bluuebunny/crossref_metadata_2025 huggingface dataset:
Stellate Ganglion Transcriptomics in Mice Reveal Sex Differences
Authors: Bayles Richard , Fei Suzanne S. , Gao Lina , Habecker Beth
Published: June 2021

Introduction

A large number of studies have demonstrated physiological sex differences in the brains of rodents, however the peripheral nervous system is understudied. Stellate ganglia are of particular interest because the majority of sympathetic neurons innervating the heart reside there. The cardiovascular risk profiles in men and women are different. Transcriptomics provide a powerful tool for identifying genomic differences that may contribute to sympathetic dysfunction in disease.

Aim

To compare the transcriptomes of healthy stellate ganglia from adult male and female mice.

Method

RNA‐sequencing was carried out on individual stellate ganglia from 18‐week male and female C57Bl6J mice (n = 6 vs 6). Sequencing was carried out in an Illumina HiSeq 2500 at the Massively Parallel Sequencing Shared Resource (OHSU). Raw sequence data was aligned to the reference genome and analysed by the OHSU ONPRC Biostatistics & Bioinformatics Core.

Results

When comparing the stellate ganglia of males and females, the overall transcriptomic profile including the most highly expressed genes was similar. A subset of genes was different between the sexes however. Following differential expression analysis, at a strict threshold of FDRp‐value <0.05, there were 12 genes with significantly greater expression in females, and 9 genes with significantly greater expression in males. While the largest differences could be attributed to sex chromosome genes, other genes were unexpected. There was a significant difference between the sexes in expression of the genes encoding the TrkC receptor (Ntrk3), the axonal guidance factor netrin G1 (Ntng1), and the Chagas' related transcription factor Cha (Tcfl5). These differences and others including a number of ion channels were confirmed by real‐time qPCR.

Conclusion

Differences in gene expression were identified between male and female stellate ganglia. These differences may translate to sex specific differences in the physiology of sympathetic nerves, with relevance to a number of cardiovascular diseases.

Support or Funding Information

Supported by: NIH OT2TR001984, OHSU School of Medicine Dean's Fund

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arcuate Nucleus Angiotensin II Increases Arterial Pressure and Sympathetic Nerve Activity in Part via Inhibition of Neuropeptide Y Projections to the Hypothalamic Paraventricular Nucleus
Authors: Shi Zhigang , Brooks Virginia L.
Published: June 2021

Angiotensin II (AngII) acts centrally to increase arterial pressure (AP) and sympathetic nerve activity (SNA) mainly via AngII type 1 receptors (AT1R). Multiple brain areas have been identified as targets of AngII, including the arcuate nucleus (ArcN). However, the neural pathway that mediates AngII actions in the ArcN remains unknown. Recent studies suggest that AT1R expression in the ArcN is restricted to Neuropeptide Y (NPY) neurons, which project to the hypothalamic paraventricular nucleus (PVN). Therefore, we hypothesized that, in the ArcN, AngII increases AP and SNA in part by suppressing the activity of tonically sympathoinhibitory NPY neurons that innervate the PVN. To test this hypothesis, in α‐chloralose anesthetized male SD rats, we determined if blockade of PVN NPY Y1 receptors (Y1R) with bilateral nanoinjections of BIBO3304 (60 pmol/60nL) elicits smaller increases in AP and SNA 60 min after nanoinjection of exogenous AngII into the ArcN, compared to 60 min after ArcN injections of artificial cerebrospinal fluid (aCSF). As we reported before, after ArcN aCSF, blockade of PVN Y1R increased (n=3; all P<0.05) mean AP (MAP, 99±4 to 111± 4 mmHg), lumbar SNA (LSNA, 99±9 to 130±12 % control) and splanchnic SNA (SSNA, 105±9 to 142±10 % control). Bilateral nanoinjection of AngII into the ArcN slowly increased (n=4; all P<0.05) MAP (101±4 to 116±3 mmHg), LSNA (100 to 153±10 % control) and SSNA (100 to 151±6 % control). However, 60 min after ArcN AngII, blockade of PVN Y1R did not further increase MAP (to 122±2 mmHg), LSNA (to 174±17 % control), and SSNA (to 176±22 % control), compared to the changes observed after PVN aCSF injections (ArcN AngII+PVN aCSF: n=5; MAP, 120±4 to 123±3 mmHg; LSNA, 160±20 to 174±22 % control; SSNA, 172±21 to 193±20 % control). In summary, ArcN injections of AngII markedly attenuated the pressor and sympathoexcitatory effects of blockade of PVN NPY Y1R. Therefore, we conclude that ArcN AngII increases MAP, LSNA, and SSNA, in part, via inhibition of NPY neuronal projections to the PVN.

Support or Funding Information

Supported in part by NIH HL128181.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Suppressor of Cytokine Signaling 3 (SOCS3) in POMC Neurons in Metabolic and Cardiovascular Regulation during Chronic Leptin Infusion
Authors: Wang Zhen , Moak Sydney P. , do Carmo Jussara M. , Bailey Kandice C. , Hall John E.
Published: June 2021

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of leptin signaling and is expressed in proopiomelanocortin (POMC) neurons. We previously showed that activation of leptin receptors in POMC neurons mediates the chronic effects of leptin on blood pressure (BP). In this study, we examined whether deletion of SOCS3 specifically in POMC neurons amplifies the chronic metabolic and cardiovascular responses to leptin. 28‐week old male (n=5) and female (n=7) SOCS3flox/flox‐POMC/cre mice in which SOCS3 was selectively deleted in POMC neurons and control SOCS3flox/flox male (n=5) and female (n=7) mice were implanted with BP telemetry probes. After recovery, 24‐hr mean arterial pressure (MAP), heart rate (HR), body weight, and daily food intake were recorded for 5 consecutive days at baseline. Then leptin was infused (2 mg/kg/day, i.p) via osmotic minipumps for 14 days followed by 6‐days of recovery.

