bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and progression of overactive bladder urinary in heart failure rat
Authors: Claudino Mário Angelo , Furquim Silas Ramos , Tartarotti Sabrina , Mora Aline Gonçalves , Priviero Fernanda , Antunes Edson
Published: November 2023

Heart failure (HF) and urinary symptoms (US) is highly prevalence and affects approximately 37% of man and 67% of woman have a significant impact on quality of life. There is evidence that HF and US share risk factors that are associated with autonomic nervous system dysfunction, which has also been implicated in overactive bladder (OAB). Thus, the aim of study was evaluated the possible relationship in the progression OAB associate to HF in rats submitted to arteriovenous fistula (HF model). After 4 or 12 weeks, cystometric parameters (ex vivo) were assessed: Threshold pressure (the intravesical pressure immediately before micturition); Post‐void pressure (the intravesical pressure immediately after micturition); Contraction micturition (the peak pressure reached during micturition); Capacity (the volume of saline needed to induce the first micturition); Frequency of non‐voiding contractions. The histomorphometry (in vitro), also were performed. After 4 weeks, HF animals have an increase of micturition contraction and threshold pressure associated with a decrease of capacity (P<0.05) when compared to group sham. Moreover, after 12 weeks, HF animals increase of micturition contraction, threshold pressure and frequency of non‐voiding contractions and keep the decreased of bladder capacity (P<0.05) when compared to Sham group. In addition, HF group (12 weeks) increase the frequency of non‐voiding contractions (P<0.05) when compare to HF group (4 weeks). Histomorphometry showed that HF group (4 weeks) present an increase of urothelium thickness (P<0.05) compared to sham group, whereas, HF group (12 weeks) present an increase in the detrusor muscle (P<0.05) when compared to sham group. Thus, HF rats, using the arteriovenous fistula, promote structural changes in the detrusor muscle contributing to the development of OAB and impaired of urinary function. Furthermore, our data suggest that OAB is progressive and is associated with the HF time course.

Support or Funding Information

Financial support: FAPESP
Betaine Supplementation Accelerates Elimination of Acetaldehyde from Blood in Rats Challenged with Ethanol Acutely
Authors: Jun Doo S , Ahn Chul W , Kim Jung I , Kim Young C
Published: November 2023

Earlier studies in our laboratory showed that betaine administration may modify the metabolic reactions for sulfur‐containing amino substances in the liver. In this study we determined the changes in elimination of ethanol from blood induced by betaine supplementation. Male adult rats were provided with betaine (1 %) in drinking water for 2 weeks prior to a challenge with an acute dose of ethanol (3 g/kg, ip). Blood ethanol and acetaldehyde levels were monitored for 330 min after ethanol treatment. Ethanol concentrations in blood reached a peak in 30 min and declined rapidly thereafter in both groups. Ethanol concentrations and its elimination rate in blood were unaffected by betaine intake. Blood concentrations of acetaldehyde, a toxic metabolite of ethanol, were elevated gradually, reaching a plateau in around 150 min. However, blood acetaldehyde concentrations appeared to be significantly lower in the rats supplemented with betaine. The area under the curve (AUC) calculated for acetaldehyde was markedly lower in the rats supplemented with betaine (1.25 ± 0.08 mg·min/ml) than in the control rats (2.07 ± 0.18 mg min/ml). Methionine, glutathione, and S‐adenosylmethionine contents in the liver were significantly decreased by ethanol treatment, which were all prevented in the rats supplemented with betaine. Lipid peroxidation in liver tissue was also inhibited by betaine intake. It is suggested that the reduction of acetaldehyde concentrations in blood by betaine intake may be associated with the enhanced production of sulfur‐containing substances in the liver.

Support or Funding Information

Supported by National Research Foundation (NRF) grants (No. 2014‐R1A2A1A11052967 and No. 2009‐0083533) funded by the Ministry of Education, Science and Technology (MEST), Korea.
Vinorelbine Inhibits Pregnane X Receptor‐Mediated Induction of Cytochrome P450 3A4 Gene Expression in Human Hepatocytes
Authors: Chaudhury Chloe , Abbott Kodye , Pondugula Satyanarayana
Published: November 2023

Pregnane X receptor (PXR), a ligand‐dependent nuclear receptor, is involved in regulating the gene expression of enzymes and proteins involved in endobiotic and xenobiotic metabolism. In particular, PXR plays a crucial role in regulating the gene expression of drug‐metabolizing enzymes, including cytochrome p450 3A4 (CYP3A4). In cancer patients, upregulation of the PXR target genes, including CYP3A4, can lead to not only adverse drug interactions but also chemoresistance. It is possible to inhibit the activity of PXR in order to overcome PXR‐mediated adverse drug interactions and chemoresistance. In this regard, a few PXR inhibitors or antagonists have been identified and characterized. However, these compounds fall short of reaching their clinical utility as they are non‐selective and/or highly‐toxic. Therefore, there is a practical need to develop a selective and less‐toxic PXR antagonist to overcome PXR‐mediated adverse drug interactions and chemoresistance. We sought to determine whether vinorelbine (VNB), a clinically‐used antimicrotubule agent, inhibits the transcriptional activity of PXR in human hepatocytes. PXR transactivation and cell viability assays were performed to determine CYP3A4 promoter activity and cytotoxicity, respectively, in HepG2 human liver carcinoma cells. Quantitative RT‐PCR assays were conducted to study gene expression of CYP3A4 in human primary hepatocytes. VNB, at its non‐cytotoxic and therapeutically‐relevant concentrations, inhibited PXR transactivation of CYP3A4 promoter activity. Additionally, VNB attenuated the PXR agonist‐induced gene expression of CYP3A4, suggesting that VNB downregulates CYP3A4 gene expression by inhibiting the activity of PXR. These results support our conclusion that VNB inhibits the transcriptional activity of PXR at its non‐cytotoxic and therapeutically relevant concentrations. Future studies will be directed to study the selectivity of VNB to PXR as well as the mechanisms of VNB inhibition of PXR‐mediated CYP3A4 induction.

