bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of Linaclotide‐induced Trafficking of CFTR and NHE3 in the Human Intestinal Cell‐lines Caco‐2 BBe and T84 cells
Authors: Ahsan Md. Kaimul , Silos‐Santiago Inmaculada , Ameen Nadia
Published: June 2021

Linaclotide (Linzess) is a potent guanylate cyclase‐C (GCC) agonist in clinical use for treatment of chronic constipation and irritable bowel syndrome. Electrophysiological studies indicated that linaclotide modulates CFTR and NHE3 ion transport. Here we investigated linaclotide‐dependent intracellular signaling pathways using human intestinal cell lines resembling crypt (T84) and villus (Caco‐2BBe) enterocytes. PCR and immunoblots were employed to examine GCC‐dependent machinery critical for CFTR and NHE3 trafficking. Linaclotide‐induced phosphorylation of VASPSer239, a substrate for cGKII, the kinase necessary for cGMP dependent trafficking CFTR and NHE3 in the intestine, was examined. Trafficking of CFTR and NHE3 into the apical membrane was examined by surface biotinylation following treatment with cGMP agonists and linaclotide in the absence or presence of H7 (PKA inhibitor) or H8 (PKGII inhibitor). Both cell lines expressed the cGMP‐dependent kinase cGKII. Protein levels of cGKII were higher in the villus resembling Caco‐2BBe cells, consistent with findings in native intestine. Apical treatment of filter‐grown polarized cells with the cGMP analog 8Br‐cGMP (0.2mM,1mM) and linaclotide (1µM, 3.27µM) resulted in phosphorylation of VASPser239, indicating activation of cGKII. Levels of surface‐biotinylated CFTR and NHE3 were consistent with the phosphorylated cGKII responses to 8Br‐cGMP and linaclotide and were reduced by both PKA and PKG inhibitors. These data indicate that linaclotide stimulates trafficking of CFTR and NHE3 into the apical membrane through cGMP dependent PKGII and cross activation of cAMP dependent PKA signaling pathways.
Osteopontin‐Stimulated Apoptosis in Cardiac Myocytes Involves Reactive Oxygen Species and Mitochondrial Pathway
Authors: Dalal Suman , Singh Mahipal , Singh Krishna
Published: June 2021

Increased osteopontin (OPN) expression associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we have shown that OPN stimulates cardiac myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. The objective of this study was to evaluate the production and source of reactive oxygen species (ROS), and their role in OPN‐stimulated myocyte apoptosis. Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) induced protein oxidation as analyzed by protein carbonylation assay. Direct monitoring of ARVMs using total ROS detection kit and DCFDA (2',7'‐dichlorofluorescin diacetate; a fluorogenic dye) staining showed increased number of ROS‐positive cells in response to OPN. OPN induced mitochondrial depolarization, and decreased the expression of mitochondrial antioxidant enzyme, manganese superoxide dismutase‐2 (SOD‐2). Transmission electron microscopy revealed that OPN induces mitochondrial abnormalities including swelling and reorganization of cristae. OPN‐mediated increase in the levels of cytosolic cytochrome c and apoptosis were significantly inhibited by N‐acetyl‐cysteine (antioxidant), apocynin (NADPH oxidase inhibitor), MnTBAP [Mn(III)tetrakis (4‐benzoic acid) porphyrin; SOD mimic] and glibenclamide (mitochondrial KATP channel blocker). These data suggest that OPN induces ROS production, and affects mitochondrial morphology and integrity. Mitochondria may serve as a source of ROS production through the opening of mKATP channels.

Supported by Department of Veterans Affairs (BX002332) and NIH
Trafficking and Membrane Targeting of NBCn1 in MCF‐7 Breast Cancer Cells
Authors: Olesen Christina , Vogensen Jens , Pedersen Stine
Published: June 2021

We previously showed that the Na+,HCO3‐ cotransporter NBCn1 (SLC4A7) is upregulated in breast cancer, and that cisplatin treatment of breast cancer cells causes pronounced loss of NBCn1 plasma membrane localization. However, essentially nothing is known regarding the mechanisms of NBCn1 trafficking and turnover. NBCn1 localized predominantly to the basolateral membrane in polarized breast cancer cells, with a highly cell‐density dependent expression profile. Removal of the cytosolic NBCn1 C‐terminal tail (NBCn1‐Ct) strongly reduced NBCn1 membrane localization in HEK293 cells. GST pull‐down in MCF‐7 cells followed by LTQ Orbitrap Mass spectrometry identified Receptor C kinase‐1 (RACK1), retromer complex components vacuolar protein sorting‐associated protein 35 (VPS35) and Sorting Nexin 27 (SNX27), and AP1 and ‐2 adaptor proteins, as NBCn1‐Ct interaction partners. Co‐immunoprecipitation and immunofluorescence analysis confirmed interaction between RACK1 and NBCn1 in MCF‐7 cells. Furthermore, siRNA‐mediated RACK1 knockdown reduced NBCn1 expression and strongly attenuated its plasma membrane localization. SNX27 and VPS35 partially colocalized with NBCn1 in breast cancer cell lines, in a punctate pattern closely adjacent to the plasma membrane, suggestive of a role in NBCn1 trafficking. In conclusion, these preliminary findings point to the involvement of RACK1 and likely also VPS35 and SNX27, in regulation of NBCn1 expression and localization in breast cancer cells.
Quantification of Mitochondrial DNA Lesions in Cultured H9c2 Cardiomyocytes Subjected to Oxidative Stress
Authors: Rodríguez‐Muñoz Adlín , Barreto‐Torres Giselle , Soto‐Hernández Jessica , Ayala‐Peña Sylvette , Javadov Sabzali , Torres‐Ramos Carlos
Published: June 2021

