bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C‐site phosphorylation of cardiac myosin binding protein‐C decreases cross‐bridge kinetics (1081.5)
Authors: Govindan Suresh , Ait Mou Younss , Lynch Thomas , de Tombe Pieter , Sadayappan Sakthivel
Published: June 2021

Cardiac myosin binding protein‐C (cMyBP‐C) phosphorylation is necessary for the regulation of sarcomeric structure and myocardial contractility in vivo. Different from skeletal isoforms, cMyBP‐C has M‐domain phosphorylation motifs in which three serines, Ser273, 282 and 302, are known to be substrates for several kinases. Phosphorylation of these M‐domain sites regulates the interaction of cMyBP‐C with thick and thin myofilaments during muscle contraction. While protein kinase C (PKC) is known to phosphorylate Ser273 and Ser302, the precise functional consequences of PKC‐mediated cMyBP‐C phosphorylation are not fully characterized. Therefore, the objective of this study was to determine how PKC‐mediated phosphorylation at Ser273/302 might control the function of cMyBP‐C in the context of thick and thin myofilament regulation and sarcomere contraction. Using recombinant proteins and yeast‐2‐hybrid (Y2H) clones expressing Ser273‐Ser282‐Ser302 (cMyBP‐CWT), Ala273‐Asp282‐Ala302 (cMyBP‐CADA) or Asp273‐Ala282‐Asp302 (cMyBP‐CDAD), cosedimentation of C0C2 and Y2H of C1C2 assays with either cardiac myosin S2 region or sarcomeric actin revealed that PKC phosphorylation of Ser273/Ser302 in cMyBP‐C M‐domain is sufficient to maintain its interaction with myosin S2 region, but diminish its interaction with actin in vitro. Furthermore, mechanical studies using skinned ventricular fibers from cMyBP‐CDAD revealed that PKC phospho‐mimetic exhibits a significant decrease in active force, cross‐bridge kinetics and Mg2+‐ATPase activity and myofilament Ca2+ sensitivity in vitro, compared to the controls. Taken together, our studies demonstrated that two phosphorylatable (by PKC) serines in cMyBP‐C M‐domain enable the interaction between cMyBP‐C protein and myosin S2, but diminishes its interaction with actin, thus affecting the ability of this protein to regulate sarcomere contractility
Prevention of ischemic vascular injury: targeting cellular stress in the endothelium with 5‐aminoimidazole‐4‐carboxamide 1‐β‐D‐ribofuranoside (1084.12)
Authors: Johnson Sarah , Banek Christopher , Gillham Haley , Needham Karen , Gilbert Jeffrey
Published: June 2021

A variety of stressors induce endothelial cells (ECs) to produce excessive oxygen radicals, triggering vascular inflammation, cellular stress and apoptotic activation which may have deleterious effects on vascular function and contribute to the pathophysiology of hypertension during preeclampsia. Although the benefits of AICAR on ischemic‐reperfusion (I/R) injury in the myocardium are well established, the role of AICAR on specifically on vascular ECs is much less clear. Thus, we hypothesized AICAR would promote cytoprotective pathways in ECs, reducing stress and attenuating apoptotic activity under hypoxic stress. To model vascular ischemia as might be found in the preeclamptic placenta, human umbilical vascular endothelial cells (HUVECs) were cultured at physiologic normoxic (8% O2) or hypoxic (1% O2) conditions and treated with AICAR (0.2, and 2mM). Cell protein and media were collected for further analysis. AICAR increased (p<0.05) pAMPKα/AMPKα in hypoxic conditions, but not in normoxia. AICAR pretreatment (2mM, 1hr) and subsequent exposure to hypoxia (24hr) showed decreased (p<0.05) BiP expression and cell‐adhesion molecule ICAM‐1 compared to normoxic control. AICAR reduced (p<0.05) cellular metabolic activity measured by MTT assay in both 8 and 1% O2. Further, ET‐1 secretion was reduced (p<0.05) following AICAR treatment (434 ±14 vs. *179 ±1pg/mL). Endothelial permeability was increased (p<0.05) with AICAR at 12 (869%) and 24 hours (315%) compared to the control. HUVEC adhesion was reduced (p<0.05) following AICAR treatment (‐91.57%), and CC blocked this effect. These data suggest by slowing cellular metabolic activity, reducing cell stress and apoptotic activity, AICAR could be a novel cytoprotective factor for improvement of vascular endothelial function in preeclampsia as well as other types of cardiovascular disease.

Grant Funding Source: Supported by National Institute of Health RO1HL114096 and AHA 10SDG2600040
Effects of long‐term aldosterone administration on mRNA expression levels in the renal distal nephron examined by FACS and RNA sequencing (1088.13)
Authors: Poulsen Søren , Praetorius Jeppe , Fenton Robert , Christensen Birgitte
Published: June 2021

The renal distal nephron [distal convoluted tubule 2 (DCT2) and connecting tubule (CNT)] is crucial for sodium and blood pressure regulation, and is tightly controlled by aldosterone. Due to difficulties in isolating the DCT2 and CNT, only few studies have examined the regulation of cellular processes in these segments. We have generated a mouse model expressing EGFP in the DCT2 and CNT, making it possible to collect EGFP cells using FACS sorting. Combined with the emergence of next generation sequencing (RNA sequencing), we utilized these tools to generate an improved catalog of aldosterone‐regulated mRNA transcripts in the distal nephron. The EGFP mice were administrated saline (n=7) or aldosterone (n=7) for six days. Following, a population of EGFP positive cells was collected using FACS sorting (purity~90%). RNA sequencing results confirmed that mRNA transcripts classically known to be regulated by aldosterone were changed in abundance [NCC and αENaC tended to be increased (p=0.058 and 0.147, respectively), Sgk1 was increased (p=0.003), and Nedd4‐2 was decreased (p=0.011)]. Based on regular p‐values (significance level set to 0.05), 562 genes differentially regulated by aldosterone were identified. In particular, genes involved in processes in the nucleus, mitochondrion, and plasma membrane were highly represented. The results provide us with the opportunity to identify novel mRNAs involved in renal blood pressure regulation.
LPA receptor 2 and 3 reversely regulate TPA‐induced megakaryopoiesis in K562 leukemia cell line (1094.3)
Authors: Ho Ya‐Hsuan , Lin Kuan‐Hung , Lin Yu‐Nung , Li Meng‐Wei , Lin Shih‐Hung , Yang Ya‐Jan , Chiang Chi‐Ling , Yao Chao‐Ling , Lee Hsinyu
Published: June 2021