Support or Funding Information

This work was supported by the Auburn University Research Initiative in Cancer Grant, Animal Health and Disease Research Grant, and Auburn University Startup Funds to Pondugula SR. The authors would like to thank Drs. Coleman, Tao, and Schwartz for sharing their research facilities.
Role of cGMP Signaling for Training‐induced Vascular and Skeletal Muscle Adaptations in Young and Older Humans
Authors: Piil Peter , Vorup Jacob , Gliemann Lasse , Egelund Jon , Hellsten Ylva , Nyberg Michael
Published: November 2023

Aging is associated with impaired blood flow regulation; however, the mechanisms underlying this impairment and to what extent a period of exercise training can improve the matching between O2 delivery and utilization remain unclear. Eight young and 6 older (24 ± 1 vs. 74 ±2 years) healthy sedentary men participated in the study. We examined the vascular response to femoral arterial infusion of the vasoactive compound ATP and the effect of inhibiting the main enzyme involved in cGMP degradation, phosphodiesterase 5 (PDE5), on hemodynamics and O2 uptake in contracting skeletal muscle before and after 10 weeks of aerobic high intensity training. Catheters were placed in the femoral artery and vein of both legs. A microdialysis probe was inserted into one of the venous catheters to allow for measurements of plasma ATP concentrations. Measurements of blood flow, intra‐arterial and ‐venous blood pressure, and arterial and venous O2 content were performed during ATP infusion and knee‐extensor exercise. As post‐intervention testing is currently being conducted, only data from pre‐intervention testing is presented in is this abstract.

At baseline, the ATP‐induced change in leg vascular conductance was lower in the older group (P<0.05). During sub maximal knee‐extensor exercise in the control setting, O2 uptake tended to be lower (−14%, P=0.093) in the older subjects compared to young subjects. After intake of the highly selective PDE5 inhibitor sildenafil, blood flow, leg vascular conductance and O2 uptake were higher (P<0.05) in the older group during knee‐extensor exercise whereas no changes were observed in the young group. These results indicate an insufficient oxygen delivery to the contracting skeletal muscle of aged individuals as a result of reduced cGMP signaling. Data from post‐intervention testing will provide insight as to whether exercise training can improve the matching of O2 delivery and utilization via improved cGMP signaling in the older subjects. Also, it is thought that ATP released from red blood cells and the endothelium play a role in blood flow regulation, and data from post‐intervention testing will elucidate if a period of exercise training increases ATP sensitivity and/or venous plasma ATP concentrations during exercise.

Support or Funding Information

Financial support for the study was provided by the Danish Council for Independent Research – Medical Sciences and Lundbeck Foundation.
Cardiac complement in Toll‐like receptor 4 KO mice subject to renal ischemia/reperfusion
Authors: da Cruz Junho Carolina Victoria , Sonoda Mayra Trentin , Ramos Marcela Sorelli Carneiro
Published: November 2023

Background

The immune system consists of an array of cells and molecules distributed throughout the organism; its function is to recognize antigens or molecular structures, generating an effective response in the pathogens destruction or inactivation. It can also act via the complement system (CS). The cardiac tissue is a main target of the immune system and several cardiovascular diseases (CVD) are preceded by general or local inflammation, contributing directly to cardiac remodeling and changes in heart tropism. It is known that renal ischemia/reperfusion (I/R) may result in the development of systemic inflammation and it can reach the cardiac tissue. Tissue response may be initiated by Toll‐like receptors [TLR‐ pathogen‐associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) recognizers] or also by complement system. Within the inflammatory process, the complement component C3a is released in abundance and binds to C3aR1 receptor, which might be involved in TLRs activation.

Aim

Since it has been demonstrated that renal I/R is able to induce CH and ablation of TLR4 prevents such CH development, the aim of this study was to evaluate if TLR4 −/− had differential expression of complement components when compared to wild type mice.

Methods

C57BL/6 male mice and C57BL/6 TLR 4 −/− were subjected to surgical occlusion of left renal pedicle for 60min followed for reperfusion (I/R) for 15 days. Morphometric analysis were performed using as parameters heart weight, kidneys weight and body weight. Real time PCR were utilized to analyze gene expression.

Results

The C3aR component in KO group after 15 days of reperfusion, showed no change. These data is in accordance with previous data where we have not observed any modulation in the WT groups after 15 days of reperfusion. However, analysis of the complement component C3 mRNA levels, showed a significant increase in the WT I/R group compared to WT sham group (p <0.05), and TLR4 absence was capable of preventing such increase. The component C5R presents significant increase after 15 days of reperfusion (p <0.05) as well as Cfb (p<0.05) in TLR4 KO I/R animals, compared to Sham.

Conclusion

Cardiac gene expression of complement components differs in WT and TLR4KO mice subjected to renal ischemia/reperfusion, indicating a possible interaction between these two main responses of innate immunity in our model.

