bluuebunny/crossref_metadata_2025 huggingface dataset:
Caffeine protects cerebral artery function in the reduced uterine perfusion pressure model of preeclampsia (1070.9)
Authors: Dennis Spencer , Balser Brittany , Powers Robert , Novak Jacqueline , Ramirez Rolando
Published: June 2021

Coffee is one of the most widely consumed beverages in the world, and caffeine is the most studied compound in coffee. Coffee consumption is associated with a decreased risk of preeclampsia. Preeclampsia is a hypertensive disorder of pregnancy, and under‐perfusion of the placenta is hypothesized to contribute to the disease. An animal model of preeclampsia is the reduced uterine perfusion pressure (RUPP) model. The focus of this study was to investigate if caffeine prevents peripheral vascular function dysfunction, specifically the posterior cerebral artery (PCA), in the RUPP model. Pregnant rats were divided into three groups: control pregnant, RUPP, RUPP + caffeine (~10µg/day via drinking water). Near term, maternal PCAs were studied in a pressurized arteriograph. The diameter of each artery was measured in HEPES buffered saline solution with and without calcium. Percent tone was calculated for each artery at each pressure step (25‐200mmHg). The tone of the PCAs isolated from controls was significantly lower (ex. 125mmHg, 8.7±1.9%) than tone exhibited by the PCAs isolated from RUPP animals (14.6±6.1%). Conversely, caffeine prevented the increase in tone induced in the RUPP model (5.1±1.9%). Caffeine treatment prevents the increase in tone induced in the RUPP model. These data suggest a protective effect of caffeine on cerebral vascular function during pregnancy, and may have relevance to preeclampsia.
Functional characterization of a newly identified LMNA mutant in HL‐1 cardiomyocytes (1073.7)
Authors: Carmosino Monica , Forleo Cinzia , Pisani Francesco , Procino Giuseppe , Torretta Silvia , Favale Stefano , Svelto Maria
Published: June 2021

A novel mutation in the Lamin A/C gene (LMNA c.418_438dup) was detected in the index patient and in additional family members with diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) and history of sudden cardiac death. The functional characterization of this LMNA mutant was performed in cultured HL‐1 cardiomyocytes expressing EGFP‐tagged wild‐type and mutated LMNA constructs and subjected to confocal microscopy analysis and hypoxic stress conditions in 100% N2 for 8h. Mutated LMNA was clearly expressed in aggregates of different sizes and not uniformly distributed along the nuclear envelope as WT LMNA. Moreover, the mutated LMNA variant causes perturbation in nuclear shape and Nuclear Pore Complexes organization.

Of note, we observed that under hypoxic conditions nuclear envelopes expressing mutated LMNA become leaky, leading a nuclear fluorescent marker to escape into the cytoplasm. This indicates that, under cell stressing conditions, the nucleo‐cytoplasmic compartmentalization is affected in cardiomyocytes expressing this LMNA mutation, inducing, as final fatal consequence, cell apoptosis.

In conclusion, we not only open new avenues to gain more insights in the pathogenesis of ARVC and to conceive novel therapeutic strategies but we also shed lights on the role of nuclear Lamin A in the physio‐pathology of cardiomyocytes.

Grant Funding Source: Supported by Fondo per gli Investimenti della Ricerca di Base‐Rete Nazionale di Proteomica (RBRN07BM
Individual differences in susceptibility to depression and cardiovascular disease comorbidity in a rat model of social stress (1074.10)
Authors: Lombard Calliandra , Melson Michael , Wood Susan
Published: June 2021

Psychosocial stress precipitates psychiatric disorders (eg, depression) and cardiovascular disease in vulnerable individuals, but underlying mechanisms remain unknown. We previously identified two distinct phenotypic responses to social stress in rats (resident‐intruder model), characterized by either passive coping, assuming defeat within a short latency (SL, 343±7) or by proactive coping behaviors and longer defeat latencies (LL, 571±53). The SL phenotype was associated with endocrine and behavioral characteristics resembling depression and decreased heart rate variability indicating greater cardiovascular disease risk. The present study characterized the impact of stress coping on susceptibility to a depressive‐like phenotype with comorbid hypertension in rats exposed to 7 (30 min/day) social defeat or control manipulations. 24‐hr ECG and pressure telemetry revealed persistent elevations in resting mean arterial pressure (MAP) selectively in the SL phenotype as early as the 4th day (change from day 0: con 0.4 ± 2, SL 7.1 ± 2, LL 2.5 ± 0.9). Interestingly, sucrose intake measured 7 days post stress was negatively correlated with changes in MAP (r= ‐0.34, p=0.03), indicating an association between anhedonia and increased MAP. These studies also sought to determine the impact of these cardiovascular changes on susceptibility to isoproterenol‐induced arrhythmias. These data suggest passive stress coping promotes vulnerability to a depressive‐cardiovascular disease‐like phenotype, while active coping confers resilience. Future studies will utilize this model to identify critical brain regions underlying the pathogenesis of depression‐cardiovascular disease comorbidity.