Erythrocytes and megakaryocytes (MK) are derived from a common progenitor that undergoes lineage specification. Recently, lysophosphatidic acid (LPA), a lipid growth factor that regulates diverse cellular responses through activating LPA receptors, is shown to be a regulator in erythropoiesis. However, whether LPA is involved during megakaryopoiesis process remains unclear. In this study, we use K562 leukemia cell line as a model to investigate the roles of LPA in megakaryopoiesis. Our data demonstrate that expression levels of LPA receptor 2 (LPA2) is relatively higher than LPA3 in K562 cells. Phorbol 12‐myristate 13‐acetate (TPA), a commonly used megakaryopoiesis inducer in K562, increases LPA2 expression levels 24 hours after treatment, and decreases significantly after 48 hours. In contrast, TPA treatment increases LPA3 expression levels continuously. By monitoring mRNA and protein expression levels of MK marker CD61, blockade of LPA1/3 signaling by 20 μM Ki16425 inhibits TPA‐induced megakaryopoiesis. Moreover, activation of LPA3 signaling by 500 nM OMPT treatment enhances megakaryopoiesis. On the contrary, activation of LPA2 signaling by 10 μM MDP treatment suppresses megakaryopoiesis. Furthermore, knockdown of LPA3 by siRNA impairs megakaryopoiesis, while knockdown of LPA2 enhances megakaryopoiesis. These results suggest that, by monitoring CD61 expression, LPA2 and LPA3 inversely regulate megakaryopoiesis in K562 cells.
Src glutathionylation and its role in oxidant and TGFβ1‐mediated Src activation (1095.13)
Authors: Zhang Hongqiao , Forman Henry
Published: June 2021

Src is a critical enzyme involved in epithelial‐mesenchymal transition that contributes to metastasis and possibly fibrosis. Previous studies have demonstrated that oxidants are involved in Src activation by various stimuli. As glutathionylation of critical cysteine residues is an important mechanism of redox signaling, we studied the glutathionylation of Src and its role in Src activation in response to TGF‐β1, hydrogen peroxide (H2O2) and α,β‐unsaturated aldehydes. Human H358 lung carcinoma cells were transfected with FLAG‐tagged human c‐Src constructs, exposed to agents or control media. Then, FLAG‐Src was immunoprecipitated with anti‐FLAG antibody. Src protein was glutathionylated upon exposure to TGF‐β1 and H2O2, but not to aldehydes. Roles of individual cysteines in c‐Src activation were examined by mutating the cysteine (to alanine), transfecting cells with mutant c‐Src, and then examining oxidant‐stimulated auto‐phosphorylation of c‐Src at Tyr418. Mutation at C248 or C501 abolished Src activation by oxidants and TGF‐β1 and glutathionylation was absent in these mutants. The data suggests that glutathionylation plays a critical role in c‐Src activation by both TGF‐β1 and oxidative stimuli.

Grant Funding Source: ES020942
Bioinformatic analysis of differential protein expression in calu‐3 cells exposed to carbon nanotubes (1095.7)
Authors: Li Pin , Lai Xianyin , Witzmann Frank , Blazer‐Yost Bonnie
Published: June 2021

Carbon nanotubes (CNT) are widely used in industry, medicine and scientific research. To examine potential impact on human health, the human airway epithelial cell line, Calu‐3, was exposed to a physiologically relevant (100 ng/mL) concentration of either single‐ and multi‐wall CNTs for 24 h. Label‐free quantitative mass spectrometry showed differential protein expression of 281‐282 proteins of which 231 were common to both nanotube exposures. The majority of the proteins showed decreases suggesting a general stress response to protect cells. Ingenuity Pathway Analysis was used for bioinformatic analysis. The STRING database was used to analyze functional protein networks. Common proteins changes occurred within the cellular functions of cell death and survival, cell‐to‐cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. Some proteins like cadherin 1, STAT1, plakoglobin, and PYCARD, appear in several functional categories and tend to be in the center of the protein networks, suggesting important roles in multiple cellular functions and activities altered in response to CNT exposure.

Grant Funding Source: Supported by Grants from the National Institutes of Health (R01GM085218 and RC2ES018025)
Epinephrine regulates aquaglyceroporin HC‐3 expression and subcellular localization in cultured erythrocytes from the freeze‐tolerant treefrog, Hyla chrysoscelis (1099.5)
Authors: Ratycz Connor , Nickell Philip , Frisbie James , Goldstein David , Krane Carissa
Published: June 2021

Cope’s gray treefrog Hyla chrysoscelis, accumulates and distributes glycerol as a cryoprotectant in anticipation of freezing. Transmembrane glycerol and water flux in H. chysoscelis erythrocytes likely occurs through HC‐3, an ortholog of mammalian aquaporin 3. HC‐3 protein is in higher abundance and is preferentially localized to the plasma membrane in RBCs from cold‐acclimated treefrogs as compared to warm‐acclimated animals. It is hypothesized that neuroendocrine agonists via receptor mediated second messenger pathways integrate signals derived from fasting, dehydration, diurnal, and/or temperature changes during cold‐acclimation to regulate HC‐3 expression as part of the mechanism of freeze tolerance. In this study, cultured H. chrysoscelis erythrocytes were exposed to 1 uM epinephrine for 30 and 60 minutes. Native HC‐3 expression increased 3 fold at 30 minutes and 5.5‐fold at 60 minutes relative to controls, whereas glycosylated HC‐3 expression increased by 1.1‐fold at 30 minutes and by 2 ‐fold at 60 minutes relative when exposed to epinephrine. Moreover, epinephrine treatment resulted in membrane localization as compared to cytosolic distribution in control cells. Erythrocytes pre‐treated with Calphostin C, a PKC inhibitor, showed no HC‐3 membrane localization, and native HC‐3 expression was reduced by 66% relative to controls and 94% relative to epinephrine‐treated cells. Thus, epinephrine begins a PKC‐dependent mechanism that results in an increase in HC‐3 abundance, HC‐3 membrane localization, and enhanced glycosylation in erythrocytes. These regulatory mechanisms are consistent with the in vivo regulation of HC‐3 expression observed in erythrocytes from cold‐acclimated treefrogs.
Altered contractile properties of fast and slow skeletal muscles from db/db mice (1102.16)
Authors: O’Connell Grant , Guo Ge , Pistilli Emidio
Published: June 2021