Support or Funding Information

FAPESP (São Paulo Research Foundation)
Effect of High Intensity Exercise Preconditioning and Training on Antioxidant Enzymes in Cardiomyocytes During Doxorubicin Treatment
Authors: Jarrett Catherine L , D'Lugos Andrew C , Mahmood Tara N , Gonzales Rayna J , Hale Taben M , Carroll Chad C , Dickinson Jared M , Angadi Siddhartha S
Published: November 2023

Doxorubicin (DOX) is a common chemotherapeutic treatment for breast cancer and has been proven to reduce recurrence and mortality. However, a well‐recognized side effect is DOX‐induced cardiac toxicity. This is typically thought to occur due to increased generation of reactive oxygen species (ROS) and oxidative damage within cardiomyocytes. Exercise training has been shown to increase expression of several key intracellular antioxidant enzymes. The purpose of this study was to examine the potential for high‐intensity interval exercise (HIIE) initiated prior to (i.e. preconditioning) and continued throughout (i.e. training) bi‐weekly DOX treatments to preserve antioxidant levels in cardiomyocytes. We hypothesized that DOX treatment would reduce the antioxidant enzymes, Cu‐Zn superoxide dismutase (SOD1), Mn SOD (SOD2) and catalase (CAT) in cardiac tissue whereas exercise would attenuate this response. Eight‐week old female ovariectomized Sprague Dawley rats (N=25) were randomized to one of the four treatments: Exercise+DOX (n=7; Ex‐DOX); Ex+Vehicle (n=6; Ex‐veh); Sedentary+DOX (n=6; Sed‐DOX); and Sed+veh (n=6; Sed‐veh). Dox (4mg/kg) or vehicle (saline) intraperitoneal injections were performed bi‐weekly for a total of 3 injections (cumulative dose 12mg/kg). HIIE consisted of 4×4 min bouts of treadmill exercise at 85–95% of VO2peak separated by 2 min of active recovery. One week prior to the first injection, exercise animals underwent training for 5 days and continued throughout the study duration. Animals were euthanized 5 days following the last injection, following which the heart was extracted and left ventricular tissue was prepared for western blot analyses. Levels of SOD1 (p=0.078) and SOD2 (p=0.052) tended to be higher in hearts from exercising rats, independent of treatment. However, DOX administration for both Ex‐DOX and Sed‐DOX groups had significantly higher CAT levels (p<0.001). These preliminary analyses suggest that exercise preconditioning and training prior to and during DOX administration may raise SOD1 and SOD2 levels in cardiac tissue, whereas DOX administration elevates CAT levels regardless of exercise.
Acute and Chronic Biological Effects of Copeptin on Vascular Function in Mice and Humans
Authors: Rasor Madalyn , Wegman‐Points Lauren , Reho John J , Santillan Mark K , Grobe Justin L , Santillan Donna , Sindler Amy L , Rahmouni Kamal , Scroggins Sabrina , Pierce Gary L
Published: November 2023

Background

Copeptin, the 39 amino acid C‐terminal segment of the precursor protein of arginine vasopressin (AVP), is secreted from the posterior pituitary in a 1:1 ratio with AVP in response to osmotic and hemodynamic stimuli. Copeptin is significantly elevated in pregnant women with preeclampsia, and predicts the development of preeclampsia as early as the 6th week of gestation. Preliminary data in a recent prospective study demonstrated that higher copeptin was associated with lower endothelial function (brachial artery flow‐mediated dilation, FMD) during the 2nd trimester in pregnant women at high risk of preeclampsia. However, whether copeptin itself has any independent biological effects on the vasculature is completely unknown.

Methods and Results

Chronic incubation of mouse aortic rings with copeptin (n=4 male mice, 1nM, 24 hours) had no effect on endothelium‐ dependent (acetylcholine) or ‐independent (sodium nitroprusside) vasodilatory responses, indicating the lack of effect on endothelial or vascular smooth muscle function. Similarly, circulating copeptin was not associated with brachial artery FMD in non‐pregnant young women (n=11, r= −0.08, P=0.81), postmenopausal women (n=15, r=0.06, P=0.82) or men (n=24, r=0.10, P=0.62). In contrast, acute incubation of aortic rings (n=6 male mice) with copeptin demonstrated a dose response vasodilation beginning at 1nM up to a maximum relaxation at 100nM (43.6 ± 15.5%, p<0.05, n=6). Co‐incubation with the nitric oxide (NO) inhibitor of L‐NG‐Nitroarginine methyl ester (L‐NAME) significantly attenuated the max vasodilatory response to 12.5 ± 0.6%, indicating that the acute vascular response to copeptin is largely NO‐mediated. Incubation of aorta with copeptin (60 min, 0.5nM) caused a significant increase in protein kinase B (AktSer473) phosphorylation (Western blot) vs. control tissue (0.63 ± 0.19 vs 1.27 ± 0.6, p<0.04, n=5) and a trend towards increased phosphorylation of eNOSSer1177 (0.69 ± 0.02 vs 0.91 ± 0.21, p<0.3, n=2).

Conclusions

These data indicate that acute exposure of aorta of mice to high copeptin results in marked NO‐mediated vasodilation. However, chronic exposure to high copeptin does not alter vascular endothelial or smooth muscle function in mice nor is circulating copeptin associated with endothelial function in males or non‐pregnant females. This suggests that adverse vascular effects associated with chronically elevated copeptin concentrations in preeclampsia are more likely a result of the pathophysiological consequences of high AVP rather than copeptin.