Grant Funding Source: Supported by NARSAD 17830 and 13BGIA 14370026 to SKW
Effect of aging on eNOS‐associated protein:protein interactions throughout the arterial network (1075.8)
Authors: Luttrell Meredith
Published: June 2021

We previously demonstrated age‐related endothelial dysfunction in oxidative soleus muscle feed arteries (SFA), while endothelial function in glycolytic gastrocnemius muscle feed arteries (GFA) is maintained with age. One mechanism that may account for endothelial dysfunction with age is alterations in eNOS‐associated protein:protein interactions with caveolin‐1 (Cav1) and calmodulin (CaM). Therefore we tested the hypothesis that Cav1:eNOS interaction increases with age while CaM:eNOS interaction decreases with age in SFA and that there would be no change in either protein:protein interaction in aged GFA. Additionally, we assessed the effects of aging on eNOS‐associated protein:protein interactions throughout the arterial network. Co‐immunoprecipitation was used to determine protein:protein interactions. Results show that Cav1:eNOS and CaM:eNOS interactions were not altered by age in SFA or GFA; Cav1:eNOS interaction was greater in GFA than SFA; the phenotype of both Cav1:eNOS and CaM:eNOS interactions was heterogeneous throughout the arterial network; and aging had a non‐uniform effect on both Cav1:eNOS and CaM:eNOS interactions throughout the arterial network. In conclusion, age had no effect on Cav1:eNOS or CaM:eNOS interactions in either GFA or SFA. Also, Cav1:eNOS and CaM:eNOS interactions throughout the arterial network and are non‐uniformly altered with age.

Grant Funding Source: Research Supported By: AHA 0765043Y, 4150031 (CRW), AHA 467371 (MJL)
Intravascular pressure stimulates functional KV1.5 surface expression in mesenteric artery smooth muscle cells (1077.4)
Authors: Kidd Michael , Leo M , Narayanan Damodaran , Bannister John , Jaggar Jonathan
Published: June 2021

Intravascular pressure stimulates arterial depolarization, leading to myocyte voltage‐dependent K+ (KV) channel activation that opposes vasoconstriction. We tested the hypothesis that membrane potential regulates smooth muscle cell KV channel surface expression to modulate Kv channel functionality in mesenteric arteries. Q‐PCR indicated predominant expression of KV1.5 and 2.1 in mesenteric arteries. Contrary to expectations, 70% of total and surface KV1.5 protein was lost within 3 hrs in non‐pressurized arteries. In contrast, KV1.5 or 2.1 mRNA and total and surface KV2.1 protein did not change over the same time course. Bafilomycin, a lysosome inhibitor, and MG‐132, a proteasome inhibitor, both prevented KV1.5 loss. Depolarization (30 mM K+) also inhibited KV1.5 loss and this was blocked by nimodipine, a voltage‐dependent Ca2+ channel blocker. Patch‐clamp studies indicated that KV currents sensitive to DPO‐1, a Kv1.5 inhibitor, were 60% smaller in myocytes isolated from arteries kept at zero pressure than controls. Kv current loss was also blocked by bafilomycin. We also show that physiological intravascular pressure maintains functional Kv1 channel activity in arteries. In summary, data indicate that intravascular pressure inhibits KV1.5 channel degradation to maintain functional surface expression of these proteins in arterial myocytes.