In the db/db mouse, leptin receptor mutation results in a model of metabolic syndrome. Initial hyperinsulinemia brought on by hyperphagia is followed by peripheral insulin resistance, elevated blood glucose and increased body weight. The purpose of this study was to determine the skeletal muscle contractile properties in the BKS.Cg‐Dock7m +/+Leprdb/J mouse model of metabolic syndrome (i.e. db/db mouse). Muscle contractile properties were determined ex vivo in the fast extensor digitorum longus (EDL) muscle and the slow soleus muscle in 17 week old mice. Body weight was 43% greater in db/db mice compared to wild type control mice. Soleus muscle mass was 49% lower and EDL muscle mass was 55% lower in the db/db mouse. EDL twitch force was 58% lower than control with an associated 20% longer contraction time and 25% longer 1/2 relaxation time. There were no differences in soleus twitch force. However, contraction time was 1.7‐fold longer and 1/2 relaxation time was 1.6‐fold longer than control. The force‐frequency relationship in the soleus muscle was significantly shifted to the left toward lower stimulation frequencies, with no differences in the EDL. The rate of force loss with repeated contractions was also consistent with a less oxidative profile in both muscle types. Collectively, these data demonstrate the effects of risk factors for metabolic syndrome on muscle contractile properties in the db/db mouse.
Control of thin filament lengths by sarcomeric tropomodulin isoforms: insights from mouse models (1102.2)
Authors: Gokhin David , Fowler Velia
Published: June 2021

The uniform lengths of skeletal muscle thin filaments are controlled by actin subunit exchange at pointed ends (P‐ends). Each P‐end is capped by two sarcomeric tropomodulin (Tmod) molecules, with Tmod1 and Tmod4 isoforms present in a 1:9 ratio. In Tmod1‐null sarcomeres, P‐ends are capped by Tmod3 (normally a sarcoplasmic reticulum‐associated Tmod isoform) and Tmod4, preserving normal thin filament lengths. In Tmod4‐null sarcomeres, P‐ends are capped solely by Tmod1, also preserving normal lengths. The mild phenotypes of Tmod1‐null and Tmod4‐null muscle highlight skeletal muscle’s ability to deploy compensatory mechanisms to specify correct thin filament lengths in the absence of one sarcomeric Tmod isoform during myofibril assembly and muscle development. To discern the effect of sarcomeric Tmod depletion from mature muscle, we measured thin filament lengths in the mdx and mdx/mTR mouse models of Duchenne muscular dystrophy, in which Tmods are calpain‐proteolyzed in a muscle use‐dependent manner. Proteolysis of Tmod1 and/or Tmod4 in mdx and mdx/mTR muscles results in 10‐12% increases in thin filament lengths, due to addition of actin subunits onto P‐ends. Collectively, these results show that Tmod1 and Tmod4 are individually dispensable for initial specification of muscle‐specific thin filament lengths, but important for maintenance of correct thin filament lengths in mature contracting myofibrils.

Grant Funding Source: Supported by NIH and MDA.
Phosphodiesterase inhibition increases PGC‐1α in muscle cells (1102.32)
Authors: Rahnert Jill , Zheng Bin , Woodworth‐Hobbs Myra , Hudson Matthew , Price S
Published: June 2021

Muscle wasting associated with chronic diseases, such as type I diabetes (DM), can be countered by restoring the reduced expression of the transcriptional coactivator PGC‐1α. Previous reports indicate that PGC‐1α transcription can be regulated by CREB in a process that requires nuclear localization of CRTC (CREB‐regulated transcription coactivator). We recently found that CRTC protein is reduced in skeletal muscle of diabetic rats whereas CREB is highly phosphorylated (i.e., activated). Nuclear translocation of CTRC is promoted by dephosphorylation via calcineurin (CnA) whereas nuclear export occurs by phosphorylation via the salt‐inducible kinases (SIK). Inhibition of phosphodiesterases (PDE), which raise cyclic AMP (cAMP) levels, results in increased PGC‐1α mRNA expression. cAMP also inhibits SIK suggesting this pathway may contribute to CRTC localization and thus PGC‐1α activity. Our experiments tested the hypothesis that inhibition of PDE increases the amount of nuclear CRTC and PGC‐1α transcription. Myotubes were transfected with luciferase reporter plasmids (Luc) and treated with the general PDE inhibitor IBMX (250uM) for 6 hours. IBMX increased PGC‐1α‐Luc promoter activity as well as the transcriptional activity of CREB. Deletion of the CRE site in PGC‐1α‐Luc prevents the increase in luciferase activity suggesting IBMX increases PGC‐1α activity via CREB. In addition, protein analysis indicates IBMX increases the level of nuclear CRTC. Together, these data imply that raising cAMP levels by inhibiting PDEs results in increased PGC‐1α transcriptional activity by a mechanism involving CREB and its coactivator, CRTC.

Grant Funding Source: Supported by NIH RO1 DK95610, NIH T32 DK007656 and VA Merit X01BX001456
Effect of meal composition on postprandial blood glucose levels in middle aged and young adults (1107.6)
Authors: Koch Carynn , Koch Jennifer , Hopper Mari
Published: June 2021

Effect of meal composition on postprandial blood glucose levels was determined in two cohorts, one middle aged (MA) (35‐55 years) and one young adult (YA) (19‐24 years). Subjects first completed an OGTT following an overnight fast. Over the next two weeks, and in random order, subjects consumed two breakfast meals varying in glycemic load: 1) high glycemic load (HGL); 2) low glycemic load (LGL). An Accucheck® glucometer determine fasted, 30, 60, and 120 minute postprandial blood glucose levels.