Support or Funding Information

APS STRIDE Undergraduate Summer Research Fellowship;AHA 15SFRN23730000;University of Iowa Center for Research by Undergraduates (ICRU); University of Iowa Center for Hypertension Research.
High Glucose Concentration Attenuates Insulin‐Stimulated Protein Kinase B Levels In Cultured Astrocytes
Authors: Yu Eric , Cohen Susan , Liu Qiuli , Ryan Robert , Gebremedhin Debebe , Harder David , Rarick Kevin
Published: November 2023

BACKGROUND

Glucoregulatory abnormalities have been implicated to contribute to the pathophysiology of Alzheimer‐type dementia. The brain requires insulin for supply of energy critical for processing of memory and cognition. Insulin acts on the insulin receptor initiating signaling cascades that activate protein kinase B (Akt) through phosphorylation (p‐Akt) in cultured astrocytes. However, it is not yet clear whether the insulin induced production of p‐Akt could be attenuated by high glucose concentrations through stimulation of an inhibitory signaling pathway involving activation of protein kinase C (PKC).

OBJECTIVE

The objective of our study is to examine the phosphorylation of Akt in cultured neonatal astrocytes following insulin stimulation and to determine if this response could be attenuated by culturing astrocytes in high glucose media via activation of PKC.

METHODS

Neonatal astrocyte cultures were derived from rat hippocampus and cultured for three passages in normal (5.5 mM) or high glucose (25 mM) media. Astrocyte cultures were stimulated with increasing concentrations of insulin (10−11 to 10−6 M) and Western immunoblotting analysis was used to determine protein levels of p‐Akt and insulin receptor. Protein levels of PKC isoforms (alpha, gamma, and delta) were determined in whole cell lysates from separate astrocyte cultures grown in normal or high glucose media.

RESULTS

Insulin‐induced a dose‐dependent increase in p‐Akt protein level in astrocytes cultured in normal glucose. However, insulin stimulation of astrocytes cultured in high glucose media resulted in an attenuated protein level of p‐Akt (100 nM insulin: 3.6 ± 1.0 vs. 8.9 ± 1.6 fold increase compared to control), requiring more insulin to increase the protein levels of p‐Akt (interaction effect: p = 0.024). The protein levels of insulin receptor in cultured astrocytes also increased in response to stimulation with insulin. Astrocytes cultured in high glucose media exhibited increased protein levels of PKC alpha (1.5 fold increase, p < 0.05) and gamma (2 fold increase, p < 0.05) but not of the PKC delta isoform compared to astrocytes cultured in normal glucose media.

CONCLUSIONS

Insulin‐dependent intracellular signaling involving phosphorylation of Akt is attenuated in astrocytes cultured in high glucose media. Astrocytes respond to increased environmental glucose with isoform specific increases in PKC protein levels which may have resulted in negative regulation of astrocyte insulin receptor signaling.

Support or Funding Information

Funding from NIH (NHLBI) T35 HL072483, R01 HL033833 and HL105997 and the Veterans Administration Research Career Scientist Award.
Beneficial effects of physical exercise under lightening condition on progressive cartilage damage
Authors: Hong Yunkyung , Jo Youngjin , Kim Hyunsoo , Hong Yonggeun
Published: November 2023

Osteoarthritis (OA) is frequently occurred in the elderly people. Physical exercise used to be recommended for OA patients, but the effects differ as to how to take exercise. Collagenase‐induced early OA rats were subjected to determine whether environmental light diminishes the beneficial effects of physical exercise on the degenerative musculoskeletal unit. Motor‐driven treadmill exercise (Ex) was applied for 30 min (22:00) on 5 days/week. Intervention was performed for 4 weeks under constant light (LL) as well as 12:12hr light‐dark (L/D). The elevated Mmp13 expression was reduced in the L/D+Ex group, but it was reversed when exercise was executed under LL condition. Exercise under L/D prevented loss of type II collagen (COL2A1) in the cartilage specimens, the effect of which was mitigated in LL condition. Concomitant with these results, the value of joint thickness was recovered in the L/D+Ex group only. Exercise activated autophagic machinery in the periarticular muscle regardless of light condition, the extent of which was higher in the L/D+Ex group than the LL+Ex group. However, CHOP protein was more present in the LL+Ex group. Taken together, musculoskeletal remodeling induced by physical exercise might be affected by lightening condition in vivo animal model.

Support or Funding Information

NRF‐2012R1A1A2005089, NRF‐2013R1A2A2A01067169, NRF‐2015R1C1A2A01055691
Liver specific depletion of mitochondrial ATP transporter enhances uncoupled respiration and prevents fatty liver and obesity
Authors: Cho Joonseok , Zhang Yujian , Sunny Nishanth E , Kim Jae‐Sung , Shiratsuchi Takayuki , Oh S. Paul , Lee Young Jae , Terada Naohiro
Published: November 2023

Adenine nucleotide translocases (ANTs), also called ADP/ATP carriers (AACs), transport ADP and ATP through the mitochondrial inner membrane and play an essential role for mitochondrial energy supply in eukaryotes. In the liver mitochondria, Ant2 is predominantly expressed among the three murine Ant paralogs Ant1, 2 and 4. When we conditionally disrupted the Ant2 gene in the liver using Alb‐Cre mice, ADP/ATP exchange capacity in the liver mitochondria was indeed inhibited over 96%.