Compared to SOCS3flox/flox controls, SOCS3flox/flox‐POMC/cre mice had similar body weight (36.5 ± 2.4 vs 38.5 ± 5.0 g in males and 24.7 ± 0.7 vs 25.2 ± 0.8 g in females) during baseline and consumed similar amounts of food (3.2 ± 0.3 vs 3.3 ± 0.4 g/day in males and 2.6 ± 0.2 vs 2.7 ± 0.1 g/day in females). MAP and HR were similar in SOCS3flox/flox‐POMC/cre and control mice in males (111 ± 5 vs 113 ± 2 mmHg; 546 ± 24 vs 543 ± 26 bpm) and females (105 ± 3 vs 106 ± 2 mmHg; 562 ± 20 vs 573 ± 25 bpm). Leptin infusion reduced body weight by 7–8% in male and female SOCS3flox/flox‐POMC/cre and control mice. Reductions of food intake were similar in SOCS3flox/flox‐POMC/cre and control mice in males (2.5 ± 0.4 vs 2.5 ± 0.7 g/day) and females (2.3 ± 0.3 vs 2.2 ± 0.4 g/day) when calculated as average daily food intake during 10 days of leptin infusion. Leptin infusion transiently increased MAP in male and female SOCS3flox/flox‐POMC/cre and control mice despite decreasing body weight and food intake. Increases of MAP and HR during leptin infusion were similar in male and female SOCS3flox/flox‐POMC/cre mice compared to control mice. Chronic leptin infusion produced similar reductions in fasting blood glucose concentration in SOCS3flox/flox‐POMC/cre and control mice.

These results provide no evidence that SOCS3 deletion specifically in POMC neurons amplified the chronic effects of leptin to reduce food intake, body weight, and fasting glucose concentration in male or female mice. Deletion of SOCS3 in POMC neurons also did not substantially amplify the chronic effects of leptin on MAP and HR.

Support or Funding Information

Funded by NHLBI PO1HL51971, NIGMS P20GM104357

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A NOVEL ROLE OF THE DESCENDING ANALGESIA SYSTEM IN THE REGULATION OF VAGAL REFLEXES
Authors: McGovern Alice , Kerr Nicole , Farrell Michael , Mazzone Stuart
Published: June 2021

The submedius nucleus of the thalamus (SubM), ventrolateral orbital cortex (VLO) and periaqueductal grey (PAG) constitute a well‐described pain modulatory pathway. Activation leads to depression of nociceptive inputs in the spinal cord via the rostromedial medulla and brainstem descending inhibitory systems. Using transynaptic anterograde viral tracing we noted a neural circuit derived from jugular ganglia vagal afferents innervating the respiratory tree projecting to the SubM, suggestive that vagal afferent pathways may be regulated by SubM‐VLO‐PAG descending modulatory system. In functional studies using urethane anesthetized Sprague Dawley rats, electrical stimulation of the larynx to activate vagal afferent fibres evoked respiratory slowing in a stimulus frequency dependent manner. Concomitant SubM activation via microinjections of 5 ug serotonin significantly inhibited reflex reductions in respiration when compared to vehicle controls (e.g., baseline respiratory rate of 117±4.7 breaths per minute (bpm), to 12±2.2 bpm and 69±5.9 bpm in vehicle and serotonin animals, respectively; p=0.0001, n=9/group). Chemogenetic activation of the SubM with the Gq‐DREADD demonstrated a similar inhibitory effect on respiratory slowing (86±7.6 bpm and 15±62.4 bpm in +m3D+1 mg/kg CNO and –m3D+1 mg/kg CNO animals, respectively; p=0.0001, n=3/group). This inhibitory effect was absent in animals receiving prior electrolytic lesion of the VLO. Moreover, lesion of the VLO facilitated respiratory slowing caused by laryngeal stimulation (6±6.6 bpm and 73.5±6.7 bpm in lesion and sham animals, respectively; p=0.0001, n=5/group), consistent with a tonic descending inhibitory influence over respiratory vagal afferent processing. Taken together, these data support the notion that the SubM‐VLO‐PAG descending modulatory system plays an import role in the regulation of bulbar visceral afferent processing.

Support or Funding Information

A McGovern a a current recipient of a Peter Doherty Fellowship (National Health and Medical Research Council, Australia).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic effects of Inflammatory Cytokines on the Protein Expression of Voltage‐gated Potassium Channels in a Dorsal Root Ganglion Cell line
Authors: Hong Juan , Adam Ryan J. , Xia Zhiqiu , Rozanski George J. , Zuker Irving H. , Wang Hanjun
Published: June 2021