Although fasted blood glucose levels were normal for all individuals, the mean value for the MA group was significantly higher than YA (87.1±0.70 vs. 82.9±0.67 mg/dl; p=0.0001). Mean blood glucose values following OGTT were normal and similar for the two groups. Following HGL and MGL meal consumption, blood glucose values for MA were higher than YA at 30 and 60 min and AUC’s were also significantly higher (13724±388 vs. 12780±354; p=0.042 and 12896±335 vs. 11703±212; p=0.003).

The MA individuals with high waist circumference (WC>88cm) demonstrated significantly higher OGTT values than MA with normal waist circumference at 30 and 60 min (149.3±4.8 vs. 128.0±8.1; p=0.0273 and 147.2±7.6 vs. 118.6±11.6; p=0.0373). Following meal consumption high waist circumference individuals had higher blood glucose values than normal waist circumference at all time points with significant differences following MGL at 30 and 60 minutes.

Grant Funding Source: Support received from the University of Southern Indiana Early Undergraduate Research Program
Forkhead box transcription factors are required for expression of the glycemia‐protective enzyme angiotensin‐converting enzyme 2 in pancreatic β‐cells (1108.10)
Authors: Pedersen Kim , Lazartigues Eric
Published: June 2021

Angiotensin‐converting enzyme 2 (ACE2) opposes Ang‐II signaling and improves β‐cell function in several diabetic animal models. We previously reported that diabetes progression reduces islet ACE2 expression and that HNF1α dose‐dependently increases ACE2 expression in pancreatic islets. Because HNF1α is not sufficient for the full ACE2 expression, we hypothesized that ACE2 expression in β‐cells is regulated by other factors binding to evolutionarily conserved motifs of the human ACE2 promoter. A putative motif for forkhead box transcription factors is located 144‐153 bases upstream of the translational start site. Mutation of the motif in luciferase reporters decreased ACE2 promoter activity in 832/13 insulinoma cells by 56% (p<0.001). Overexpression of FOXO1 slightly increased (< 3‐fold) promoter activity and led to FOXO1 binding to the motif as observed by EMSA. However, the endogenous complexes in 832/13 cells binding to the motif contained FOXA proteins with the most abundant of these complexes containing FOXA1/2. Furthermore, ENCODE data suggest that an additional FOXA1/2 binding site is located further upstream in the ACE2 promoter. We conclude that forkhead box transcription factors, in particular of the FOXA class, are required for expression of ACE2 in β‐cells. Depletion of nuclear FOXA2 occurring in islets of type 2 diabetics may thus contribute to reduced islet ACE2 levels.

Grant Funding Source: Supported by NIH/NIDDK (DK084466)
Proteomic profiling of ubiquitinated proteins in human urinary exosomes (1109.1)
Authors: Huebner Alyssa , Cheng Lei , Fenton Robert , Pisitkun Trairak
Published: June 2021

Urinary exosomes are small (20‐100 nm) membrane vesicles that contain various molecular constituents of every epithelial cell type facing the urinary space. Based on our previous proteomic studies of urinary exosomes, ubiquitin was identified throughout the molecular weight range of the analysis, suggesting the presence of multiple ubiquitinated proteins in urinary exosomes. Ubiquitination is the classical signal that marks plasma membrane proteins for incorporation into multivesicular bodies where exosomes are originated as internal vesicles. We postulated that ubiquitinated plasma membrane proteins are present in urinary exosomes, potentially providing non‐invasive clues for ubiquitin‐dependent signaling processes in renal epithelial cells. Immunoelectron microscopy revealed ubiquitin labeling on many, mostly inside, urinary exosomes. LC‐MS/MS analysis of urinary exosomes with and without enrichment by di‐glycine‐lysine antibody identified 747 ubiquitinated peptides corresponding to 593 proteins, out of 5007 total proteins. Ubiquitinated proteins were mainly associated with plasma membrane including transporters, channels, and receptors and comprising a number of novel ubiquitinated sites. All but one polyubiquitin chain linkages were identified, with K63‐linked chains being the most frequently observed suggesting non‐proteolytic roles of urinary exosomal ubiquitination.

Grant Funding Source: Supported by the Lundbeck Foundation, AU Research Foundation, Danish Medical Research Foundation
Biphasic effect of PGF2a on the basolateral 40 pS K channel (a heterotetramer of Kir4.1 and Kir5.1) in the early distal convoluted tubule (DCT1) (1109.9)
Authors: Wang Lijun , Zhang Chengbiao , Su Xiaotong , Wang Wen‐Hui
Published: June 2021

We used the patch‐clamp technique to examine the effect of PGF2α on the basolateral 40 pS K channel in the DCT1. Application of PGF2α at concentrations lower than 500 nM stimulated the 40 pS K channel. The stimulatory effect of PGF2α was abolished by inhibiting src‐family protein kinase (SFK) and NADPH oxidase. Moreover, adding 100 μM H2O2 mimicked the effect of PGF2α and increased the 40 pS K channel activity in DCT1 and augmented whole cell K currents in HEK293 cells transfected with KCNJ10/16 which are known to form the heterotetramer in the basolateral membrane of DCT1. The inhibition of SFK also blocked the stimulatory effect of H2O2 on the basolateral 40 pS K channel in DCT1. The role of SFK in mediating the effect of H2O2 on the 40 pS K channel was further indicated by the observation that H2O2 failed to stimulate the K channels in cells transfected with KCNJ16+KCNJ10Y9F in which tyrosine phosphorylation site was mutated. This suggests that PGF2α and H2O2 stimulate the basolateral 40 pS K channel in DCT1 by SFK‐induced phosphorylation of KCNJ10. While PGF2α stimulates the K channels at low dose, PGF2α at high dose (>1 uM) inhibited the basolateral K channels. The inhibitory effect of PGF2α was abolished by inhibiting PKC. We conclude that PGF2α at low doses stimulates the basolateral 40 pS K channel by a SFK‐dependent pathway, while at high concentrations it inhibited the K channel in the DCT1 by a PKC‐dependent pathway.