Oxidative stress plays a crucial role in the pathogenesis of cardiovascular diseases such as myocardial infarction, hypertrophy and heart failure. ROS can induce a variety of DNA lesions in nucleus and mitochondria. However, quantitative analyses of these DNA lesions require methodical approaches that can discriminate between nuclear and mitochondrial DNA (mtDNA) lesions. In this study, we aimed to apply a PCR‐based (QPCR) assay for the quantification of mtDNA lesions in cultured cardiomyocytes subjected to oxidative stress. In this assay, lesions that blocks the progression of the PCR polymerase result in decreased amplification of the target sequence, giving a relative measure of the lesions present. H9c2 embryonic rat cardiomyocytes cultures were treated for 1 h with increasing concentrations of H2O2 followed by DNA isolation and QPCR analysis. Compared to untreated cells, cells treated with 75, 100 and 200 µM of H2O2 showed a significant 60%, 80% and 90% reduction in the amplification of a mtDNA target respectively. These results indicate that the QPCR assay can be applied to detect mtDNA damage in rat cardiomyocytes after acute oxidative stress. Future experiments involve: (1) the determination of the repair kinetics of mtDNA damage and (2) the effects of mtDNA lesions in mitochondrial function. Supported by NIH grants SC1HL118669 and G12RR03051.
Is P53 Activated in Response to Airborne Hazards Exposure in Deployed Military?
Authors: Chen Yang , Klein Jacquelyn , Ndirangu Duncan , Condon Michael , Falvo Michael
Published: June 2021

The tumor suppressor p53 plays a critical role in mediating cell cycle, DNA repair and apoptosis in response to environmental stress. Increased levels of p53 have been observed secondary to airborne hazards exposure, including polycyclic aromatic hydrocarbons (PAH) and particulate matter (PM). Exposure to airborne hazards has been shown to induce systemic oxidative stress and promote mitochondrial damage and we hypothesize that p53 will be involved in with this associated repair. Military personnel deployed to Iraq and Afghanistan were likely exposed to high levels of airborne hazards during their service, including PM and PAH, and are therefore a unique population for investigation. Twenty deployed veterans volunteered for this study and were assigned to two groups (蠄12 months) or (>12 months) based on total deployment length. All subjects completed maximal exercise testing and provided blood samples for measurement of p53 plasma protein level via ELISA as well as lactate. We found deployment length was positively correlated to p53 plasma protein level (r=0.56, p< 0.01). Veterans with longer deployment lengths (>12 months) demonstrated a non‐significant trend towards higher p53 plasma protein levels (p = 0.12, Cohen's d = 0.76). In addition, post‐exercise lactate production showed a greater trend (p = 0.066) in veterans with longer deployments. Increased levels of plasma p53 may be an adaptive response to airborne hazards exposure during military deployment. As p53 appears to be uniquely involved in mitochondrial regulation of reactive oxygen species, additional studies to directly assess mitochondrial damage and function in this population are warranted.
Mechanism of Cell Death Induced by in Vitro Hypoxia/Reoxygenation Using Glucose Oxidase in Human Cells
Authors: Dubick Michael , Barr Johnny , Bowman Phillip
Published: June 2021

Ischemia/reperfusion injury (I/RI) is associated with trauma and various diseases including stroke and heart attack. This study investigated the mode of cell death induced in an vitro system of hypoxia/reoxygenation that may be useful for screening for drugs to reduce I/RI. The usual method of subjecting cells to reduced oxygen (O2) by purging with nitrogen is a slow process and does not reproduce the rapid local reduction in O2 that occurs in vivo during ischemia. Glucose oxidase (GOX), however, rapidly oxidizes extracellular glucose producing a very low O2 level (< 0.4 µM) within 5 min with only minor reductions in glucose levels. Human umbilical vein endothelial cells (HUVEC) and HeLa cells were subjected to GOX‐induced hypoxia for 10 min and returned to medium and normoxia for up to 6 hrs. Cells loaded with tetramethylrhodamine ethyl ester (TMRE) and subjected to hypoxia showed that the mitochondrial permeability transition pore was compromised by 3 hrs and mitochondria were losing the ability to retain TMRE. Significant propidium iodide (PI)‐positive HUVEC cells, an indicator of cell death,were evident by 6 hrs. Similar responses were observed in HeLa cells indicating a generalized necroptotic cell death. When catalase was included in the medium following GOX‐induced hypoxia, cell death was prevented implicating hydrogen peroxide as an important contributor to this injury. This in vitro system can be used to further understand responses of cells to I/RI and identify drugs and potential therapeutic windows in vitro that may be translated to ameliorate ischemia/reperfusion injury in vivo. Funded by US Army Medical Research Materiel Command.
Temporal Heterogeneity In Apoptosis Execution
Authors: Vorobjev Ivan , Barteneva Natasha
Published: June 2021

Apoptotic process is highly heterogeneous, and a long‐standing question is what parameters define time and reversibility of the apoptotic response at a single cell level. Using multicolor fluorescence assay we characterized at a single‐cell and population levels sequence of apoptotic events induced by extrinsic (TRAIL) and intrinsic (staurosporine, actinomycin D and etoposide) apoptotic stimuli in Jurkat and HeLa cells. Regardless of the stimulus first visible event on a single cell level was rapid apoptotic volume decrease (AVD) and Na+ influx tightly coordinated with mitochondria outer membrane permeabilization (MOMP) and mitochondrial depolarization. These events happened largely asynchronous in the cell population. Activation of caspases 3/7 is slow process at a single cell level. It always starts after MOMP with significant delay and caspase activity linearly accumulates in a given cell for several hours.