An exaggerated exercise pressor reflex (EPR) causes excessive sympatho‐excitation and exercise intolerance in the chronic heart failure (CHF) state. A recent preliminary study from our laboratory suggested that a functional loss of voltage‐gated potassium (Kv) channels in muscle afferent neurons plays a critical role in mediating the exaggerated EPR and afferent sensitization in CHF. However, the molecular mechanisms underlying the Kv channel dysfunction in muscle afferent neurons in CHF remains unclear. Since CHF is associated with chronic inflammation, we investigated the potential effects of chronic inflammation on protein expression of Kv channels in the 50B11 DRG cell line. We used pro‐inflammatory cytokines Interleukin‐6 (IL‐6), Interleukin‐1b (IL‐1β) and tumor necrosis factor alpha (TNFα) to create an inflammatory environment. We investigated the effects of TNFα, IL‐1β and IL‐6 on multiple Kv channel isoforms (Kv1.4, Kv3.4, Kv4.2 and Kv4.3), respectively. Three day exposure of TNFα (10 ng/ml) decreased protein expression of Kv3.4, but increased Kv1.4 protein expression without any change in the protein expression of Kv4.2 and Kv4.3. Similarly, 3‐day exposure of of IL‐1β (10 ng/ml) decreased the protein expression of Kv3.4, but had no effect on protein expression of Kv1.4, Kv4.2 and Kv4.3 in 50B11 cells. Finally, 3‐day exposure of IL‐6 (10 ng/ml) decreased the protein expression of Kv3.4, Kv4.2 and Kv4.3 without any effect on Kv1.4 (Table 1). These data suggest that chronic exposure of inflammatory cytokines cause the reduction of Kv channels in DRG neurons. However, each cytokine may have a slightly different impact on individual Kv channel isoforms.

Support or Funding Information

This work was supported by grants from the National Heart, Lung, and Blood Institute (R01HL121012‐A1 to HJW and in part, by R01HL126796‐A1 to HJW and IHZ).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Neurogenic CGRP on Renal Afferent Peptidergic Neurons
Authors: Rodionova Kristina , Raschke Giulia , Ditting Tilmann , Hindermann Martin , Ott Christian , Schmieder Roland , Amann Kerstin , Veelken Roland
Published: June 2021

Although release of the proinflammatory vasodilator CGRP from peptidergic afferent nerves is generally unquestioned the matter is far less investigated with respect to afferent renal innvervation that is said to be involved in sympathetic control and blood pressure regulation. Furthermore, it is not known in how far released neurogenic CGRP will affect renal afferent innervation in return. Hence we wanted to test the hypothesis that CGRP elicits action potential production related to TRPV1 receptor stimulation in cultured neurons with afferent projections from the kidney in vivo.

Cultured dorsal root ganglion neurons (Th11‐L2) of rats with renal afferents in vivo were investigated in current clamp mode to asses action potential generation or in voltage clamp mode to investigate inward currents during stimulation of TRPV1 receptors with acid of pH 6 (−80 mV holding voltage) and/or administration of CGRP (0,5 μmol). Furthermore, renal slices were incubated during baseline, experimental stimulation period and recovery to study CGRP release due to TRPV1 receptor stimulation (capsaicin 10−6 M, increased proton concentrations). CGRP concentrations in the organ bath were assessed with a commercial kit. Capsazepine was used for TRPV1 receptor blockade

More than 90 DRG neurons with renal afferents were tested. Addition of CGRP did not change action potential generation nor inward currents. Proton stimulation (ph 6) of TRPV1 markedly increased long‐term inward currents (baseline −361,7 +/− 89,6 pA vs. −1393,3+/−337,3 pA, p<0,05, mean +/− SEM). The co‐stimulation of renal neurons with protons (pH 6) and CGRP led to an impairment of sustained inward current as compared to proton stimulation alone (baseline − 478,2 +/−54,4 pA vs. −767,6+/−89,6 pA, p<0,05, mean +/− SEM). Baseline CGRP release of renal slices (2.5 to 6 pg/ml) increased significantly after TRPV1 receptor stimulation (on average: 21+4 pg/ml CGRP).

In contrast to our hypothesis CGRP could not elicit action potentials in afferent neurons related to the kidney but even impaired electric currents in general after TRPV1 receptor stimulation. Hence, renal CGRP secretion from peptidergic afferent nerves might decrease further neuronal CGRP release.

Support or Funding Information

DFG Deutsche Forschungsgemeinschaft VE 104/4‐1

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ALTERATIONS IN THE ENDOCANNABINOID SYSTEM IN ASTROCYTES RELATED TO COMMERCIAL SWEETENER INTAKE
Authors: Aguirre‐Fuerte María , Contreras Irazú , Estrada José Antonio
Published: June 2021

Chronic non‐communicable diseases (NCDs) have prevalence rates of up to 60% in some countries, with obesity and diabetes corresponding to the highest number of cases worldwide. Presently, non‐nutritive sweeteners are used as a therapeutic alternative for these pathologies, as well as being commonly used by the healthy population. Recent studies have shown that human health can be impacted by frequent intake of non‐nutritive sweeteners, so it is important to determine the molecular mechanisms involved. The endocannabinoid system (ECS) has gained relevance due to its roles in the regulation of homeostasis, energy metabolism and behavior, through effects on a variety of tissues, including the central nervous system (CNS). Although in the CNS these processes are governed primarily by neurons, astrocytes play an important role in their regulation by modulating neuronal function, in part through the ECS. The objective of this project is to evaluate the effect of frequent intake of non‐nutritive sweeteners on the expression of endocannabinoid CB1 receptors in astrocytes in vivo. Adult Balb/C male and female mice were supplemented with commercially available sucralose, steviol glycosides or sucrose in their drinking water, daily, for 8 weeks. After treatment, their brains and spinal cords were dissected and analyzed for CB1 expression in astrocytes by immunofluorescence. Preliminary results show increased CB1 expression in astrocytes from mice supplemented with sucralose, whereas there was a decrease in CB1 expression in astrocytes from mice supplemented with either steviol glycosides or sucrose, compared to controls without sweetener supplementation. Our results suggest the presence of alterations in CB1 expression in astrocytes related to frequent intake of commercial sweeteners, which in turn may be related to changes in feeding behavior that may be relevant to human health.