Grant Funding Source: NIH
The role of mitofusin 2 in saturated fatty acid induced ER stress in H4IIE liver cells (1116.4)
Authors: Moran G. , Wei Y. , Wang D. , Pagliassotti M.J.
Published: June 2021

Genetic ablation of mitofusin 2 (Mfn2) in mice results in ER stress and insulin resistance in the liver. In liver cells, saturated fatty acids (SFA) induce ER stress and activate c‐Jun NH2 terminal kinase (JNK) which can mediate Mfn2 degradation. We hypothesized that SFA would reduce Mfn2 expression. We examined the effects of the unsaturated fatty acid oleate (O250µM) and the SFA palmitate (P250 µM) on Mfn2 mRNA, translation and protein expression in H4IIE liver cells (n=3). O250 µM and P250 µM had no significant effect on Mfn2 mRNA. P250 µM significantly increased Mfn2 translational efficiency based on polysome profiling, however no increase in Mfn2 protein expression was observed. JNK‐mediated degradation of Mfn2 involves serine phosphorylation (pSer) and ubiquitination (Ub), however P250 µM did not increase pSer or Ub of Mfn2. These data suggest that either the increase in translational efficiency was not sufficient to alter Mfn2 protein levels or that Mfn2 degradation occurs via mechanisms independent of pSer and Ub. We utilized si‐RNA mediated knockdown of Mfn2 to determine whether Mfn2 could protect against palmitate‐induced ER stress (n=3). Knockdown of Mfn2 had no significant effect on thapsigargin (Thap) or P250 µM ‐induced ER stress in H4IIE cells. These findings indicate that Mfn2 does not appear to play a protective role in Thap or P250 µM mediated ER stress in H4IIE cells.

Grant Funding Source: NIH DK 072017
ErbB4 deletion compromises the murine small intestinal stem cell niche and sensitizes the epithelium to TNF‐induced apoptosis (1119.6)
Authors: Almohazey Dana , Bernard Jessica , Frey Mark
Published: June 2021

The ErbB4 receptor tyrosine kinase is induced by inflammation in intestinal epithelial cells (IECs). Its ligand, NRG4, promotes survival signaling in colonocytes. However, the effects of ErbB4 loss on the small intestine or the intestinal stem cell (ISC) niche have not been defined. We asked whether ErbB4 deletion enhances tumor necrosis factor (TNF)‐induced apoptosis, and if it compromises the ISC niche. METHODS: Epithelial enteroid cultures from ileal crypts of ErbB4flox/flox (WT) and ErbB4flox/flox; Villin‐Cre (ErbB4KOIE) mice were given TNF (200 ng/ml) daily for 5 days, then expression of Paneth cell (Lyz1) and ISC (Lgr5) markers were tested by qPCR. WT and ErbB4KOIE mice were injected with 250 μg/kg TNF; after 24 h, ileums were fixed, sectioned, and apoptosis detected by In Situ Oligo Ligation (ISOL). RESULTS: ErbB4KOIE enteroids were dysmorphic, with 64% fewer buds/enteroid than WT, and had lower expression of Lyz1 (57% reduction, p<0.05) and Lgr5 (75% reduction, p<0.01). ErbB4‐null enteroids were highly sensitive to TNF, losing all crypt bud architecture after exposure. In vivo, ErbB4KOIE mice had increased TNF‐induced ileal epithelial apoptosis (1.8‐fold increase in ISOL+ cells, p=0.001) than WT. CONCLUSIONS: ErbB4 loss exacerbates TNF‐induced IEC death both in vivo and in vitro. This is associated with loss of PC and ISC markers, suggesting a role for ErbB4 in regulating the ISC niche.

Grant Funding Source: Supported by NIH grant R01DK095004 and ACS grant 124678‐RSG‐13‐199‐01‐DDC
The brain renin‐angiotensin system suppresses digestive efficiency (1126.13)
Authors: Weidemann Benjamin , Littlejohn Nicole , Cole Renee , Grobe Justin
Published: June 2021

The renin‐angiotensin system (RAS) contributes to metabolic control through local actions within the brain and adipose. All components of the RAS are also expressed in the gastro‐intestinal (GI) tract, leading to the hypothesis that the RAS controls digestive efficiency. Mice with transgenic hyperactivity of the brain RAS (sRA) were generated through neuron‐specific expression of human renin, and expression of human angiotensinogen via its own promoter. sRA exhibit reduced body mass and elevated resting metabolic rate through elevated sympathetic nervous activity and a chronic suppression of the circulating RAS. Caloric densities of food/feces were assessed by bomb calorimetry. sRA had increased caloric intake (15.4±0.7 kcal/day, n=8) vs littermate control mice (13.0±0.7, n=7, P<0.01) as well as an increase in caloric loss to feces (6.3±0.8 vs 2.9±0.4 kcal/day, P<0.01). Thus, sRA had decreased digestive efficiency (59±5 vs 78±2 %, P<0.01) versus littermates on chow diet (Teklad 7013), but total daily caloric absorption (9.1±0.8 vs 10.0±0.5 kcal/day, P=0.36) was normal. Infusion of the angiotensin (Ang) II type 2 receptor agonist CGP‐42112a (50 ng/kg/min, s.c., 8 weeks) had no effect on efficiency or absorption (sRA 63±6 vs control 80±1 %; 9.3±0.8 vs 9.8±1.0 kcal/day). We conclude that the brain and circulating RAS help control digestive efficiency, and hypothesize a role for GI tract Ang II type 1 receptors.

