amplitude from baseline to 60 minutes post-sAIH) were different in aged versus young rats (120 ± 22% vs. 98 ± 17%, respectively). Thus, aged male rats preserve the capacity for adenosine driven phrenic motor plasticity. Although we cannot conclude that sAIH-induced pLTF is actually increased, age effects on adenosine versus serotonin-driven plasticity are clearly different.

NIH HL148030.

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.
Potassium Ion Channel Modulation as a Potential Remedy for Cadmium-induced Hypertension
Authors: McCalla Garsha , Brown Paul , Nwokocha Chukwuemeka
Published: May 2024

Objective: Hypertension remains a chief non-communicable lifestyle disease that is negatively associated with dietary cadmium (Cd). The exact mechanism of Cd toxicity and the mitigating role of potassium supplementation are not fully known, hence, this study aimed to investigate their effects on the aorta. Hypothesis: We hypothesized that potassium supplementation can modulate Cd-induced vascular dysfunction. Method: Six cohorts of control, Cd-exposed, and potassium supplemented male Sprague-Dawley rats were selected. Hypertension was induced using cadmium chloride (2.5 or 5 mg/kg b.w.). Blood pressure was measured twice weekly. Reactivity to pharmacological agents with and without potassium ion channel modulation was assessed in thoracic aortic rings after eight weeks, using an organ bath set-up. Data: Statistical analysis was done using one-way analysis of variance and the results were reported as mean ± standard error of the mean. The Games-Howell or Bonferroni post hoc test was used for multiple comparisons. Summary of Results: Cadmium significantly (p<0.05) elevated systolic, diastolic, mean arterial, and pulse pressures, reduced (p<0.001) phenylephrine-induced contraction mostly via BKCa channels, and augmented (p<0.05) sodium nitroprusside-induced relaxation via K-ATP, KV, and KIR channels, without affecting acetylcholine-induced relaxation. Potassium supplementation ameliorated these effects via KIR and BKCa channels. Conclusions: The results indicate that modulation of potassium ion channels in the vasculature might provide therapeutic management for environmentally-induced hypertension and should be further explored.

Funding source: The Mona Campus Committee for Research and Publications and Graduate Awards, School for Graduate Studies and Research, University of the West Indies.

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.
Genetically-targeted Recordings from Respiratory Chemoreceptor Neurons in the Retrotrapezoid Nucleus of Freely Behaving Mice by Fiber Photometry
Authors: Souza George , Stornetta Daniel , Abbott Stephen
Published: May 2024

Central respiratory chemoreceptors regulate breathing homeostasis in relation to arterial pH/PCO2. Neurons located in the retrotrapezoid nucleus (RTN) are considered to function as central respiratory chemoreceptors and/or to be part of the neural network that drives the central chemoreflex. RTN neurons are sensitive to changes in pH in vitro under synaptic blockade and in anesthetized preparations with artificial ventilation. However, there is limited information about their activity in vivo in non-anesthetized conditions. Recent studies have shown that Nmb-expressing neurons in the RTN have acid-sensitive properties and are necessary for the respiratory response to CO2. In this study, we hypothesized that RTN- Nmb neurons are activated during hypercapnia in freely behaving condition. To test this hypothesis, we measured the dynamic activity of RTN neurons using a genetically encoded calcium indicator (GCaMP7s) via fiber photometry in non-anesthetized mice. Expression of GCaMP7s was genetically targeted to RTN neurons by microinjection of a Cre-dependent viral vector (AAV1-Syn-Flex-jGCAMP7s) in transgenic Nmb-Cre mice. One month later, the animals were placed in a plethysmography chamber to simultaneously record breathing and GCaMP7s fluorescence from RTN- Nmb neurons in freely behaving conditions. During hypercapnia, RTN neurons were markedly activated, and the effect size was graded to FICO2 (DF/F0 from baseline: 1.9 ± 1.0 % at 0.03, 8.0 ± 4.0% at 0.06 and 12.0 ± 5.0% at 0.09, n=6), and significantly different for each level of CO2 (RM One-way ANOVA, p=0.0012). Importantly, the increase in RTN neuron activity correlates with changes in the FICO2 (p<0.0001, R2=0.97) and respiratory frequency (p<0.0001, R2=0.85). This study shows that RTN neurons are potently activated by hypercapnia in freely behaving conditions, and the response of RTN neurons to CO2 aligns with changes in breathing. These results are consistent with the concept that RTN neurons monitor arterial PCO2/pH and are essential for the hypercapnic ventilatory response.

This study was supported by National Institutes of Health (HL148004 ) to Stephen BG Abbott.