Cell‐to‐cell variability of the beginning of MOMP is well approximated by Gaussian distribution with CV about 35‐40%, while the γ‐distribution model describes cell‐to‐cell variability in the pre‐MOMP and MOMP‐to‐caspase activation stages. Sorting different subpopulations after induction of apoptosis we show that cells with intact mitochondria can recover after washout of apoptotic stimulus, cells after MOMP recover rarely and cells with caspase 3/7 activity can not recover at all. We propose a double‐stroke model for apoptosis execution/cell recovery.
Striatin is cleaved during apoptosis in a caspase‐3 mediated manner: a step towards novel biomarkers for apoptosis.
Authors: Nader Moni , Al‐Gohi Amani , Hoilat Judie , Khalil Bariaa , Kvietys Peter , Bakheet Dana , Dzimiri Nuduna
Published: June 2021

Recent reports showed that striatin (STRN) is a substrate for calpain2 and caspase‐3 in a model of traumatic brain injury, but its role in apoptosis remains uncertain. We found that striatin (110 kDa) is cleaved under different apoptotic stimuli (Staurosporine, etoposide, and TRAIL) in Hela cells and this cleavage (~60kDa) appeared mainly in the cytosolic fraction when compared to non‐cytosolic extracts (including mitochondria). Interestingly, staurosporine challenge of Hela cells decreased STRN levels in the non‐cytosolic fraction compared to the cytosolic fraction implying a shuttling of STRN under during apoptosis. Pharmacological treatment with the general caspase inhibitor z‐VAD.fmk abolished the cleavage of striatin in total cell lysates and in the cytosolic fraction of apoptotic Hela cells. While staurosporine was unable to significantly cleave STRN in cells lacking caspase‐3 (MCF‐7), overexpression of active or mutant (inactive) form of caspase‐3 resulted in cleavage of STRN (Western blot). Interestingly, this cleavage was also abolished by the pretreatment of MCF‐7 cells with z‐VAD.fmk. Collectively, our data suggest that STRN is cleaved during apoptosis in a caspase‐3 related manner and emerges as a novel marker for apoptosis.
PPARγ‐Independent Side Effects of Thiazolidinediones on Mitochondrial Redox State in Rat Isolated Hearts
Authors: Salzman Michele , Cheng Qunli , Weihrauch Dorothee , Camara Amadou , Stowe David , Riess Matthias
Published: June 2021

The effect of anti‐diabetic thiazolidinediones (TZDs) on contributing to heart failure and cardiac ischemia/reperfusion (IR) injury is controversial. Peroxisome proliferator‐activated receptor‐gamma (PPARγ) agonists are under scrutiny because of concerns about their cardiovascular safety. Acute administration of rosiglitazone, for example, was reported to differentially aggravate IR injury in a consomic rat heart model. In this study we investigated the effect of select TZDs on mitochondrial function in rat isolated hearts. Different concentrations of the TZDs rosiglitazone and pioglitazone were administered in the presence or absence of the specific PPARγ antagonist GW9662, and their effects on mitochondrial redox state were measured by online NADH and FAD autofluorescence. Both TZDs resulted in dose‐dependent, reversible increases in mitochondrial oxidation that were not attenuated by GW9662. Taken together, these data suggest excessive mitochondrial uncoupling by a PPARγ‐independent mechanism. If translated clinically, this may explain in part how TZDs adversely enhance cardiac IR injury and adverse events in susceptible patients. Supported by Department of Veterans Affairs (CARA‐026‐10F), NIH and institutional funds.
Truncation of GSK‐3β in Cardiac Mitochondria is the Master Switch of the mPTP
Authors: Hurst Stephen , Gomez Ludovic , Jhun BongSook , O‐Uchi Jin , Sheu Shey‐Shing
Published: June 2021

Glycogen Synthase Kinase‐3β (GSK‐3β) is proposed as one of the key molecules that regulate cell death during ischemia reperfusion (IR) injury in the heart, largely via modulating mitochondria permeability transition pore (mPTP) activity, where both Ca2+ and oxidative stress are at peak levels. However, the exact mechanism of how GSK‐3β controls cardiomyocyte survival and death has remained elusive. In non‐cardiac cells, μ‐Calpain activated by high levels of Ca2+ and ROS can truncate GSK‐3β at both the N and C termini to generate a constitutively‐active GSK‐3β by removing the inhibitory phosphorylation sites. Therefore, we hypothesize that truncation of GSK‐3β in cardiac mitochondria can modulate mPTP function via the phosphorylation and binding affinity of mPTP component proteins.

Using mitochondrial fractionation and proteinase‐K digestion, we found that GSK‐3β is truncated by μ‐Calpain in the mitochondria matrix. Using co‐immunoprecipitation (IP) we determined that truncated GSK‐3β associates with and phosphorylates cyclophilin D (mCypD). In addition, P‐mCypD increases the binding affinity to ATP‐Synthase, a component of the mPTP pore. Overexpression of a mitochondrial targeted Calpain inhibitory peptide based on the Calpastatin inhibitory domain in H9c2 cardiac myoblasts significantly blocked ionomycin‐induced cell death, as measured by Caspase 3/7 activation, to a greater extent than pretreatment with cyclosporine A, a potent mPTP inhibitor. In conclusion, Calpain‐mediated truncation of GSK‐3β in the mitochondrial matrix leads to phosphorylation of mCypD, allowing P‐mCypD to bind to and open the mPTP pore complex in cardiac cells.

Funding Source: NIH Grant 5T32AA007463‐28
PAH Exposure Impacts Cardiac Function in Xenopus laevis Embryos
Authors: Pinette Julia , Sestak Madison , Johnson Kirsten , Whittemore Susan
Published: June 2021

Using Xenopus laevis, a well‐established model system for vertebrate heart development, we assessed the impact of three common polyaromatic hydrocarbons (PAHs): phenanthrene (PHE), pyrene (PYR), and fluoranthene (FLA) on heart rate (HR), interbeat variability (IBV), incidence of atrioventicular (AV) block, and stroke volume (SV) and compared effects with the well‐characterized PAH benzo(a)pyrene (BaP) and with DMSO (vehicle) controls.