Grant Funding Source: Supported by NIH: HL098276, HL084207.
Brain endoplasmic reticulum stress mediates sympathetic and hemodynamic responses to blood‐borne TNF‐α in rats (1130.14)
Authors: Yu Yang , Zhang Zhi‐Hua , Wei Shun‐Guang , Felder Robert
Published: June 2021

The pro‐inflammatory cytokine tumor necrosis factor (TNF)‐ α acts upon the CNS to augment sympathetic drive in normal and pathophysiological conditions, including heart failure. The underlying mechanisms are poorly understood. We hypothesized that TNF‐ α induces endoplasmic reticulum (ER) stress in the brain, upregulating inflammatory mechanisms that drive sympathetic activity. In anesthetized rats, a centrally directed intracarotid artery (ICA) injection of TNF‐α (0.5 µg/kg) increased (p<0.05, vs baseline) renal sympathetic nerve activity, mean blood pressure and heart rate. These responses were attenuated by continuous intracerebroventricular (ICV) infusion of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 5 µg/hr). ICA TNF‐α induced molecular markers of ER stress, reactive oxygen species (ROS), NF‐кB, cyclooxygenase‐2 (COX‐2) and phosphorylation of p44/42 and p38 mitogen‐activated protein kinase (MAPK) in the subfornical organ (SFO), a CNS site at which circulating proinflammatory cytokines act to elicit sympathetic responses. TUDCA significantly reduced TNF‐α induced indicators of ER stress, phosphorylation of p44/42 and p38 MAPK and NF‐кB and COX‐2 activity in SFO, but not ROS generation. The data suggest that ER stress induced in brain tissues by blood‐borne TNF‐α contributes to central inflammatory mechanisms driving sympathetic excitation, likely via MAPK and NF‐кB signaling.

Grant Funding Source: supported by NIH Grants HL‐073986 and HL‐096671 and the Department of Veterans Affairs
Vagal nerve stimulation can elicit both activation and inhibition of brown adipose tissue sympathetic nerve activity (1131.6)
Authors: Morrison Shaun , Madden Christopher , Tupone Domenico
Published: June 2021

The dependence of BAT thermogenesis on the availability of metabolic substrates suggests that metabolic signals from the viscera could influence the sympathetic outflow driving BAT thermogenesis. Viscerosensory afferents in the proximal end of the sectioned left cervical vagus nerve were activated with electrical stimulation in anesthetized, ventilated rats while recording the SNA to the contralateral interscapular BAT pad. Paired‐pulse VNS delivered once every 5 sec evoked excitatory evoked potentials in BAT SNA with a peak latency of ~ 180 ms, followed by a ~2 s period of quiescent BAT SNA. Within a few seconds of stimulus onset, continuous, single‐pulse VNS at 2 Hz produced a complete inhibition of cold‐evoked BAT SNA that was sustained for at least 2 hrs of maintained VNS. Similarly, activation of neurons in the intermediate nucleus of the solitary tract (iNTS), at the level of the area postrema, with nanoinjections (60 pmol) of bicuculline also inhibits the VNS‐evoked excitation of BAT SNA. Inhibition of neurons in the rostral ventromedial medullary raphe pallidus region also eliminates the VNS‐evoked excitation of BAT SNA. These data support the existence of populations of vagal afferents whose stimulation can (1) increase BAT SNA via activation of BAT sympathetic premotor neurons or (2) inhibit BAT SNA via activation of BAT sympathoinhibitory neurons in the iNTS (see Am. J. Physiol. Regul. Integr. Comp. Physiol., 299:R277‐R290, 2010. PMC2904145).

Grant Funding Source: Supported by NIH NS40987
The role of activation of chemokine receptor 2 in TRPV1‐mediated renal protection during salt‐sensitive hypertension (1136.2)
Authors: Wang Youping , Zhu Mingjun , Liu Weihong , Wang Xiaoxiao , Cui Lin , Shen Si , Xu Hui , Wang Donna
Published: June 2021

To determine whether deletion of TRPV1 exaggerates salt‐sensitive hypertension‐induced renal injury via activation of chemokine receptor 2 (CCR2), wild type (WT) and TRPV1‐null mutant (TRPV1‐/‐) mice were subjected to uninephrectomy and DOCA‐salt treatment for 4 weeks with or without selective CCR2 antagonist, RS504393. DOCA‐salt treatment increased systolic blood pressure (SBP), urinary excretion of albumin and 8‐isoprostane, and decreased creatinine clearance compared with the control. DOCA‐salt treatment also caused obvious glomerulosclerosis, tubulointerstitial injury and monocyte/macrophage infiltration compared with the control. The changes in the parameters were greater in TRPV1‐/‐ mice compared with WT mice with the exception of SBP. Blockade of the CCR2 with RS504393 (4 mg/kg) had no effect on SBP. However, treatment with RS504393 prevented renal functional and morphological damage, and reduced the increase in monocyte/macrophage infiltration in DOCA‐salt hypertensive WT and TRPV1‐/‐ mice with the greater in the latter. Our results suggest that TRPV1 attenuates salt‐sensitive hypertension‐induced renal damage possibly via suppression of CCR2. (This work was supported by grants from the National Natural Science Foundation of China (No. 81170243) and Henan Provincial Science and Technology Innovative Program for Outstanding Scholarship (No. 124200510007).

Grant Funding Source: NNSF of China (No. 81170243), Henan Provincial STlPOS(No.124200510007).
Identification of protein kinases that phosphorylate the urea channel protein UT‐A1 (1137.11)
Authors: Hwang Gloria , Chou Chung‐Lin , Hoffert Jason , Knepper Mark
Published: June 2021

Vasopressin regulates the urea channel protein UT‐A1 by phosphorylation at Ser84 and Ser486. What kinases are responsible for these phosphorylation events remains an open question. To identify protein kinases that interact with UT‐A1 in IMCD, we employed crosslinking with BSOCOES, UT‐A1 immunoprecipitation, SDS‐PAGE separation, and quantitative mass spectrometry of trypsin digests of gel pieces. The peptides passing stringent spectral quality thresholds were quantified (label‐free) to identify those with high signal:noise (UTA1 antibody/preimmune IgG>4). This identified four protein kinases bound to the UT‐A1 (Phosphorylase Kinase [PhK], Calmodulin‐Dependent Kinase 2δ□[CAMK2], CDC42‐Binding Protein Kinase β□[CDC42BPK] and mTOR). In vitro phosphorylation assays were carried out by incubating these kinases or Protein Kinase A (purified, active recombinant) with synthetic peptides containing the known phosphorylation sites in UT‐A1. Phosphorylation was determined by immunoblotting using phospho‐specific antibodies. Four of the five tested kinases phosphorylated Ser486 (PKA > CDC42BPK > PhK > CAMK2). Kinetic studies of Ser486 phosphorylation (1 min‐24 hr) also showed PKA > CDC42BPK > PhK. In contrast, only CDC42BPK phosphorylated Ser84‐UT‐A1. mTOR did not phosphorylate either site. These results provide important data for modeling vasopressin signaling pathways.