Support or Funding Information

This project is funded by grant 4320/2017/CI from the Autonomous University of the State of Mexico (UAEM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MASP‐3 aggregation and its blood to cerebrospinal fluid diffusion.
Authors: Padrón‐González Alexander Ariel , González‐Losada Cristobal , Lumpuy‐Castillo Jairo , Rodriguez‐Pérez Jose Alejandro , Ramos‐Robledo Alejandro , Castillo‐González William , Dorta‐Contreras Alberto Juan
Published: June 2021

Introducction

MASP‐3 functions are not clarified totally, in spite of that it is a regulatory protein from the lectin pathway. It can be found as complexes and its molecular weight is around 105 KDa.

Materials and methods

MASP‐3 and albumin from serum and cerebrospinal fluid were quantified by immune‐enzimatic test in a 60 control group without inflammatory process. Q (cerebrospinal fluid/serum) ratio was performed. QMASP‐3 was order and its relative frequency was calculated.

Results

From the different inflexion points of the relative frequency was found five different molecular orders and it's identified with a same number of aggregation forms. Blood‐cerebrospinal fluid allows the protein diffusion according to it molecular size and its relationship with their molecular flow.

This five aggregation forms was associated with the molecular flow of this structures throughout blood‐cerebrospinal barrier. It takes into account that the structure that diffuses more rapidly should be the 105 KDa native structure y it is possible to associate this aggregation with native multiple structures and its molecular flow.
Developmental changes in hypoglossal motoneuron morphology in neonatal rat
Authors: Williams Paul Allen , Bellinger Denise , Wilson Christopher G.
Published: June 2021

The autonomic brainstem generates and modifies breathing rhythm by integrating inputs from chemo‐ and mechanosensors in the viscera while coordinating descending outputs from higher CNS structures. Hypoglossal motoneurons (XII MNs) receive inputs from respiratory premotor neurons in the medulla. Previous studies in rodents have demonstrated significant changes in breathing control during the first three weeks of life, with a putative sensitive period at 10 to 13 days post‐birth (P10–P13) characterized by pronounced changes in neurotransmitters, receptors, excitation‐inhibition balance, and breathing physiology [Li et al., J Physiol, 577:957–770, 2006]. However, age‐dependent morphological changes of XII MNs during the first three weeks post‐birth and especially during this sensitive period, have not been thoroughly studied. In this study, we comprehensively characterize and quantify the postnatal morphological changes in rat XII MNs. We hypothesized that morphological changes occur in XII MN morphology and arbor complexity corresponding to the functionally‐defined sensitive period observed at P10–P13. To test this hypothesis, we used innovative statistical approaches to quantify and compare developmental changes in Golgi‐Cox stained XII MNs at nine postnatal ages between P1–P21. Soma size increased ~40% from P1 to P21, with no significant change in shape. However, dendritic arborization increased in extent and complexity. Dendritic branching of developing neurons significantly increased from P1 through P13, with the greatest increase at P10–P13 based on the Sholl method. For many parameters, three age groups 1) P1–P5, 2) P7–P12, and 3) P13–P21 were used as possible windows of development. We also found that at specific ages certain parameters such as soma size and dendrite distance were non‐normally distributed. Our detailed characterization of XII MN morphological development establishes a foundation for the study and elucidation of morphological changes caused by maternal and perinatal conditions using a rigorous statistical approach.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipid raft uptake at diaphragm muscle axon terminals
Authors: Gonzalez Porras Maria A. , Fogarty Matthew J. , Sieck Gary C. , Mantilla Carlos B.
Published: June 2021

Lipid rafts are structurally unique components of plasma membranes, crucial for neural development and function. The uptake and transport of lipid raft associated molecules in motor neurons is poorly characterized under normal and pathologic conditions, including advanced aging effects. Lipid rafts are particularly important for motor neurons as they shuttle cargo between peripheral and central compartments. The cholera toxin subunit B (CTB) binds the cell‐surface ganglioside GM1, which is internalized and transported via lipid rafts in motor neurons, and thus is commonly used to investigate lipid rafts. We hypothesized firstly, that axon terminal cargo loading into lipid rafts will be different across fiber types and secondly, that uptake at axon terminals is not affected by activity at the neuromuscular junction (NMJ).

Ex vivo diaphragm muscle and phrenic nerve preparations from 6‐month old female and male Fischer 344 rats were harvested. Morphological analyses of pre‐ and post‐synaptic structures at the NMJ were conducted using confocal imaging of fluorescently labeled anti‐synaptophysin and alpha‐bungarotoxin, respectively. NMJs were identified at type I or IIa vs. type IIx and/or IIb fibers using validated morphological characteristics. Quantitative analyses of CTB fluorescence at each type‐identified NMJ were conducted. CTB fluorescence was identified in all NMJs, and there were no fiber type differences in CTB uptake. Phrenic nerve stimulation had no effect on any parameters of CTB uptake, consistent with the exclusive involvement of lipid rafts rather than synaptic vesicle membrane uptake. Taken together, we conclude that lipid rafts are an important constituent of axon terminal membranes, that there are no differences in lipid raft uptake across motor unit types, and that lipid raft uptake is not affected by motor unit activity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of BDNF on Mitochondrial Morphology and Protein Expression in NSC‐34 Cells
Authors: Duffey Christopher Aaron , Nicholls Shannon , Mantilla Carlos Bernardo , Fogarty Matthew Joseph , Sieck Gary Charles
Published: June 2021