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 and Characterization of Potential Anti-Hypertensive Therapeutic Candidates in Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Axis
Authors: Kumar Gaurav , Vazirabad Ibrahim , Golosova Daria , Chaihongsa Nisita , Muskus Patricia , Wackman Kelsey , Lu Ko-Ting
Published: May 2024

Blood pressure (BP) is regulated by a variety of cellular mechanisms. We previously reported the regulation of endothelial nitric oxide (NO)-dependent vessel tone and BP through RhoBTB1-CUL3 mediated proteasomal turnover of Phosphodiesterase 5 (PDE5). We determined that the C-terminal half of RhoBTB1 consisting of two BTB-domains (B1 & B2) and the C-terminal (C) domain of RhoBTB1 can deliver PDE5 to proteasomal pathway. The cellular expression of RhoBTB1 and CUL3 is ubiquitous whereas expression of PDE5 is cell specific. Moreover, vessel tone is regulated through NO-independent mechanisms. Therefore, we hypothesized that RhoBTB1 may have other interacting partners, potentially functioning as regulators of vessel tone and BP in vascular smooth muscle (VSM) cells, through a similar mode of binding as PDE5. Therefore, we utilized ascorbate peroxidase (APEX2) proximity labelling system comprising APEX2 tagged B1B2C domain (B1B2C-APEX2) to identify unknown potential regulators of vasomotor tone in RhoBTB1-CUL3 axis in A7R5 VSM cells. The background of the proteomic screen was minimized by utilizing APEX2-B1B2 which lacks the final C-terminal region of the protein and cannot bind PDE5. We validated the interaction of several identified proteins with RhoBTB1 and the B1B2C domains using co-immunoprecipitation (Co-IP). Our Co-IP data corroborated the proteomic screen as several proteins demonstrated differential interaction with B1B2C and B1B2. Among them, we examined RbFox2, a regulator of mRNA splicing for further investigation. Further data established that RbFox2 is upregulated under the following conditions: 1) pharmacological inhibition of the proteasomal pathway, 2) in CRISPR edited CUL3KO HEK293 cells, 3) in aorta of VSM cell-specific CUL3 deleted mice, 4) by siRNA mediated knock-down of RhoBTB1 in SMCs, and 5) in Ang-II treated SMCs and aorta of Ang-II treated animals. We hypothesized that RbFox2 could modulate actin and actinin-dependent cytoskeletal dynamics, an important regulator of arterial stiffness. First, Co-IP data suggest an interaction between RbFox2 and SMC-specific alpha actinin. Next, siRNA-mediated knock down of RbFox2 altered the actin and actinin-dependent cytoskeletal dynamics in SMCs. Collectively, this establishes the potential regulation of RbFox2 through the RhoBTB1-CUL3 proteasomal pathway and that RbFox2 may play a mechanistic role in arterial stiffness in hypertension.

Funding: NIH R35 HL144807 (to Dr. Curt D. Sigmund).

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.
pHire: A Novel Red Intracellular pH Sensor
Authors: Desrochers Kate , Holmberg Shannon , Holmes Heather , Zhang Lili , Foresto Neto Orestes , Reynolds Carmen , Romero Michael
Published: May 2024

Maintaining pH is critical for proper cellular and organ function. To measure intracellular pH, we made a novel genetically encoded protein, pHire, that increases red fluorescence with increasing pH. Thus, pHire allows for the coupling of multiple fluorescent tags without spectral overlap. We have expressed pHire in mammalian epithelial cells, Drosophila, and mice. In this study, our goal was to characterize pHire’s spectral properties by expression in human cells (HEK293) and in E.coli. To biophysically characterize pHire, we made a maltose binding protein (MBP)-pHire fusion protein to purify from E.coli. Pelleting individual colony growths revealed pink bacterial pellets which fluoresced with mCherry filters. Rather than purifying the MBP-pHire protein, equal volumes of bacteria were permeabilized with nigericin and high K+ solution for pH 5 to 9. These aliquots were then used to gather the fluorescence excitation and emission spectra of pHire, allowing us to determine the excitation and emission peaks of pHire as a function of pH. Our results showed peaks at Ex579 nm and Em611 nm, or roughly mCherry. Pelleted bacteria could also be fixed in perfusion chambers, revealing the same calibration curve as in mammalian cells and flies. Next, stable HEK293-pHire cells were selected to assay acid-base transport using NH4Cl or CO2/HCO3− in perfusion experiments. These HEK-pHire cells have less fluorescence than MBP-pHire bacteria but show the same Ex/Em peaks. However, an in-frame ATG 5’ to pHire in a mouse (adding 23 amino acids), removed pHire pH-dependence and decreased bacterial fluorescent output. Together these results indicate that pHire is biophysically similar in cells tested but that unstructured N-terminal additions block pH-responses.

Funding: R25-DK101405, R21-DK129897.

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.
Alleviating renal injury and mitochondrial dysfunction following hemorrhagic shock
Authors: Kennard Cianna , Calderon Alfredo , Klemcke Harold , Mdaki Kennedy , Marshall Stephanie , Meledeo Michael , Xiang Lusha
Published: May 2024