Grant Funding Source: Supported by NHLBI
The use of bioinformatics to identify potentially damaging BK channel and palmitoylation enzyme polymorphisms as novel research targets (1138.14)
Authors: Adeyileka Bernadette
Published: June 2021

Large conductance Ca2+ and voltage‐gated potassium (BK) channels, coded for by the KCNMA1 gene, are found ubiquitously in humans and couple excitation‐contraction and stimulus‐secretion. BK channel dysfunction leads to pathophysiology affecting multiple body systems and processes (2). The BK channel undergoes palmitoylation, a post‐translational modification (PTM) that alters the trafficking, targeting and function of the protein. Palmitoylation is undertaken by a group of 23 palmitoyl‐acyl‐tranferase (PAT) enzymes coded for by the zDHHC genes. BK channel palmitoylation via PAT enzymes is crucial for BK channel function and thus has also been investigated as a novel therapeutic target. This study uses a bioinformatics approach to screen the genomic and proteomic data available for human KCNMA1 and zDHHC23, to identify and prioritise isoforms for future laboratory and clinical investigation. NCBI, Ensembl and Uniprot databases were utilised to identify: (a) splice variant information; (b) the number of non‐synonymous single‐nucleotide polymorphisms (nsSNPs); (c) nsSNP co‐location with key PTM and functional sites; (d) the damaging probability of prioritized nsSNP. Screening of KCNMA1 and zDHHC23 gene canonical proteins produced 154 and 63 unique nsSNPs respectively, with prioritisation of 13 KCNMA1 and 3 zDHHC23 nsSNP as amino acid (AA) co‐ordinates matched those of PTM residues or functionally important sites. nsSNP within 3 AA of key sites were recorded, with 64 (KCNMA1) and 22 (zDHHC23) nsSNP identified. The damaging probability of each prioritised nsSNP was assessed using SIFT and PolyPhen predictive software, with COSM196758, rs202024249, COSM920646, COSM332559 producing the highest scores. Prioritised SNPs will be investigated for functional impact in laboratory based assays. nsSNPs found to alter BK channel or PAT function will then be examined with respect to their distribution and incidence within appropriate populations and disease groups.

Grant Funding Source: Supported by RGU IHWR
Brazilian epidemiologic study about urinary tract infection using urine culture (1139.14)
Authors: Saldanha Fabiana , Duarte Janaina , Mergulhão Eliane , Hyodo Vivian
Published: June 2021

The urinary tract infection is characterized by the presence of pathogenic bacteria in the urinary tract that affects men and women. Some anatomical factors allow a greater incidence of infection in women. The present study aimed to determine the prevalence of urinary infection pathogens in a laboratory of the central region of Sao Jose dos Campos‐SP. It was analyzed 5360 urine culture sample, between September of 2003 and September of 2013. During this period were held 1231 urinary cultures. At the positive cultures were found the following etiological agents: Escherichia coli (74%);Enterobacter sp (6%); Negative Staphylococcus coagulase (5%); Citrobacter sp (4%); Staphylococcus aureus (2%); and others bacterias (9%).The higher prevalence of infection was found in women (25%). Escherichia coli and Enterobacter sp kept the first and second place for both sexes. However the percentage of Escherichia coli is greater among women (78%) than in men (53%). The results obtained in the current study have great importance as it will allow more appropriate treatment, and prevent complications and relapses.
Characterization of UDP‐glucuronosyltransferase 2B expression and regulation by nuclear signaling pathways in HepaRG cells (1141.6)
Authors: Barrett Kathleen , Duniec‐Dmuchowski Zofia , Kocarek Thomas , Runge‐Morris Melissa
Published: June 2021

HepaRG cells are bipotent human liver progenitor cells that can be differentiated into hepatocyte‐like cells that retain important liver functions. We evaluated the expression and regulation of the UDP‐glucuronosyltransferase (UGT) 2B subfamily of conjugating enzymes (UGT2B4, 2B7, 2B10, 2B11, 2B15, 2B17, and 2B28) in HepaRG cells. During proliferative culture conditions, expression of all UGT2Bs increased with cell confluency. After 2% DMSO‐induced differentiation, expression of UGT2B7and 2B17 initially decreased, but levels of all UGT2B mRNAs increased with continued differentiation. Treatment of differentiated HepaRG cells with the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) agonist, phenobarbital, increased expression of all UGT2B genes except UGT2B17. Rifampicin (PXR) increased expression of all UGT2B genes except UGT2B7. CITCO (CAR) increased UGT2B15 and decreased UGT2B17. GW3965 (liver X receptor) decreased all UGT2Bs except UGT2B11, while GW4064 (farnesoid X receptor) increased UGT2B10, 2B11, and 2B15 but decreased UGT2B4, 2B7 and 2B17. Treatment with ciprofibrate (peroxisome proliferator‐activated receptor α) increased UGT2B4, 2B7, and 2B15 but decreased UGT2B10 and 2B17. Treatment with 2,3,7,8‐tetracholorodibenzo‐p‐dioxin (Ah receptor) increased UGT2B4, 2B10, 2B11, 2B15, and 2B28. In conclusion, UGT2B genes are expressed in differentiating HepaRG cells and are differentially regulated by nuclear signaling pathways.

Grant Funding Source: Supported by NIH grant ES005823
Prophylactic effect of calcium channel blockers against oxaliplatin‐induced acute peripheral neuropathy (1143.8)
Authors: Kawashiri Takehiro , Tsutsumi Kuniaki , Ushio Soichiro , Egashira Nobuaki , Masuda Satohiro
Published: June 2021

Although oxaliplatin (L‐OHP) is an important anticancer agent against colorectal cancer, severe acute and chronic peripheral neuropathies are frequently occurred. We peviously reported that oxalate derived from L‐OHP is involved in acute cold hyperalgesia. In the present study, the mechanism of L‐OHP‐induced acute neuropathy was examined with the effects of calcium channel blockers in the rat model and primary cultured dorsal root ganglia (DRG).