Age related loss of larger phrenic motor neurons (PhMNs) may underlie diaphragm muscle sarcopenia. We found increased mitochondrial fragmentation in larger PhMNs, similar to that reported to be associated with motor neuron loss in amyotrophic lateral sclerosis (ALS). In rodents, we observed diminished brain‐derived neurotrophic factor (BDNF) signaling via its high affinity TrkB.FL receptor in older animals. TrkB.FL is expressed on the outer mitochondrial membrane, and in ALS models, BDNF/TrkB.FL signaling promotes motor neuron survival and mitochondrial fusion. In the present study, we hypothesized that enhancing BDNF/TrkB.FL signaling increases Mfn2 expression, thereby promoting mitochondrial fusion. Studies were conducted using a mouse motor neuron‐like cell line (NSC‐34), which were differentiated by serum depletion until neurite structures were observed. The differentiated NSC‐34 cells were treated with BDNF (12.5 ng/ml for 1 h) and then loaded with MitoTracker Green to image mitochondria using a Nikon A1R confocal microscope (60× oil immersion 1.4 NA). BDNF treatment increased both form factor and aspect ratio of mitochondria by ~ 20% and 15%, respectively, while also causing a threefold increase in expression of Mfn2, indicating mitochondrial fusion. These experiments were performed in six different trials with two replicates each. The observed relationship between BDNF/TrkB.FL signaling and mitochondrial fusion/fragmentation proteins supports our hypothesis. These results are consistent with diminished BDNF/TrkB.FL signaling underlying mitochondrial fragmentation and dysfunction in age‐related PhMN loss.

Support or Funding Information

Supported by NIH grant AG44615

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lung Immune Regulation by Vectorial Transport of Endothelial Mitochondria
Authors: Islam Mohammad Naimul , Rowlands David John , Gusarova Galina , Bhattacharya Jahar
Published: June 2021

The mitochondrial regulation of lung immunity and repair remains unclear. Here, we consider the role of vectorial mitochondrial transport in endothelial cells (EC) of alveolar capillaries. In axons, which have high length‐diameter ratios, the mitochondrial Rho‐GTPase, Miro determines microtubule‐guided, vectorial mitochondrial transport. Cytosolic Ca2+ elevation increases Ca2+ binding to Miro, causing mitochondrial transport arrest. To determine whether similar mechanisms occur in lung ECs in situ, which are linearized cells with 30–50 μm long cytoplasmic extensions, we viewed live capillaries by confocal microscopy of isolated, blood‐perfused mouse lungs. To visualize EC mitochondria, we microinfused the capillaries with the fluorescent dye, mitotracker green (MTG) that specifically localizes to mitochondria. By vascular transfection, we induced EC expression of a Miro mutant, Mirokk, which does not cause Ca2+‐dependent mitochondrial transport arrest. EC mitochondria were evident as distinct, fluorescent spots of diameter 2–3 um, each representing a cluster of 1–3 mitochondria, that distributed linearly along the cell length. Time‐dependent imaging revealed that the clusters were in continuous motion at 1 um/min, such that a single cluster traversed the distance from the nucleus to the cell periphery in ~40 min. Vascular microinfusion of tumor necrosis factor‐alpha (TNFα) elevated EC cytosolic Ca2+, arrested mitochondrial transport and induced peri‐nuclear mitochondrial clustering. By contrast, Mirokk expression inhibited the transport arrest, while decreasing lung inflammation, as reflected in leukocyte counts in the broncho‐alveolar lavage, and mouse mortality. We show for the first time, that vectorial mitochondrial transport occurs along the cytoplasmic length of capillary EC. We report a novel, Miro‐dependent mechanism of lung immune regulation in which agonist‐induced cytosolic Ca2+ elevation blocks the mitochondrial transport, promoting lung inflammation and injury. Miro may be a therapeutic target for lung repair (support HL122730).

Support or Funding Information

support HL122730 to JB and AHA Grand‐in‐Aid to MNI

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Coexpression of proximal tubule claudins‐2 and ‐10a increases conductance and decreases permselectivity in a renal epithelial cell line
Authors: Curry Joshua N. , Li Jiahua , Zhuo Min , Yu Alan
Published: June 2021

The mammalian nephron is composed of a series of tubule segments with distinct patterns of tight junction morphology, ion selectivity, and protein expression. Paracellular permeability of epithelial tissues is determined in part by claudins, a family of tight junction membrane proteins. The most abundantly expressed claudins in the proximal tubule (PT) are claudin‐2 and ‐10a. In general, claudin‐2 is expressed in leaky epithelia and functions as a cation‐permeable paracellular channel, whereas claudin‐10a has been shown to form anion‐selective pores. We hypothesized that coexpression of these PT claudins leads to distinct paracellular permeability properties, and contribute to ion transport in this segment.

We established a double‐inducible, titratable gene expression system using Madin‐Darby canine kidney cells (MDCK I). Utilizing tetracycline‐controlled transcriptional activation (Tet‐Off), we developed stable cell lines which expressed claudin‐2 in the absence of tetracycline antibiotics. From this parent line, we generated stable transfectants expressing claudin‐10a under the control of a cumate‐inducible switch (SBI). Induction of claudin‐2, ‐10a, or both isoforms, all reduced TER compared to cells with no claudin overexpression (p<0.0001 using 2‐way ANOVA). We found a marked increase in PCl in cells induced to express claudin‐10a alone (17.4 ± 1.7 * 106 cm/s vs. 0.55 ± 0.06 * 106 cm/s with no induction), and a marked increase in PNa in cells induced to express claudin‐2 alone (12.2 ± 0.6 * 106 cm/s vs. 0.59 ± 0.07 * 106 cm/s with no induction). When both claudin‐2 and ‐10a were expressed, PCl and PNa were both increased (to 9.5 ± 1.5 * 106 cm/s and 6.2 ± 0.4 * 106 cm/s, respectively) and PNa/PCl was unchanged (0.76 ± 0.10 vs. 1.07 ± 0.05 with no induction). Calcium transport is believed to occur via a paracellular route in the PT, and so we measured the effect of PT claudin overexpression on unidirectional 45calcium radiotracer flux. Claudin‐2 expression increased calcium flux by over 7‐fold (p<0.0001), while claudin‐10a had no effect compared with no expression (p=0.99). No difference was detected in calcium flux between claudin‐2 expression and claudin‐2 and ‐10a in combination (p=0.89). These results further support claudin‐2 as the mediator of calcium reabsorption in the PT, and suggest that an interaction between PT claudin isoforms reduces the permselectivity of the PT to sodium and chloride.