Surprisingly, the mice appeared normal though they had a noticeably leaner phenotype compared to WT mice. These null mice did not show signs of liver dysfunction for over 10 months suggesting mitochondrial ATP supply is largely dispensable for basal liver function and homeostasis. Upon closer examination of Ant2 depleted liver mitochondria, both outer and inner membranes were intact, and the membrane potential was maintained normally. Notably, mitochondrial mass was significantly increased in Ant2‐depleted hepatocytes, where basal respiration was upregulated due to an increase in uncoupled respiration. In addition, liver specific Ant2 knockout mice demonstrated low blood glucose, cholesterol, and insulin levels and improved glucose tolerance. These data indicate that the Ant2 depleted mitochondria in the liver become highly uncoupled and consequently energy‐inefficient, but remain intact yielding no apparent adverse effects to the organ or systemically to the body. Indeed, when mice subjected to high fat/fructose diet, the liver specific Ant2 knockout mice developed significantly less fatty liver changes and less obesity than controls. In addition, when wild type mice were subjected to a low dose of the general Ant inhibitor, carboxyatractyloside, during high fat/fructose diet regimens, the drug also partially mitigated fatty liver changes and obesity. Of interest, liver Ant2 expression in wild type mice is significantly decreased by high fat/fructose diet but increased by fasting. Further, In vitro treatment of a hepatic cell line with a high concentration of fatty acids demonstrated reduced human ANT2 expression. These indicate that there is a physiological mechanism to control Ant2 expression under high‐fat diet conditions which may help prevent obesity and fatty liver.

Given the beneficial effects that we identified here with liver‐specific Ant depletion and systemic Ant inhibition combined with high fat diet, targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of Ant2, could represent a novel strategy to prevent fatty liver and obesity.

Support or Funding Information

research grant from Otsuka Maryland Medicinal Laboratories
Plasma Soluble Erythropoietin Receptor is Decreased During Sleep in Andean Highlanders with Chronic Mountain Sickness
Authors: Corante Noemi , Anza‐Ramirez Cecilia , Figueroa‐Mujica Romulo , Vizcardo‐Galindo Gustavo , Mercado Andy , Macarlupú Jose Luis , León‐Velarde Fabiola , Villafuerte Francisco
Published: November 2023

Excessive erythrocytosis (EE) is the main sign of Chronic Mountain Sickness (CMS), a maladaptive syndrome with significant prevalence in high‐altitude inhabitants. Although chronic hypoxemia represents its underlying stimulus, the fundamental pathophysiological mechanism is still debatable. Studies during sleep in CMS patients consistently find lower average pulse O2 saturation (SpO2) compared to healthy highlanders (HH). Although, whether accentuated hypoxemia during sleep has a significant impact on the Epo system that could explain the occurrence of EE is still unclear. We have recently shown that morning soluble Epo receptor (sEpoR), an endogenous Epo antagonist, is decreased in CMS patients suggesting increased circulating Epo availability (increased Epo/sEpoR). The aim of the present study was to characterize the concentration profile of sEpoR and serum Epo during night‐time, and their relationship with SpO2 and hematocrit (Hct) in HH and CMS patients, as well as the association between testosterone concentration and Epo and sEpoR at early morning. Thirty‐nine male participants (CMS, n=23; HH, n=16) from Cerro de Pasco, Perú at 4340m, between 21 and 65 years old and with normal iron status participated in the study. Blood samples were taken every 4h from 6pm–6am for Epo and sEpoR measurements; the morning sample was also used for evaluation of serum iron, ferritin, transferrin and testosterone concentrations. SpO2 was assessed at 6pm and continuously monitored from 10pm–6am. Mean SpO2 during sleep was lower in CMS patients compared to HH (75.2±1.5 vs 79.9±0.9%), and the percentage of sleep‐time spent with SpO2 below 80% was higher (67.2±7.2 vs 37.9±8.5%). The CMS group showed higher Epo from 6pm–2am (24.1±2.6 vs 14.0±2.2, 26.4±4.4 vs 10.5±0.9 and 25.3±3.8 vs 11.3±1.3mIU/mL, respectively) and lower sEpoR from 10pm–6am (1.4±0.1 vs 2.2±0.1, 1.5±0.2 vs 2.2±0.2 and 1.5±0.1 vs 2.2±0.2 ng/mL, respectively). Consequently, the Epo/sEpoR was significantly higher in the CMS group from 6pm–6am (15.8±2.5 vs 7.7±1.0, 20.9±3.7 vs 5.2±0.7, 20.5±3.8 vs 5.7±0.8 and 20.7±3.9 vs 6.8±0.8 mIU/ng, respectively). Multiple regression analysis showed mean sleep‐time SpO2 and Epo/sEpoR as significant predictors of Hct corrected for potential confounders (age, body mass index and serum testosterone). Total and free testosterone levels were not associated to Hct, nor to the erythropoietic factors. In conclusion, our results show a sustained erythropoietic stimulus driven by the Epo system in CMS patients, further enhanced by the continuous exposure to accentuated hypoxemia during sleep.

Support or Funding Information

Supported by The Wellcome Trust grant 097275/Z/11/Z
Tumor Necrosis Factor‐α Receptor 1 (TNFR1) Knockdown in Subfornical Organ (SFO) Attenuates Central Expression of Excitatory Mediators and Ameliorates Peripheral Manifestations of Heart Failure in Rats after Myocardial Infarction
Authors: YU YANG , Wei Shun‐Guang , Weiss Robert M , Felder Robert B
Published: November 2023