A 72‐hr exposure (during heart valve formation) was associated with tachycardia for all PAHs at all doses (0.25‐25 μM). Increased HRs ranged from 20‐35% (p<0.05‐0.001). With the exception of 25 μM PHE and FLA, PAH‐induced tachycardia persisted at 96 hr (during atrial partitioning), while bradycardia was observed in embryos with a mature heart phenotype after 120 hr exposure to 25 μM FLA and PHE (14% and 17% decrease in HR; p<0.05, 0.01, respectively).

While PAH exposure did not alter IBV, an indicator of ventricular arrhythmia, 25 μM FLA and PHE treatment did interfere with normal cardiac conduction. The incidence of AV block with FLA (25 μM) was 40%, with similar, but less dramatic, dose‐dependent responses observed for PHE. In contrast, none of the DMSO or 0.5 μM BaP‐treated animals exhibited AV block, with 5% incidence for 5 μM BaP. SV was unaffected by 120 hr exposure to any of the PAHs at any dose.

The embryonic heart generates the hemodynamic forces needed for development of the circulation. These results suggest that early exposure to these common PAHs compromises cardiac function and may impact circulatory development. All three PAHs have been detected in significant amounts in human milk and cord blood, underscoring the need to gain a better understanding of their potential for cardiotoxicity.
Expression of Hemoglobin Related Proteins in Nucleated and Enucleated Red Blood Cells
Authors: Götting Miriam , Nikinmaa Mikko
Published: June 2021

In vertebrates a total of eight different globins are known which are expressed in a highly species‐ and tissue‐specific manner. Globins are small molecules with a heme prosthetic group, by which they reversibly bind O2 and other gaseous ligands. They are all involved in cellular processes related to O2 transport (e.g. hemoglobin) and storage (e.g. myoglobin), detoxification of reactive oxygen and nitrogen species, sensing and signaling or may have yet unknown functions. The functions of hemoglobin and myoglobin are well understood and known since several years, but data on the other globins are fragmentary. In particular, the factors controlling globin mRNA stability, gene transcription and translation of these globins as well as their physiological functions are currently unknown. Here we examined the expression of globin genes in nucleated and enucleated erythrocytes of different vertebrate model species by quantitative real‐time reverse transcription PCR (qRT‐PCR). Furthermore we studied the transcription rate and mRNA levels of globin genes and other genes (e.g. hypoxia inducible factor 1α, sodium/proton exchanger) under hypoxia, temperature stress and adrenergic stimulation by the nuclear run‐off assay in rainbow trout red blood cells. We surprisingly detected transcripts of the whole gnathostome globin superfamily in mature circulating red blood cells of all vertebrate species but the mRNA expression levels varied among species. The transcription rate of the genes depended on the treatment. Our results add new and unexpected aspects to the current knowledge on the expression specificity of globins in vertebrate tissues and organs.
Adult nephrogenesis and persistence of Six2 expressing progenitor cells in the Reptilian kidney
Authors: Camarata Troy , Vasilyev Aleksandr
Published: June 2021

Kidney formation proceeds in reptiles, birds, and mammals through the sequential development of the pronopheros, mesonephros, and finally the metanephros. Metanephric nephron formation, termed nephrogenesis, ceases in mammals around birth and is completely absent in adults. We investigated the presence of adult nephrogenesis in reptiles by examining adult kidneys from several species including T. scripta, C. picta, B. constrictor, T. tegu, A. carolinensis, and A. mississipiensis. We found that all major reptilian groups (Testudines, Archosauromorpha and Squamates) showed the presence of adult nephrogenesis. The total amount of nephrogenesis varied greatly between species with turtles and alligators displaying the highest density of nephrogenesis. The nephron is derived from progenitor cells which express the transcription factor Six2. In mammals, Six2 expression is downregulated as the progenitor cell population is exhausted and nephrogenesis ends. Since we detected nephrogenesis in adult reptiles, we postulated that Six2 expression persists in these species. Unlike in mammals, we were able to detect Six2 protein expressing condensing mesenchyme in adult alligator kidneys, correlating with areas of nephrogenesis. This suggests that reptiles have maintained the ability to continually grow new nephrons during adulthood due to the presence of a Six2‐expressing progenitor cell population.
Depression‐like Symptoms are Associated with Dysbalance in Vascular Norepinephrine Uptake. This is Worsening by Antidepressants and Increases the Risk for Hypertension.
Authors: Matchkov Vladimir , Wiborg Ove , Bouzinova Elena , Aalkjaer Christian
Published: June 2021

Antidepressants affecting norepinephrine (NE) reuptake are known to increase the risk for hypertension. Anhedonia ‐ a decreased ability to experience pleasures ‐ is a central symptom in depression and can be evoked in preclinical models exploring the role of the stressful environment in mediating affective signs and autonomic function.

Some rats exposed to Chronic Mild Stress (CMS) developed depression‐like symptoms (i.e. anhedonia, measured by sucrose intake) while others were resilient to 8 wks of CMS. Normal blood pressure and decreased cardiac function suggested increased peripheral resistance in rats with depression‐like symptoms. Chronic treatment with escitalopram (SSRI) did not affect blood pressure but normalized behavior in ~50% of treated rats, e.g. responders.

Resistance (small mesenteric and femoral) arteries from CMS and non‐stressed rats responded similarly to NE in vitro. Inhibition of neuronal reuptake with cocaine increased NE sensitivity more in rats with depression‐like symptoms than in resilient and non‐stressed groups. Corticosterone‐sensitive extra‐neuronal catecholamine uptake was diminished in rats exposed to CMS. These changes were associated with upregulation of the neuronal NE transporter and reduced expression of the extra‐neuronal transporter (OCT‐2) in arteries from 'depressed' rats. Escitalopram further upregulated neuronal NE reuptake in all groups.