Support or Funding Information

Supported in part by NIH grant F30DK109605

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Scleraxis Regulation of Snail1 and Twist1 Gene Expression in Epithelial – Mesenchymal Transition
Authors: Czubryt Michael Paul , Al‐Hattab Danah Sufian , Safi Hamza Ayoub , Nagalingam Raghu Sundaresan
Published: June 2021

Epithelial‐Mesenchymal Transition (EMT) is a process by which epithelial cells lose their connectivity with each other and convert to a migratory, mesenchymal phenotype, and plays an important role in development as well as pathogenesis of diseases such as cancer and fibrosis. EMT is induced in part by the up‐regulation of the transcription factors Twist1 as well as Snai1, which directly represses transcription of E‐cadherin, an integral component of adherens junctions: tight cell‐cell connections prominent in epithelial cell sheets. Our previous data in mice demonstrated that gene knockout of the transcription factor scleraxis, which is involved in cell phenotype conversion, resulted in fewer cardiac fibroblasts, which arise developmentally from proepicardial cells undergoing EMT. We observed an increase in epithelial markers concomitant with a loss of mesenchymal markers in scleraxis null hearts, suggesting that EMT was impaired by scleraxis deletion. Our data indicated that scleraxis directly transactivates the Twist1 and Snail1 gene promoters, and that scleraxis was sufficient to induce EMT in A549 epithelial cells, but the mechanistic details of this regulation were unclear.

Here we examined the Twist1 and Snail1 gene promoters for E‐boxes – CANNTG sequences to which scleraxis binds. By in silico analysis, we identified one and four putative E‐boxes in the Twist1 and Snail1 proximal promoters, respectively. Using luciferase reporter assays, we found that scleraxis directly transactivated both promoters. The single E‐box in the Twist1 promoter was required for this effect, as mutation of the E‐box abolished scleraxis‐mediated reporter induction. Conversely, three out of four E‐boxes within the Snail1 promoter were similarly required. Results for both studies were confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays, which demonstrated that scleraxis directly interacted with the E‐boxes in question. Scleraxis over‐expression resulted in up‐regulation of both Snail1 and Twist1, and increased migration of A549 epithelial cells as measured via scratch assay, indicating progression of EMT. TGFβ1, a known inducer of both EMT and scleraxis expression, increased Snail1 and Twist1 expression, but scleraxis knockdown via shRNA attenuated this induction, demonstrating that TGFβ1‐mediated EMT requires scleraxis. These findings suggest that scleraxis is an important factor in driving EMT, and may be a novel therapeutic target to arrest cell conversion in EMT‐dependent pathological processes.

Support or Funding Information

DSA was supported by funding from the GETS program of the University of Manitoba. MPC was supported by an Open Operating Grant from the Canadian Institutes of Health Research (MOP136862).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IRBIT expands signaling repertoire: Regulation of the Ca2+‐activated Cl− channel Bestrophin 2 by synergy between Ca2+ and cAMP at membrane contact sites
Authors: Chung Woo Young , Park Seonghee , Muallem Shmuel
Published: June 2021

The scaffolding protein IRBIT (Inositol 1,4,5‐Trisphosphate (IP3) Receptor Released with IP3) regulates the surface expression and activates several transporters, including NBCe1‐B, NHE3, CFTR, and SLC26A6, to coordinate secretory cells fluid and HCO3− secretion. IRBIT also mediates the synergy between cAMP and Ca2+ signaling. One mechanism involves PKAmediated phosphorylation of the IP3Rs that dissociate IRBIT from the IP3Rs and makes it available to activate the transporters. However, whether IRBIT affect Ca2+ and cAMP activation of the transporters and the molecular mechanism of such a regulation is not known. Here, we discovered that IRBIT is a potent activator of the Ca2+‐activated Cl− channel Bestrophin 2 (Best2), but not of Best1. IRBIT achieves this by two mechanisms. IRBIT recruits Best2 to the ER/PM junctions to increase surface expression of Best2 and increase the Ca2+‐activated Clcurrent density. At the ER/PM junctions IRBIT assembles a complex of Best2‐Adenylys cyclase 8‐PKA to confer Best2 regulation by the cAMP/PKA pathway. PKA‐mediated phosphorylation prominently increases Best2 apparent affinity for Ca2+. Best2 regulation by PKA requires intact ER/PM junctions with dissociation of the junction by knockdown of ER/PM junctions tether proteins eliminates activation of Best2 by IRBIT and regulation by PKA. Notably, activation of Best2 by Ca2+‐mobilizing receptors is markedly reduced in the salivary gland ducts of IRBIT−/− mice. These findings show the importance of localization in the ER/PM junctions for synergy in signaling and how IRBIT expands signaling repertoire by converting purely Ca2+‐activated protein to a Ca2+‐activated and cAMP/PKA regulated protein.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IRBIT stimulates zebrafish NBCe1 (Slc4a4) activity and stimulates functionally impaired human NBCe1‐B cSNP activity
Authors: Chang Minhwang , Wang Yi‐Fang , Romero Michael F.
Published: June 2021