Myocardial infarction (MI)‐induced heart failure (HF) is a systemic inflammatory state characterized by increased circulating proinflammatory cytokines (PICs). Circulating PICs up‐regulate inflammation and renin‐angiotensin system (RAS) activity in cardiovascular regions of the brain, contributing to sympathetic excitation and the progression of HF. We recently demonstrated that the subfornical organ (SFO), a forebrain circumventricular organ that lacks a blood–brain barrier, is a predominant site in the brain at which circulating PICs act to elicit cardiovascular and sympathetic responses. Here we tested the hypothesis that tumor necrosis factor‐α receptor1 (TNFR1) knockdown in the SFO would modulate the MI‐induced expression of excitatory mediators in the SFO and downstream in the hypothalamic paraventricular nucleus (PVN) that mediate hemodynamic and sympathetic responses, and would ameliorate the peripheral manifestations of HF after MI. Rats received SFO microinjections of a TNFR1 shRNA or scrambled shRNA lentiviral vector carrying green fluorescent protein (GFP), or vehicle. One week later, transfection potential of the lentiviral vector was examined in some rats receiving SFO TNFR1 shRNA; other rats underwent coronary ligation to induce HF confirmed by echocardiography, or a sham operation. One week after SFO microinjections of TNFR1 shRNA, GFP fluorescence was visualized in the SFO but not in other brain areas. Four weeks after MI, HF+vehicle rats had increased mRNA of TNFR1, NF‐κB p65, cyclooxygenase (COX)‐2, angiotensin II type 1 receptor (AT1R) and c‐fos, and decreased mRNA of IkB‐α in both SFO and PVN, compared with SHAM+vehicle rats. HF+TNFR1 shRNA rats, but not HF+scrambled shRNA rats, had reduced mRNA of TNFR1 in the SFO, associated with decreased mRNA of NF‐κB p65, COX‐2, AT1R and c‐fos and elevated mRNA of IkB‐α not only in SFO but also in PVN. HF+vehicle rats also had increased plasma norepinephrine levels, lung/body weight and right ventricle/body weight ratios, which were attenuated in HF+ TNFR1 shRNA but not in HF+scrambled shRNA rats. The results suggest that HF‐induced increases in circulating TNF‐α act upon TNFR1 in the SFO to upregulate excitatory mediators in the SFO and downstream in the PVN, contributing to sympathetic excitation and cardiac dysfunction in HF. TNFR1 in the SFO may be a novel target for therapeutic intervention in inflammatory cardiovascular conditions like heart failure and hypertension.

Support or Funding Information

Supported by NIH Grants HL‐073986 and HL‐096671 and Department of Veterans Affairs.
Hypoventilation in the Mdx Mouse Model of Duchenne Muscular Dystrophy
Authors: Burns David P , Arijit Roy , McDonald Fiona , Wilson Richard J , O'Halloran Ken D
Published: November 2023

Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by a lack of the structural protein dystrophin. Patients have severe muscle wasting and chronic respiratory insufficiency. Hypoventilation and sleep disordered breathing are both common features of the disease and are known to culminate in periods of hypoxaemia and hypercapnia. There is a paucity of information regarding the control of breathing in animal models of DMD. We measured ventilation, oxygen consumption (VO2) and carbon dioxide (VCO2) production in freely behaving mdx (dystrophin deficient; n=11) and wild‐type (WT; n=10) mice at 8 weeks of age. qRT‐PCR was used to examine inflammation and hypoxia associated gene expression in diaphragm muscle from WT and mdx mice. Minute ventilation (VE; whole‐body plethysmography) was significantly reduced in mdx mice compared with WT controls (30% reduction). There was no significant difference in VO2 and VCO2 when expressed in absolute terms. Carotid sinus nerve unitary discharge was significantly depressed during normoxia in mdx (n=6) compared with WT (n=6) ex vivo perfused preparations. NF‐kB and TNF‐α mRNA expression were both significantly increased in mdx diaphragm compared with WT. HIF‐1α was significantly increased, while HIF‐2α was significantly decreased in mdx diaphragm muscle. Young mdx mice hypoventilate during air breathing (decreased VE/VCO2), which likely relates to reduced carotid body discharge during normoxia. Mdx diaphragm muscle is in a pro‐inflammatory state and there is differential expression of hypoxia associated genes. We reveal that respiratory control is altered in the mdx mouse model of DMD at a young age.

Support or Funding Information

Funded by the Department of Physiology, School of Medicine, University College Cork and Muscular Dystrophy Ireland.
Rewiring of spinal respiratory neural network via cervical glutamatergic interneurons preserves respiratory function in progressive cervical spinal cord compression injury (CSM)
Authors: Satkunendrarajah Kajana , Karadimas Spyridon K , Khazaei Mohamad , Samson Patricia , Fehlings Michael G
Published: November 2023

Cervical spinal cord injury (SCI) disrupts the spinal respiratory neural circuitry leading to the loss of diaphragmatic and intercostal muscle function. While acute traumatic SCI leads to significant ventilatory impairment, CSM, in which the cervical spinal cord is progressively compressed over time, results in milder respiratory dysfunction despite the significant disruption of the cervical neural network. The chronic and progressive nature of this disease modifies the respiratory neural network to limit respiratory insufficiency.

Using a unique mouse animal model of CSM, we found a significant loss of PMNs (retrograde labeled with CTb) that innervate the main inspiratory muscle, yet these animals do not exhibit severe respiratory insufficiency similar to what is observed in human CSM patients. Progressively increased Vglut2 positive boutons on the preserved PMNs indicates that despite the significant loss in the number of PMNs, respiratory motor output is maintained via compensatory and progressive increases in glutamatergic input onto preserved PMNs. While the PMNs were decreased following CSM there was an increase in the number of prephrenic cervical interneurons labeled with pseudorabies virus‐152 (PRV‐152). We set out to specifically delineate the extent of glutamatergic presynaptic inputs directly arising from rVRG neurons and those that are relayed via prephrenic cervical interneurons in CSM. To assess direct monosynaptic excitatory glutamatergic projections arising directly from rVRG and cervical spinal cord onto PMNs, a modified rabies virus (Rb) missing the glycoprotein but carrying a GFP protein (Rb‐ΔG‐GFP) with adeno associated virus expressing the Rabies‐Glycoprotein (RG, essential for subsequent trans‐synaptic transfection), was injected into the diaphragm of CSM and sham Vglut2::cre;tdtomato mice. We observe significantly altered input from glutamatergic monosynaptic inputs from rVRG neurons and those from prephrenic cervical interneurons onto PMNs during CSM. In conclusion, this study provides novel insights into the alterations in spinal respiratory networks that occur in the setting of CSM and a greater understanding of the neural control of breathing and the compensatory changes that occur in neural circuits with chronic, graduated compression.