Our results indicate that depression‐like symptoms are associated with changes in NE uptake in the vascular wall and could explain pro‐hypertensive action of some antidepressants.
The Role of Nucleus Tractus Solitarii (nTS) Neurons in Cardiorespiratory Dysfunction Associated with Alzheimer's Disease
Authors: Ostrowski Tim , Kline David , Hasser Eileen , Ostrowski Daniela
Published: June 2021

Alzheimer's disease (AD) is associated with dysfunction of cardiorespiratory control. The mechanisms behind these alterations are not understood. The nTS is critical in cardiorespiratory homeostasis and reflex function, and may play a crucial role in AD dysautonomia. Here we examined autonomic and respiratory regulation, and neuronal alterations in the nTS of an established rodent AD model (CNS Streptozotocin (STZ)‐induced). Results are compared to saline‐injected control rats. In vivo plethysmography revealed a strong trend for blunted hypoxia/chemoreflex‐induced increases in breathing (p=0.068). Furthermore, baroreflex‐mediated sympathoexcitation to hypotension was markedly blunted in AD rats (p蠄0.01), consistent with orthostatic hypotension observed in AD patients. To examine the neuronal alterations in the nTS, patch clamp recordings were made in in vitro slices. When compared to control, AD rats show significantly increased network activity (p蠄0.01), spontaneous spike discharge and enhanced discharge to current injection (~40% increase vs. control; p蠄0.001). Our results suggest increased basal excitation of the nTS neuronal network and blunted cardiorespiratory reflex responses, which may play an important role in the autonomic and respiratory dysfunction observed in AD patients.

Supported by: RO1 HL098602, AHA 12POST11670002
Distinct Organization of Upper Airway Nodose and Jugular Vagal Circuits in the Brain: Evidence from Conditional Anterograde Transneuronal Tracing and Physiology
Authors: McGovern Alice , Driessen Ally , Simmons David , Farrell Michael , Davis‐Poynter Nicholas , Mazzone Stuart
Published: June 2021

Irritations of the airways evoke brainstem‐mediated reflex changes in breathing and complex sensorimotor responses dependent upon ascending inputs to higher brain centers. Airway vagal afferents terminate in the nucleus of the solitary tract (Sol) and the paratrigeminal nucleus (Pa5) however, whether these nuclei are comparable in terms of afferent inputs, central ascending projections or physiological function is unknown. In Sprague‐Dawley rats (n=4) retrograde tracing with fluorescently tagged cholera toxin B revealed neurons in the jugular vagal ganglia project predominately to the Pa5, while nodose vagal ganglia neurons terminate in the Sol. Electrical stimulation of the trachea in urethane anesthetized rats evoked stimulus dependent apneic responses (control n=4; Emax reduction in respiratory rate = 3±2 breaths/min) which were abolished after inhibition of the Sol by microinjection of GABAA agonist, muscimol (n=4, Emax = 62±5, p=0.001), but only partially inhibited from the Pa5 (n=5; Emax = 36±6, p=0.01). To determine the ascending projections of airway specific Sol and Pa5 neurons we constructed a conditional HSV‐1 H129 virus that switches fluorescent reporters in the presence of Cre allowing the connectivity of subsets of neurons to be visualized. Following inoculation of the airways with HSV‐1 H129, the Sol (n=8) projected to the zona incerta, paraventricular and lateral hypothalamic nuclei, while Pa5 (n=7) neurons terminated in the submedius and ventrobasal thalamus. Taken together, these data suggest distinct pathways governing airway sensations exist, likely playing differing roles in the processing of respiratory responses.
Brain Region‐Specific Expression of Angiotensin AT1A Receptors in Microglia
Authors: Voong Susan , Claflin Kristin , Grobe Justin
Published: June 2021

Through its actions at the AT1A receptor in the hypothalamus, the renin‐angiotensin system contributes to the control of resting metabolic rate and blood pressure. Activation of microglia is required for angiotensin‐dependent hypertension, and exposure to high fat diet increases microglia infiltration in the hypothalamus. We therefore hypothesized that angiotensin AT1A receptors are expressed on microglia within the hypothalamus, and that these cells may play a role in the development of obesity‐hypertension. Brains from mice expressing green fluorescent protein via the angiotensin AT1A promoter (“NZ44” mice from the GenSat Project) were sectioned and immunostained to label microglia via antibodies against allograft inflammatory factor 1 (IBA1) conjugated to red fluorescent protein. When mice were maintained on standard chow diet, we observed dual‐fluorescent cells within the ventromedial hypothalamus, but not in the arcuate nucleus, median eminence, paraventricular nucleus, dorsomedial hypothalamus, or subfornical organ despite substantial numbers of singly‐fluorescent cells in each of these regions. Together, these data support a region‐specific expression of AT1A in microglia. Ongoing studies are focused on investigating the effects of dietary interventions (e.g. – high fat diet) and hypertensive stimuli (e.g. – deoxycorticosterone acetate (DOCA)‐salt treatment) upon region‐specific microglial infiltration, activation, and expression of AT1A.
Central Inhibition of Bradykinin B1 Receptor Attenuates DOCA‐Salt Mediated Hypertension, Dysautonomia and Hypertrophy
Authors: Sriramula Srinivas , Lazartigues Eric
Published: June 2021