Administration of L‐OHP or oxalate to rats induced cold hyperalgesia, and increased in transient receptor potential melastatin 8 (TRPM8) mRNA levels in DRG. In addition, the treatment of L‐OHP or oxalate induced Ca²⁺ influx and nuclear factor of activated T‐cell (NFAT) nuclear translocation as well as up‐regulation of the TRPM8 mRNA levels in cultured DRG. These oxalate‐induced changes were inhibited by nifedipine, diltiazem and mexiletine. Comparable to the in vitro results, administration of these calcium channel blockers prevented the L‐OHP‐induced cold hyperalgesia in rats.

These results suggest that the L type calcium channels/NFAT/TRPM8 pathway is a downstream mediator for L‐OHP‐induced cold hyperalgesia, and that calcium channel blockers may have prophylactic potential against L‐OHP‐induced acute neuropathy.
Effects of therapeutic and abusive doses of methylphenidate on neurogenesis and neuronal survival rates in murine brain (1145.4)
Authors: DeVee Carley , Farmer Brandon , Hanley Angela , Pond Brooks
Published: June 2021

Methylphenidate (MPH) is a psychostimulant that blocks reuptake of dopamine and norepinephrine via their transporters. Currently, MPH is used for the treatment of narcolepsy and is the most common medication used for the treatment of Attention Deficit Hyperactivity Disorder. It is also misused and abused due to its ability to increase attention, decrease appetite, and when injected, create a “high”. Various psychoactive medications, such as antidepressants, opioids, and other psychostimulants have been shown to alter the rate of neurogenesis or the “birth” or new neurons in the dentate gyrus of the hippocampus. Therefore, our lab postulated that long‐term exposure to MPH may also affect neurogenesis rate. In these studies, 25 day old Swiss‐Webster mice received twice daily injections of saline, 1 mg/kg, or 10 mg/kg MPH for 28 days; the 1 mg/kg MPH and 10 mg/kg MPH represent therapeutic and potential abusive doses, respectively. Three days after the final MPH injection, mice were injected with 5‐ethinyl‐2’‐deoxyuridine (EdU), a thymidine analog that incorporates into the DNA of dividing cells. In order to determine both the rate of neurogenesis and the long‐term survival of the new cells, mice were sacrificed at either 1 day post EdU injection or 28 days after EdU injection. Brains were sectioned on the cryostat and mounted on slides. Slides were stained using a Click‐iT EdU imaging kit, and neurons were double‐labeled using immunohistochemistry for the neuronal marker NeuN. Neurons within the dentate gyrus were counted to create a ratio of EdU+ /NeuN+ (or newly produced neurons to total neurons). Our preliminary data suggests that low dose (1 mg/kg) MPH has no effect on neurogenesis rate. However, high dose (10 mg/kg) MPH produces a higher ratio of newly synthesized neurons. Additionally, it appears that these neurons are maintained 28 days post injection. These effects may be relevant to the therapeutic action of MPH and/or its propensity for abuse.
Vasopressin instead of epinephrine enhances efficacy of CPR without causing tachycardia (1150.5)
Authors: Cherry Brandon , Nguyen Anh , Williams Arthur , Scott Gary , Hollrah Roger , Ryou Myoung‐Gwi , Hoxha Besim , Olivencia‐Yurvati Albert , Mallet Robert
Published: June 2021

Survival from cardiac arrest is highly dependent on the arterial pressure generated by cardiopulmonary resuscitation (CPR). To increase efficacy of precordial compressions, the potent vasoconstrictor epinephrine (EPI) is administered. However, EPI also elicits a robust, β1‐adrenoceptor‐mediated tachycardia following defibrillation, depleting the myocardium of the energy reserves it requires for recovery. We proposed that the adrenoceptor‐independent vasoconstrictor vasopressin (AVP) increases arterial pressure as effectively as EPI without producing tachycardia. After 6 min pacing‐induced cardiac arrest, domestic swine (25‐35 kg; 10 boars, 11 sows) received precordial compressions (100/min) for 4 min, and either EPI (0.1 mg; n=5) or AVP (10 U; n=16) was injected iv at 1 min CPR. EPI and AVP similarly increased mean arterial pressure from 31±3 to 66±4 mmHg vs. 34±3 to 59±3 mmHg at 4 min CPR. Countershock energy to achieve cardioversion was 16±4 J/kg in EPI‐ vs. 12±2 J/kg in AVP‐treated pigs. Post‐arrest tachycardia was less intense in AVP‐ (133±11 bpm) than EPI‐treated (174±14 bpm) pigs (P<0.05). Thus, AVP augments CPR as effectively as EPI but avoids tachycardia. Support: NINDS R01NS076975, NIA T32AG020494

Grant Funding Source: Supported by: NINDS R01NS076975, NIA T32AG020494
Rebuilding a heart: complete regeneration after myocardial infarction in the axolotl (1151.1)
Authors: Lauridsen Henrik , Pedersen Michael
Published: June 2021

Intro: Cardiac failure resulting from myocardial infarction is a leading cause of death. This is the fatal result of the lack of regenerative capacity of the mammalian heart. Salamanders such as the axolotl possess unmatched regenerative capacity, mastering the ability to regenerate internal organs and whole limbs. To draw basic information about molecular and cellular mechanisms in cardiac repair, we have established and validated a myocardial infarction model in the axolotl.

M&M: Myocardial infarction in the axolotl was induced by cryoinjury. Regeneration was described histologically at 4, 7, 14, 31, 40 and 94 days post infarction (dpi) and 4 days post sham (dps). Concurrently functional restoration of cardiac output was monitored using echocardiography and MRI. Protein profiles of myocardial tissue 4, 14 and 30 dpi and 4 dps were produced using LC‐MS/MS.

Results & Discussion: The axolotl restores complete myocardial structure and functionality within 2‐3 months after a 25% myocardial infarction. Interestingly, the initial response to injury is fibrosis forming a scar as found in mammals. However, this response is replaced within a few weeks when cardiomyocytes are deposited in the fibrotic scaffold aiding a gradual recovery of cardiac output.

This model can potentially improve our understanding of heart regeneration, and we seek to identify signaling events at the protein level using the proteomics data.