Support or Funding Information

PVA
The Hairpin‐Hinge Model of Tat Protein Transport
Authors: Hamsanathan Shruthi , Bageshwar Umesh , Musser Siegfried
Published: November 2023

The bacterial twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane. Biochemical evidence indicates that the TatBC receptor complex contains the signal peptide binding site and the interaction of the signal peptide with this complex triggers the assembly of a translocation channel. However, it is unclear how the signal peptide interacts with the receptor complex and how it enables the movement of cargo through the channel. Here we show using fluorescence quenching and disulfide crosslinking that TatBC complex inserts pre‐SufI as a hairpin that extends about half‐way across the membrane. Photo crosslinking studies indicated that the c‐terminal of the hairpin interacts with TatB. Furthermore, computational analysis of ~500 Tat signal peptides and docking simulations of TatBC and pre‐SufI revealed 3 common features of the hairpin structure: 1) a helical structure; 2) a helix destabilizing residue; and 3) a relatively disordered domain. A hinge motion at the hairpin bend would allow the signal peptide to be bound to TatC surface via double arginines (n‐terminal) and allow the cleavage site (c‐terminal) to migrate to the periplasmic side. Disulfide crosslinking of n‐ and c‐terminal hairpin ends inhibited the transport of the cargo suggesting a novel Hairpin‐Hinge Translocation for Tat pathway

Support or Funding Information

NIH Grant GMO65534
Student Exploration Teaching Cardiovascular Concepts at a Title I School during PhUn Week
Authors: Evans LaShauna Chante
Published: November 2023

Physiology Understanding (PhUn) Week is a community outreach activity in which scientists present an educational supplement for students through partnership with local K‐12 teachers. I designed a cardiovascular presentation and simultaneous demonstration for 7th graders at Irving Academy which is located in one of the poorest zip codes in San Antonio. In addition, Irving Academy is a Title I school meaning the student population is from a low‐income household and at high‐risk of educational failure. By paralleling the school curriculum in which students were currently learning the circulatory system, this 30 minute activity sought to create greater understanding of the subject material, as well as, expose the students to scientific careers and professionals. First, students were taught oxygenated vs deoxygenated blood and blood flow through the body. Next students used a stethoscope to measure resting vs exercising heart rate and squeezed a stress ball to understand the differences between skeletal vs cardiac muscle. Lastly, students were shown examples of a human heart from a normal vs obese person to demonstrate the effect of obesity on the human body. The goal of this activity is for students with the greatest educational need to receive in depth instruction about physiology and related career options to facilitate the learning process, as well as, create interest in scientific topics.
Integration of Echocardiography, Simulation and Traditional Teaching of Cardiovascular Physiology to First Year Medical Students
Authors: McDonough Kathleen H. , Levitzky Michael G. , Gardner Jason , Lofaso Daryl , Pellett Andrew
Published: November 2023

We have combined the use of echocardiography, high fidelity simulation, and team‐based learning into a traditional course in which traditional didactic processes formerly predominated, in order to advance the teaching of the cardiovascular system to first year medical students. In previous years, clicker technology was used to determine not only the ability of the students to demonstrate knowledge of physiology but also as a tool to determine gaps or deficiencies in understanding. Upon reorganization of the medical curriculum to present “thinking like a doctor” courses in the first year curriculum, we added more clinical components to the Medical Physiology course. During the cardiovascular section, echocardiography was incorporated into the Cardiac Cycle and the Heart Sounds and Murmurs lectures. In addition, high fidelity simulation was included in two of the cardiovascular laboratory sessions. The ECG laboratory included small groups of students obtaining ECGs on each other and an interactive session with larger groups of students discussing arrhythmias. Similar arrhythmias were then produced on a simulation manikin (CAE Healthcare) so that students could feel pulses and note hemodynamic changes resulting from the arrhythmias on a clinical monitor. In addition, the simulation manikin was used to present real time changes in cardiovascular parameters induced by drug administration, vagal stimulation, and occlusion of the inferior vena cava. This was followed by a computerized small group exercise in which medical students and CRNA students worked together to interpret the cardiovascular changes due to similar types of manipulations in an instrumented conscious dog (Comish K, Zucker I, MedEd PORTAL Publications; 2009). Group learning and interprofessional education was incorporated into the interpretation of mechanisms of responses to the cardiovascular manipulations. Thus we were able to successfully integrate several clinically relevant concepts to the understanding cardiovascular function by first year medical and CRNA students.
OMENTAL HIGH ENDOTHELIAL VENULES AS CRITICAL NEUTROPHIL EXIT SITE IN PERITONITIS
Authors: Buscher Konrad , Wang Huiyu , Zhang Xueli , Ley Klaus , Sorokin Lydia , Song Jian
Published: November 2023

Neutrophil recruitment from the blood into the peritoneal cavity is critical in acute peritonitis but precise mechanisms of extravasation are largely unknown. In this study we identified the greater omentum as major exit site in early phases of mono‐ and polymicrobial peritoneal inflammation. A novel intravital microscopy technique was developed to image the inflamed omentum showing that high endothelial venules, known for their role in constitutive lymphocyte homing, are the preferred vascular bed for neutrophil recruitment with a unique requirement of adhesion receptors. Specific inhibition of the omental pathway in mice leads to delayed neutrophil deployment in the peritoneum, increased bacterial load, and aggravates septic progression of bacterial peritonitis.