Resuscitation with polydatin (PD) has been shown to alleviate mitochondrial damage and ischemia reperfusion injury following hemorrhagic shock. However, whether PD has the same beneficial effect under prolonged hemorrhagic shock with delayed or limited resuscitation is unclear. Using a previously developed rat model of acute kidney injury (AKI) induced by prolonged hemorrhagic shock, we tested the hypothesis that PD preserves renal mitochondrial function, delays the onset of AKI, and decreases mortality following prolonged hemorrhagic shock without full resuscitation. Additionally, we determined whether polyethylene glycol-20k (PEG) can enhance the beneficial effects of PD via improved capillary perfusion and PD delivery into the ischemic tissues. Rats were randomized into 4 groups (n=8/group) based on treatment: saline (vehicle), PD, PEG, and PD+PEG. Trauma was induced in Inactin anesthetized rats with muscle injury and fibula fracture, followed by pressure-controlled hemorrhagic shock (mean arterial pressure (MAP) = 55 mmHg) for 45 minutes. Animals then received an intravenous injection of saline (vehicle), PD, PEG, or PD+PEG. MAP, blood gases, hematocrit, renal blood flow (RBF), and oxygen delivery were monitored for another 2 hours. Glomerular filtration rate (GFR), urinary sodium uptake, renal mitochondrial function, and mortality were recorded at the end of the experiment. The saline group exhibited AKI, renal mitochondrial dysfunction, and 100% mortality. PD alone showed no effect on mitochondrial function, renal injury, or mortality (100%). PD+PEG restored both complex II and IV activities, while PEG improved mitochondrial complex II function but showed no effect on complex IV. Nevertheless, PEG and PD+PEG similarly improved MAP, renal blood flow, renal oxygen delivery, GFR, sodium uptake, and mortality (PEG: 12.5%; PD+PEG: 0%). Our results suggest that polydatin requires suffcient tissue perfusion to exert its beneficial effects on the mitochondrial function of hemorrhagic shock animals. We used clinically meaningful endpoints to demonstrate the utility of PEG as a therapeutic tool for optimizing drug delivery to treat hemorrhagic shock.

DoD disclaimer: The views expressed in this article are those of the author(s) and do not reflect the offcial policy or position of the U.S. Army Medical Department, Department of the Army, DoD, or the U.S. Government. Funding: This study was funded by US Army Medical Research and Development Command.

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 Influence of Early Menopause on Baroreflex Sensitivity in Healthy Females
Authors: Anselmo Miguel F. , Nathe Stephany C. , Stokes William , Koebernick Ella , Tahsin Chowdhury Tasnova , Lee Emma J. , Teshome Soliana
Published: May 2024

Introduction: The risks of hypertension (HTN) and cardiovascular disease (CVD) rise dramatically in females around the typical age of menopause (TM; menopause age ≥46yr). Early menopause (EM; menopause age <46yr) may substantially increase this risk. Attenuated baroreflex sensitivity (BRS) can be indicative of autonomic dysfunction and has been shown to be a marker of CVD risk. Thus, we aimed to investigate how EM would influence BRS. We hypothesized that females who experienced EM would exhibit attenuated BRS compared to females who experienced TM. Methods: 32 females completed two visits: Visit 1: informed consent, medical and health questionnaires to confirm eligibility, including the Minnesota Leisure Time Physical Activity Questionnaire to assess activity levels. Visit 2: Participants rested in a supine position for 10 min. Non-invasive beat-to-beat blood pressure (BP), heart rate (HR), and muscle sympathetic nerve activity (MSNA) were recorded. Blood was drawn to measure sex steroid levels. Cardiovagal (cBRS) and sympathetic (sBRS) BRS were calculated using the sequence method and Ensemble software, respectively. Both parametric and non-parametric methods were used for the statistical analysis. Results: Results are presented as mean ± SD. Participants in the TM (n=16; age: 63±3yrs, body-mass index (BMI): 25±4kg/m2) and EM (n=16; age: 62±4yrs, BMI: 26±4kg/m2) groups were matched for age and BMI. TM and EM had similar levels of estradiol (TM: 3.9±2; EM: 4.4±4pg/mL) and activity levels (TM: 299±177; EM: 208±147kcal/day), but significantly different menopause ages (TM: 51±3; EM: 43±2yrs; p<0.001). TM and EM had similar resting systolic BP (TM: 129±13; EM: 128±20mmHg), diastolic BP (TM: 79±10; EM: 82±14mmHg), HR (TM: 59±7bpm; EM: 62±9bpm), and MSNA (TM: 51±16; EM: 45±11bursts/100heartbeats), p>0.05 for all. Further, cBRS and sBRS were similar between TM (cBRSup: 9.7±3; cBRSdown: 12.3±7; cBRStotal: 12.3±7ms/mmHg; sBRS: -2.7±1bursts/100Hb/mmHg) and EM (cBRSup: 8.7±4; cBRSdown: 11±5; cBRStotal: 10.4±4ms/mmHg; sBRS: -2±1bursts/100Hb/mmHg; p>0.05 for all). Conclusion: Contrary to our hypothesis, these findings suggest that EM may not influence BRS. These findings indicate that completing menopause early may not disrupt autonomic baroreflex function, suggesting that other mechanisms need to be explored to better understand the increased CVD risk in postmenopausal females who complete menopause prematurely or early.

This study was supported by a NIH 1 K01 AG064038-01A1 (MLKR), UMN Grant-in-Aid (MLKR), F32HL160012 (EL), and NIH National Center for Advancing Translational Sciences grant UL1TR002494.

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.