We previously reported that DOCA‐salt hypertension is attenuated in bradykinin B1 receptor (B1R) knockout mice. To further examine the role of B1R, a specific antagonist R715 was used to inhibit B1R in the brain in wild‐type (WT) mice with or without DOCA‐salt hypertension. Autonomic function was analyzed using a pharmacological method (propranolol, 4 mg/kg, ip and atropine, 1 mg/kg, ip). Hypertrophy was assessed after 3 weeks using organ weight/tibia length. Compared to WT+DOCA, R715‐treated mice (70 µg/kg/day, icv) had a blunted hypertension (Telemetry; 143±2 vs. 122±4 mmHg, P<0.01) and an improved baroreflex gain (1.2±0.1 vs. 2.4±0.3 msec/mmHg, P<0.01). WT+DOCA mice dysautonomia, illustrated by reduced tachycardia to atropine (parasympathetic tone: +91±3 bpm) and increased bradycardia to propranolol (sympathetic tone: ‐145±21 bpm), was normalized in mice with R715 infusion (+174±15 and ‐81±9 bpm, respectively, p<0.05 vs. WT+DOCA), while urine norepinephrine levels were decreased (163±19 vs. 299±23 pg/ml, p<0.01). Moreover, DOCA‐salt treatment resulted in cardiac (7.2±0.2 vs. 9.2±0.3 mg/mm) and renal hypertrophy (9.7±0.3 vs. 21.1±0.7 mg/mm) in WT mice, which was significantly reduced in WT+DOCA+R715 mice (heart: 7.4±0.3 mg/mm; kidney: 18.1±0.9 mg/mm, P<0.01 vs. WT). In DOCA‐treated mice, expression of the pro‐inflammatory ADAM17 protein in the hypothalamus was increased by 2.4 fold (n=6, p<0.01), which was blunted by R715 infusion.Together, our data provide novel evidence to support the role of central B1R in dysautonomia, hypertension and organ damage in low‐renin hypertension. Funding: AHA 12EIA8030004 and 13POST16500025.
Influence of oxygen enriched gases during decompression on bubble formation and endothelial function
Authors: Valic Zoran , Segrt Ivana , Bozic Josko , Glavas Duska , Obad Ante , Glavicic Igor , Valic Maja
Published: June 2021

Breathing oxygen enriched gases during decompression procedure leads to decreased time spent at specific decompression stop and ultimately to shorter dives with same amount of time on the bottom. However, breathing oxygen can damage endothelial function with reduction in flow mediated vasodilation (FMD). Five female and five male experienced SCUBA divers, with a mean age of 39±3 years (mean±SEM), were included in this investigation. Each diver performed three dives to 45 meters of sea water and 20 minutes bottom time using air. Air, nitrox 50 or nitrox 99 were used during decompression stops in accordance to V‐planner software. Order of the dives was randomized, with one day of recovery between each dive. Brachial artery function (FMD) was determined before and after each dive using ultrasound. Same device was used to measure number of venous gas bubbles after diving. Total diving times using air, nitrox 50 or nitrox 99 were 65, 45 and 43 minutes, respectively. Bubble grades after corresponding dives were: 1.4±0.4, 1.9±0.4 and 0.9±0.2, and did not show statistical difference. FMDs after corresponding dives were: 7.6±3.9%, 1.0±8.6% and 7.4±3.7, and did not show statistical difference although function of the arteries was greatly affected if divers used nitrox 50 as gas during decompression. In conclusion, we would recommend protocol which employs breathing nitrox 99 during decompression since it leads to the shortest dive with no significant change in both bubble number and FMD comparing to dive with air.

This study was supported by Croatian Ministry of Science, Education and Sports.
Attenuating hypothalamic neuron cell damage by hyperbaric oxygen in diabetic rats with heat stroke
Authors: Niu Ko‐Chi , Lin Kao‐Chang
Published: June 2021

Alternating hypothalamus‐pituitary‐adrenal axis mechanisms would lead to multiple organ dysfunction or failure. Herein, we attempted to assess whether hypothalamic cell damage caused by heatstroke can be affected by hyperbaric oxygen (HBO2) therapy in streptozotocin‐induced diabetic rats. In this study, anesthetized diabetic rats, immediately after the onset of heatstroke, were divide into two major groups and given the normobaric air (21% O2 in 1.0 atmospheres absolute) or HBO2 (100% O2 at 2.0 atmospheres absolute). Heatstroke was induced by exposing the animals to heat stress (43oC). Another group of anesthetized rat was kept at normothermic state and used as control. The survival time for the HBO2 –treated heatstroke‐diabetic rats increased from the control values 78‐82 minutes to new values of 184‐208 minutes. HBO2 therapy caused a reduction of heatstroke‐induced cellular ischemia, hypoxia, inflammation, and oxidative damage in the hypothalamus of the diabetic rats. Our results suggest that, in diabetic animals, HBO2 therapy may improve outcomes of heatstroke in part by reducing heat‐induced hypothalamic cell damage and other brain regions.
Measurements of Chemical and Mechanical Properties of Rats and Mice Bones under Simulated Space‐Like Environment
Authors: Mehta Rahul , Ali Nawab , Chowdhury Parimal
Published: June 2021

Changes in elemental composition and mechanical properties of leg bones were determined following hind‐limb suspension (HLS) and irradiation of rats and mice. Tibia and Femur were bent with an applied force and corresponding elastic modulus determined. The bones were cross sectioned for elemental composition using Scanning Electron Microscopy with Energy Dispersive capabilities (EDX). The effect of microgravity on bones was evident after 4 days of suspension. There was a strong relationship between the compositional ratios of calcium, carbon, phosphorus and oxygen with the location on the leg. Two methods were used to measure mechanical strength of the bones; a LASER based bending setup (Cantilever) and a 3‐point bending setup with sensor to measure bending steps and stepping mechanism. Results showed that the femur bones had Young's modulus between 6 and 18 GN/m. For control groups, it was ~2.5 times larger than that for the HLS group. This clearly points to a less elastic nature of leg bones exposed to HLS. Also the trend of the stress versus strain graph (beyond the linear range) indicates a substantially lower breaking point for HLS leg bones. The results for tibia and femur provided similar conclusion. The elemental compositions in combination with mechanical properties indicated weakening of the bones under space‐like conditions of microgravity. These changes were even larger when radiation effects were taken into account. Supported by Arkansas Space Grant Consortium
Walking‐mediated upregulation of follistatin‐like 3 expression is insufficient to increase muscle contractile force
Authors: Blazek Alisa , Beck Eric , Li Jackie , McElhanon Kevin , Hewett Timothy , Agarwal Sudha , Weisleder Noah
Published: June 2021