We previously cloned and analyzed the electrogenic Na+‐Bicarbonate Cotransporter from zebrafish (zNBCe1; 80% identity to mammalian NBCe1; Sussman 2009, PMC2770747). As other fish NBCe1 clones, zNBCe1 is the only NBCe1 isoform, verified by functional data, and is most similar to the pancreatic form of NBCe1 (Slc4a4‐B). IRBIT, an inositol 1,4,5‐trisphosphate (IP3) receptor‐binding protein, specifically binds to and activates this N‐terminus (B & C isoforms) but not the shorter kidney‐type/A‐isoform. This binding depends on IRBIT phosphorylation at multiple serine residues. We tested the hypothesis that zNBCe1, as a NBCe1‐B, could functionally interact with zIRBIT rather than no regulatory interaction observed in NBCe1‐A isoforms. The zIRBIT cRNA was co‐expressed with zNBCe1 or an N‐terminal truncation (Δ91aa) of zNBCe1 in Xenopus oocytes and functionally tested using two‐electrode voltage clamp. We also investigated whether IRBIT binding would activate NBCe1‐B specific human cSNPs (identified NHLBI 6500 genome) in the autoinhibitory domain (AID). Our data demonstrate that zNBCe1 is a functional ortholog of mammalian NBCe1‐B, which both binds IRBIT via the N‐terminus and stimulates transport activity. Nevertheless, zNBCe1 transport activity was not augmented by AID removal at SPAAER motif (Δ91aa) as observed in mammalian NBCe1‐B or NBCe1‐C. Moreover, while human NBCe1‐B specific coding SNPs (G50A, N63S) show diminished transport function (vs. WT NBCe1‐B), IRBIT can still stimulate transport to NBCe1‐A levels. Together these data implicate that IRBIT regulation of NBCe1‐function is evolutionary preserved in teleost precedes NBCe1‐A appearance and that this regulation fine‐tunes NBCe1‐B transporting capacity in the presence of certain AID‐cSNPS. This IRBIT‐control might also play a physiological role when NBCe1‐A is deleted from particular cells or tissues.

Support or Funding Information

Mayo Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA Profile in Swimming Trained Female Rats and its Therapeutic Potential in Spontaneously Hypertensive Rats
Authors: Soci Ursula Paula Renó , Nobrega Clara Paula Renó , Fernandes Tiago Menezes , Barauna Valério Garrone , Irigoyen Maria Cláudia Menezes , Oliveira Edilamar Menezes
Published: June 2021

Introduction

Both aerobic exercise training (ET) and Hypertension (HY) induce different cardiac hypertrophy (CH) phenotypes which molecular differences and may lead to new targets for therapies in cardiovascular disease, as microRNAs (miRNAs).

Methods

We selected 15 miRNAs that were differentially expressed by ET from miRNAarray data and compared them with other from SHR (Spontaneously Hypertensive Rats) miRNAarray data. Four miRNAs were selected for qRT‐PCR confirmation: miRNA‐27a, 27b, 126 e 29c. Among then, miRNA 29c was chosen to be modulated by lentiviral vector due its role in fibrosis regulation. Intramuscular cardiac injection of the lentiviral vector particles 0.6×109 vp/was performed for two different times (7 and 14 days) (SHR7 and SHR14). The transduction efficiency was assessed by GFP expression by Western Blot. Blood pressure (BP) was measured by caudal plethysmography, CH was analyzed by ratio LVW/BW (left ventricle weight/body weight), HW/BW (heart weight/body weight) and by cardiomyocytes diameter (dCMO). qRT‐PCR was used to assess miRNA‐29c expression and its targets COLIAI and COLIIIAI gene expression. The LV collagen content was assessed by histology (Picrossirius red), by collagen volume fraction, and by hydroxiproline concentration.

Results

Both groups that received the low doses were GFP positive in the heart and liver tissue. BP was increased to SHR7, but it was prevented by miR‐29c lentivirus treatment to SHR14. The miRNA‐29c expression increased in all treated groups versus their control (CSI). COLIAI expression decreased in treated groups, while COLIIIAI did not change. Collagen volume fraction decreased in all treated groups, which shows that the treatment was efficient to decrease the cardiac collagen. HW/BW ratio decreased 7–11% in SHR14, while the dCMO was increased in all treated groups.

Conclusions

These results suggest that miR‐29c lentivirus treatment induces beneficial cardiac remodeling in SHR, increasing dCMO and decreasing cardiac fibrosis. These results open perspective for further investigation of antifibrotic therapies for cardiovascular diseases.

Support or Funding Information

FAPESP, CNPq, CAPES and NAPmir

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tpcn2 Knock‐Out Mice Have Improved Insulin Sensitivity and Are Protected Against High‐Fat Diet Induced Weight Gain.
Authors: Woods Leah Solberg , Holl Katie , He Hong , DeBehnke Sarah , Yeo Chay Teng , Hansen Polly , Gebre Abraham , Leone‐Kabler Sandra , Ruas Margarida , Parks John , Parrington John
Published: June 2021