Support or Funding Information

‐ German Research Foundation grant to LS (CRC1009/A02, So285/9‐1, EXE1003)‐ German Research Foundation grant to KB (BU 3247/1)‐ Cells‐in‐Motion Cluster of Excellence Flexible Funds grant (FF‐2014‐07) to JS and KB‐ Grant of the Interdisciplinary Center for Clinical Research (IZKF) of the Medical Faculty of the University of Muenster to KB (SEED 04/12)
Regulation of the Nuclear PKA Holoenzyme
Authors: Clister Terri , Zhang Jin
Published: November 2023

cAMP and PKA activity is regulated in time and space to ensure proper cellular responses. Recently a novel pool of activatable PKA holoenzyme was discovered to reside in the nucleus, expanding the traditional model of PKA signaling. Live‐cell imaging suggested that the regulation of the nuclear PKA holoenzyme is dependent on A‐Kinase Anchoring Proteins (AKAPs) and phosphordiesterases (PDEs). Specifically, we hypothesized that PDEs anchored to AKAPs create a local microdomain with a lower concentration of cAMP, thus controling the activation of anchored nuclear PKA. Co‐IP experiments using nuclear extracts revealed that both the RIIα subunit of the PKA holoenzyme, and PDE4D isoforms interact with AKAP95, supporting this hypothesis and providing specific candidates to further explore this regulation. Our ICUE3 cAMP biosensor was localized to AKAP95 to measure the levels of cAMP directly around AKAP95 in response to differently localized sources of cAMP. We are able to generate cAMP in different cellular compartments using targeted soluble adenylyl cyclase (sAC) and activating it in a dose‐dependent manner with sodium bicarbonate. When a low concentration of cAMP is generated, only cAMP produced in the nucleus can be detected in the vicinity AKAP95, whereas cAMP produced at the plasma membrane was not detected within this microdomain. Conversely, the AKAP95 microdomain was able to sense a large cAMP signal regardless of whether it was produced at the plasma membrane or in the nucleus. These results support the hypothesis that AKAP95 and PDE4Ds create a microdomain that prevents the activation of scaffolded nuclear PKA holoenzyme except by local cAMP signals.
Ras1 Phosphorylation and its Role in Nutrient Signaling
Authors: Sethupathi Sheetal Kasi , Jin Xin , Wang Yuqi
Published: November 2023

Ras1 is a small GTPase found in Saccharomyces cerevisiae that regulates nutrient signaling and is known to undergo phosphorylation. Studies were conducted to gain a better understanding of the functional consequences and the regulation of Ras1 phosphorylation. Site‐directed mutagenesis was used to mutate critical serine residues to alanine residues. A serine residue at the C‐terminal region of Ras1 was identified as an important residue for Ras1 phosphorylation as mutating this serine residue to alanine drastically diminished the level of Ras1 phosphorylation. Additionally, phosphorylated Ras1 accumulates as cells approach the stationary phase of growth. Similarly, cells exposed to nitrogen starvation exhibit increased levels of phosphorylated Ras1. By blocking the phosphorylation of Ras1, we find that the level of autophagy decreases. Together, these findings reveal phosphorylation of Ras1 as a mechanism in regulating cellular responses to nitrogen starvation.
The Role of BCAR1 and FYN on CD133+ GBM stem cells
Authors: Yukselten Yunus , Bal Merve Gulsen , Ozkanca Seyma , Yildiz Dilara Akcora , Bunsuz Merve , Ugur Hasan Caglar , Sunguroglu Asuman
Published: November 2023

OBJECTIVE

Glioblastoma Multiforme (GBM) is the most prevalent and aggressive primary brain tumor. Glioblastoma stem cells (GSCs) are thought to play important roles in tumor initiation, establishment, and dynamic evolution. CD133 (prominin‐1), a transmembrane glycoprotein, has been shown to mark stem like cells in colon, prostate and GBM. The integrin signaling adaptors including BCAR1 (p130 Crk‐associated substrate; p130CAS and FYN (FYN Proto‐Oncogene, Src Family Tyrosine Kinase) are important players in cell invasion and migration. Moreover, BCAR1, p130CAS and FYN promotes cell proliferation, survival and motility, indicating a connection with tumorigenesis. In this study, we investigated the gene expression levels of cell adhesion molecules in both CD133+ GSCs and CD133− GBM cells.

MATERIAL METHODS

GBM primary cells were freshly isolated from glioblastoma tissue samples and cultured in DMEM supplemented with 10% fetal calf serum and 1% penicillin‐streptomycin at 37 °C in 5% CO2‐humidified incubator. We isolated CD133+ and CD133− cells from 10 GBM primary cells using MACS system. Following RNA isolation from those positive and negative cells, cDNA synthesis was performed. Finally, according to microarray protocol, cell adhesion array was applied. Gene expression levels were analyzed using the ‐delta delta Ct method. One‐sample t‐test was used to determine the mean fold changes (increase/decrease) of genes of interest between CD133+ and CD133− cells. Statistical analysis was performed using SPSS software for windows version 13.0.

RESULTS & CONCLUSION

According to our results, compared to CD133‐GBM cells BCAR1 and FYN gene expression levels were found to be 3.96 and 6.05‐fold increased respectively in CD133+ GSCs (p<0.05).

In summary, we found increased transcript levels of BCAR1 and FYN genes in CD133+ GSCs compared with CD133− cells. A deeper understanding of the gene expression signature of CD133+ GCSs might be helpful to improve the therapies against this tumor.

This research has been supported by The Scientific and Technological Research Council of Turkey (No:114S189).