Molecular mechanisms coordinating muscle and bone strength and growth with exercise (EX) are undefined. EX upregulates follistatin‐like 3 (FSTL3) in mouse and human skeletal muscle, bone and serum, and FSTL3 is required for EX‐driven bone formation (Nam, 2014, in press). FSTL3 is known to bind and inhibit myostatin. Thus, our purpose was to determine if FSTL3 increases muscle hypertrophy and force generation through this mechanism to collectively regulate musculoskeletal function. Wild‐type (WT) or Fstl3‐/‐ C57/Bl6 mice (n=30) were treadmill walked for 45 min/day at 8 m/min for 6 weeks, a protocol that increases bone formation. Muscles were mounted between stimulating electrodes, equilibrated at max force, then stimulated from 1‐150 hz to generate force vs. frequency curves. No differences in force were observed in WT EX and WT NoEX, indicating that walking was not sufficiently rigorous to produce a training effect on muscle force. No force differences were observed in Fstl3‐/‐ EX and Fstl3‐/‐ NoEX. Endogenous FSTL3 may not be sufficient to alter muscle contractile force generation in response to walking. FSTL3 regulates bone health in response to walking EX; however, walking is insufficient to increase muscle force and hypertrophy. While walking may be prescribed as a treatment regimen for increasing bone growth in humans, clinicians should be aware that walking may need to be supplemented with a resistance EX program to increase muscle strength/hypertrophy.
Is Hif‐1α Essential and Sufficient for Heat Acclimation Cross‐Tolerance?
Authors: Horowitz Michal , Alexander‐Shani Rivka
Published: June 2021

During heat acclimation (AC) HIF‐1α is constitutively elevated in the heart and the brain. HIF‐1α plays a role in cardioprotection and in spontaneous recovery from traumatic brain injury in the AC phenotype via cross‐tolerance mechanisms (HACT). In this study, we aimed to elucidate whether constitutive vs. transient HIF‐1α up‐regulation is an essential component of HACT. Acriflavine, an inhibitor of HIF‐1 transcriptional activation, was administered (IP) either daily during AC (30d, 34ºC) or as a bolus 2h before Ischemic insult. Cardiac infarct size, rigor contraction of isolated cardiomyocytes and levels of HIF‐1 target proteins or mRNA were measured. The HSF1 ‐ HSP72 cytoprotective cascade was also examined. Daily administration of acriflavine abolished protection in the hearts and cardiomyocytes. Bolus administration of acriflavine only slightly interfered with the protected cardiophenotype. Both treatments attenuated [HIF‐1 α]‐[HIF‐1β] dimerization (CIP), abolished PDK1 translation and up‐regulated GLUT1 levels. HSF1, P‐GSK and HSP72 were up‐regulated. To study the effects of HIF‐1 on COX 4.1 and COX 4.2 Complex IV subunit isoforms in the AC heart, mRNA levels of COX4.2 and LON (protease, COX 4.1 regulator) were measured. AC up‐regulated COX 4.2 and decreased LON levels while the daily acriflavine‐treated group demonstrated a reciprocal outcome. Taken together the similarity in the effects of daily and bolus acriflavine administration on PDK1 and GLUT1 levels and the maintenance of cardioprotection in the bolus group suggests that constitutive HIF‐1 inhibition is required to interfere with HACT. High HSP levels did not abolish this effect, further supporting the essential role of HIF‐1α during the course of AC in HACT. Under these conditions, HSP is not an essential component.
Prostaglandins (PGs) Contribute in an O2 Dependent Manner to Reflex Vasoconstriction in Exercise
Authors: Junejo Rehan , Ray Clare , Marshall Janice
Published: June 2021

Our previous studies indicated that the contribution of locally released PGs to exercise hyperaemia is O2‐dependent. PGs have also been implicated in triggering the exercise reflex that induces sympathetic vasoconstriction in resting muscle. We have now tested whether this role is O2‐dependent in healthy young males (18‐21 yrs; n=11) who performed two 2‐min periods of isometric handgrip contractions at 60% MVC while breathing air after placebo (P), after COX blockade with 600 mg aspirin p.o. (A), or breathing 40% O2 in a cross‐over design. Contraction was followed by 2‐min post exercise circulatory occlusion (PECO) to trap metabolites, or no occlusion (NO‐PECO). Mean arterial blood pressure (ABP) and heart rate (HR) were monitored by Finometer and ipsilateral calf blood flow (CBF) by venous occlusion plethysmography at approximately 1‐min intervals during and after contraction. Baseline Calf vascular resistance (CVR: ABP/CBF) was not significantly different between conditions. In PECO trials, CVR increased by 17.84±6.84 RU during contraction with air breathing and P, but by only 5.49±1.77* and 6.69±2.64* RU with air/A and 40% O2/P respectively (*P < 0.01; vs. P.; Factorial ANOVA). During PECO the ΔCVR was maintained at 11.30±3.47, 4.34±1.49* and 7.35±2.27 RU (*P < 0.03). In NO‐PECO trials, CVR increased by 17.13±5.97, 5.83±1.92* and 6.23±3.16* RU with air/P, or air/A, and 40% O2/P respectively (*P < 0.03). During NO‐PECO, ΔCVR fell to 4.26±1.55, 4.47±1.64 and 4.74±2.94 RU for air/P, or air/A, and 40% O2/P respectively (P < 0.95). Our findings allow the novel proposal that the contribution of PGs synthesised in exercising muscle to the exercise reflex, notably to vasoconstriction in resting muscle, is O2‐dependent.
Aerobic Training Prevents Impairment in Vascular Reactivity in Rats Under High Risk of Metabolic Syndrome
Authors: Medeiros Renata , Bento‐Bernardes Thais , Gaique Thaiane , Britto Fernanda Carla , Motta Nádia Alice , Oliveira Karen , Nóbrega Antonio Claudio
Published: June 2021