Type 2 diabetes is a complex disorder affected by multiple genes and the environment. Our laboratory has shown that in response to a glucose challenge, two‐pore channel 2 (Tpcn2) knock‐out mice exhibit a decreased insulin response but normal glucose clearance, suggesting they have improved insulin sensitivity compared to wild type mice. We tested the hypothesis that improved insulin sensitivity inTpcn2 knock‐out mice would protect against the negative effects of a high fat diet. Male and female Tpcn2 knock‐out (KO), heterozygous (Het) and wild‐type (WT) mice were fed a low fat (LF) or high fat (HF) diet for 24 weeks. HF diet significantly increases body weight in WT mice relative to those on the LF diet, whereas HF diet consumption does not significantly affect body weight in Het or KO mice. We confirm that male KO mice have improved insulin sensitivity relative to WT mice. Unfortunately, because HF diet does not lead to insulin resistance in any of the mice, we are unable to claim a direct relationship between diet‐induced protection from insulin resistance and weight gain in the KO mice. Male KO mice exhibit increased fasting plasma total cholesterol and triglyceride concentrations relative to WT mice on the LF diet, but this difference disappears in HF diet‐fed mice where there is increased cholesterol and triglycerides across all genotypes. Fasting glucose, insulin and glucose tolerance levels do not differ by genotype or diet in male mice. In contrast, females consuming the HF diet exhibit glucose intolerance and hyperinsulinemia relative to females on the LF diet, but these effects are independent of genotype. These data demonstrate that male KO mice have improved insulin sensitivity and both male and female KO mice are protected against diet‐induced weight gain independent from changes in glucose tolerance or lipid levels.

Support or Funding Information

by R01 DK088975, R01 DK106386, Department of Pediatrics at the Medical College of Wisconsin

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ALDH2 rs671 polymorphism is associated with athletic status and muscle phenotypes in the Japanese population
Authors: Kikuchi Naoki , Tajima Takafumi , Yamanaka Yoshiaki , Menuki Kunitaka , Okamoto Takanobu , Sakamaki‐Sunaga Mikako , Sakai Akinori , Hiranuma Kenji , Nakazato Koichi
Published: June 2021

Mitochondrial aldehyde dehydrogenase‐2 (ALDH2) scavenges reactive aldehyde products, such as 4‐hydroxy‐2‐nonenal. Previous studies confirmed that the ALDH2 rs671 polymorphism is associated with hypertension and cancer, probably because of increased oxidative stress. Undesirable effects of ALDH2 rs671 might also be expected in skeletal muscles. The purpose of the present study was to investigate the association of ALDH2 re671 polymorphism with athletic status and muscle phenotypes in the Japanese population.

In the present study, 1,464 athletes (672 sprint/Power athletes, 631 mixed athletes, and 161 endurance) and 1,341 non‐athletes (538 men and 803 women) were included in the analysis. Saliva samples were collected with the Oragene DNA self‐collection kit. The ALDH2 rs671 polymorphism was genotyped using a TaqMan SNP genotyping assay. The non‐athletes completed a questionnaire about exercise habits and were subjected to a battery of tests to assess their muscular functions (including grip strength and the 30‐second chair stand test)

The ALDH2 genotype frequencies among athletes and non‐athletes were in Hardy‐Weinberg equilibrium. The frequencies of the ALDH2 genotypes GG, GA, and AA were 56%, 37%, and 7% in the athletes, and 54%, 37%, and 9% in the non‐athletes, respectively. There was a significant difference of genotype frequencies between the athletes and the non‐athletes (p=0.008). Among the non‐athletes, those with GG and GA genotypes showed higher fitness score of grip strength (p=0.001) and the 30‐second chair stand test (p=0.013) than those with the AA genotype after adjusting for age, sex, and exercise habits.

Our study shows that ALDH2 rs671 polymorphism is associated with athletic status and muscle phenotypes in the Japanese population, suggesting that impaired ALDH2 activity might attenuate muscular function.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
G‐protein‐Coupled Bile Acid Receptor Attenuates Liver Injury in a Murine Model of Acute Parenteral Nutrition
Authors: Gomes Charles Klazer , Rao Prahlad , Stephenson Erin , Puchowicz Michelle , Makowski Liza , Han Joan K. , Yin Dengping , Chang Eugene B. , Pierre Joseph F.
Published: June 2021

Parenteral Nutrition (PN) provides essential caloric support in patients unable to feed enterally. Unfortunately, PN without enteral feeding is associated with hepatic injury, especially in neonates where PN associated liver disease (PNALD) affects 60–70% of recipients. PN alters intestinal physiology and host‐microbial interactions, including bile acid enterohepatic circulation, which regulates metabolism through nuclear and cell membrane bile acid receptors. Here, we examined the role of G‐protein coupled bile acid receptor deficiency (TGR5−/−) upon hepatic phenotype in a murine model of acute PN. TGR5−/− and wild type (WT) received PN for 96 hours. PN significantly elevated serum triglycerides, resistin, PAI‐1 and hepatic fatty acid synthesis gene expression in both genotypes. However, PN‐treated TGR5−/− animals had significantly larger liver weights compared with WT, but displayed no changes in triglycerides. Histological analysis instead suggested edema. Accordingly, TGR5−/− animals on PN displayed elevated hepatic IL‐6 expression and significantly higher serum AST and ALT levels compared with WT. Furthermore, TGR5−/− animals on PN also displayed significantly decreased bile acid synthesis genes, including Cyp7A1, Cyp8B1, Cyp7B1, and BAT, compared with WT. Therefore, we examined serum bile acid composition. PN elevated total conjugated primary bile acid levels, specifically TCA, TCDCA, TbMCA, and TaMCA, in both genotypes compared with chow. However, TGR5−/− on PN displayed markedly higher levels of the unconjugated primary bile acids, CDCA, aMCA, and the secondary bile acids, oMCA and DCA, compared with WT on PN. Predictive metagenomics of 16S sequencing suggested altered microbial enzymes in TGR5−/−, including BSH and 7alpha hydroxylase, consistent with serum bile acid changes. These results suggest a protective role for TGR5 in liver function during PN that potentially includes direct attenuation of hepatic inflammation or indirect manipulation through microbiota‐bile acid interactions. Greater mechanistic understanding of TGR5 signaling during PN might improve interventions to treat or prevent PNALD.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.