It is still unknown the whole mechanisms through which the aerobic training could prevent the development of metabolic syndrome. So the aim of this study was to investigate the vascular mechanism of aerobic training on vascular reactivity of rats under high risk of metabolic syndrome. Wistar rats (n=7/group), males, adults, were divided in Control group (C; drinking water), Fructose group (F; water with 10% fructose), both for 10 weeks; Control Training group (CT) and Fructose Training group (FT), both same as C and F groups, respectively, adding moderated aerobic training on a treadmill (04 times/week) in the last 8 weeks. There were no differences in body mass gain, caloric intake, glycaemia, blood pressure and heart rate between groups. Triglycerides, insulin and isoprostane were higher only in F group and was similar between FT, CT, and C groups. Nitric oxide was higher in trained groups when compared to sedentary groups. In addition, the F group exhibited increased vasoconstrictory reactivity to phenylephrine when compared to C and FT. The F group also presented an impaired endothelial vasodilator response to acetylcholine compared to C and FT vasodilatory response was similar to C and CT groups. F group had lower eNOS expression when compared to C group. The groups that trained had higher values when compared to their sedentary controls; there was no difference between CT and FT groups. In relation to eNOS (phospho S1177) it was observed that the trained groups had higher values when compared to sedentary groups. In conclusion aerobic exercise prevents damage in vascular reactivity, increases eNOS and peNOS expression in this model.
Macronutrients and Proteins in Cheese Reduce Acute Dietary Sodium‐induced Endothelial Dysfunction in the Cutaneous Microcirculation
Authors: Stanhewicz Anna , Alba Billie , Kenney W. , Alexander Lacy
Published: June 2021

Chronic dairy consumption is associated with improved cardiovascular outcomes, but a high dietary sodium (Na) intake induces endothelial dysfunction, evidenced by reduced nitric oxide (NO)‐dependent vasodilation (VD). It is unclear if increased Na intake in dairy products (cheeses) may have a benefit over other Na sources due to the moderating actions of dairy macronutrients and proteins. We hypothesized that acute cheese ingestion would attenuate reductions in NO‐dependent VD observed with an equal dose of non‐dairy Na. On 4 separate days, subjects ingested 3 oz. cheddar cheese (560 mg Na), 2.3 oz pretzels (560 mg Na), 6 oz. cheddar cheese (1120 mg Na), or 4.6 oz pretzels (1120 mg Na). An intradermal microdialysis fiber was placed in the forearm skin of 7 subjects (60±2 years) for local delivery of Ringer's solution. Red cell flux was measured by laser‐Doppler flowmetry (LDF) during standardized local heating protocol (42°C). After cutaneous vascular conductance (CVC = LDF/MAP) plateaued, 20mM L‐NAME was perfused to quantify NO‐dependent VD. Data were normalized as %CVCmax (28mM SNP). The local heating plateau was not different between treatments. NO‐dependent VD was greater following cheese consumption compared to Na alone for both 560 mg Na (53±6 vs. 41±4%; p=0.06) and 1120 mg Na (54±7 vs. 38±4%; p=0.03). Na intake in dairy products (cheese) augmented NO‐dependent VD compared to Na ingestion alone, suggesting that macronutrients and proteins in cheese may reduce acute dietary Na‐induced endothelial dysfunction.
TRB3 Inhibits Brown Adipocyte Differentiation and Function by Suppressing Insulin Signaling
Authors: Jeong Ha‐Won , Choi Ran Hee , McClellan Jamie , Tseng Yu‐Hua , Goodyear Laurie , Koh Ho‐Jin
Published: June 2021

Recent studies have demonstrated that adult humans have substantial amounts of functioning brown adipose tissue (BAT), the activity of which inversely correlates with obesity and type 2 diabetes. Since BAT has been implicated as an anti‐obese and anti‐diabetic tissue, it is important to understand the signaling molecules that regulate BAT function. Our hypotheses were that TRB3 mediates obesity‐ and diabetes‐induced impairments in BAT differentiation and function, and that inhibition of TRB3 improves BAT function. Here, we report that TRB3 expression in BAT of high‐fat fed mice and ob/ob mice was significantly increased by 56% and 110%; and this coincided with 56% and 58% decreases in the expression of BAT specific uncoupling protein 1, UCP1. Overexpression of TRB3 in brown preadipocytes significantly impaired cell differentiation as assessed by Oil‐Red O staining and expression of genes involved in BAT differentiation and function (UCP1: 73%; PRDM16: 32%; PPARγ: 25%). Consistent with these findings, TRB3KO mice exhibited higher UCP1 mRNA (80%) and protein (780%) expression in BAT, and were cold resistance (rectal temperature at 4oC for 2hrs; WT: 34.3oC ± 0.4 vs. 35.7oC ± 0.1). Based on previous studies showing that deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function, we assessed insulin signaling in brown preadipocytes and BAT in vivo. Overexpression of TRB3 in cells impaired insulin‐stimulated IRS1 Y612 (38%) and Akt S473 (45%) phosphorylation, whereas TRB3KO mice displayed improved IRS1 Y612 (70%) and Akt S473 (135%) phosphorylation. These data demonstrate that TRB3 regulates BAT differentiation and function presumably by insulin signaling regulation.