Grant Funding Source: NHLBI/NIH
A pharmacologic activator of endothelial KCa channels improves coronary function in the hearts of type 2 diabetic rats (1078.10)
Authors: Mishra Ramesh , Wulff Heike , Cole William , Braun Andrew
Published: June 2021

Endothelial dysfunction is a common early pathogenic event in patients with type 2 diabetes (T2D) who exhibit cardiovascular disease. In the present study, we have examined the effect of SKA‐31, a positive modulator of endothelial Ca2+‐activated K+ (KCa) channels, on total coronary flow in isolated hearts from Goto‐Kakizaki (GK) rats, a non‐obese model of T2D and age‐matched controls. Total coronary flow and left ventricular developed pressure were monitored simultaneously in spontaneously beating, Langendorff‐perfused hearts. Acute, bolus administrations of bradykinin (BK, 1 μg) or adenosine (ADO, 10 μg) increased coronary flow, and these responses were blunted in diabetic hearts at 10‐12 and 18‐20 weeks of age. In contrast, SKA‐31 dose‐dependently (0.01‐5 μg bolus exposures) increased coronary flow to comparable levels in both control and diabetic rat hearts at both ages. Flow responses to sodium nitroprusside were not different between control and diabetic hearts, suggesting normal coronary smooth muscle function. Importantly, continuous exposure to a sub‐threshold concentration of SKA‐31 (i.e. 0.3 μM) did not alter basal coronary flow or heart rate, but did ameliorate the blunted BK and ADO‐evoked flow responses in diabetic hearts. In summary, these data demonstrate that SKA‐31 is an effective coronary vasodilator in a rat model of T2D exhibiting endothelial dysfunction and metabolic syndrome, and further rescues impaired vasodilatory responses to BK and ADO in the coronary circulation.

Grant Funding Source: Supported by CIHR
Antiphospholipid antibodies, necrotic trophoblasts and preeclampsia? (1084.3)
Authors: Lau Sien Yee , Chen Qi , Chamley Larry , Barrett Carolyn , Guild Sarah‐Jane
Published: June 2021

Preeclampsia is characterized by de novo hypertension, proteinuria and systemic inflammatory response during pregnancy which is preceded by endothelial cell activation. It is triggered by a placental factor(s), one of which may be necrotic trophoblast debris that is shed from the placenta into the maternal blood. Clearance of necrotic trophoblast debris results in endothelial cell activation in vitro and results in a relative increase in blood pressure in vivo. There are also maternal risk factors for preeclampsia, the strongest of which are antiphospholipid antibodies (aPL). We present a two part model observing the interaction between aPL and necrotic trophoblast debris on blood pressure, markers of endothelial cell activation and urinary protein.

Pregnant female Wistar rats instrumented with radio‐telemetry blood pressure devices were received necrotic trophoblasts and aPL (n=10) or control antibodies (n=5). Fifteen further rats received only aPL (n=10), or control antibodies (n=5), in the absence of necrotic trophoblasts.

Mean arterial blood pressure was not different between rats that received control antibodies or aPL with necrotic trophoblasts. However, heart rate was lower and urinary protein levels were elevated in rats that received necrotic trophoblasts and aPL. Serum levels of interleukin‐6, the proposed spreading factor for endothelial cell activation, were also elevated in rats receiving necrotic trophoblasts.

In conclusion, antiphospholipid antibodies, the strongest maternal risk factor in preeclampsia, may interact with necrotic trophoblasts to heighten the systemic inflammatory response which precedes the clinical onset of hypertension in preeclampsia.
Cholesterol regulates both store‐ and agonist‐induced Ca2+ entry in pulmonary arterial endothelium (1089.9)
Authors: Zhang Bojun , Riddle Melissa , Naik Jay , Resta Thomas , Walker Benjimen
Published: June 2021

Endothelial Ca2+ entry is decreased in pulmonary arteries from rats exposed to chronic hypoxia (CH) (4 wk, PB = 380 torr). In addition, membrane cholesterol (Chol) is diminished in these cells and Chol supplementation restores ATP‐induced Ca2+ entry. Since both agonist‐induced and store‐operated Ca2+ entry (SOCE) are decreased after CH, we hypothesized that both require functional membrane Chol. To test this hypothesis, we administered epicholesterol (EpiCh; epimeric form of cholesterol) or vehicle to pulmonary arterial endothelial cells (PAEC) from control and CH rats to replace native Chol. The efficacy of EpiCh to diminish membrane Chol was confirmed by filipin staining in cultured ECs. EpiCh treatment greatly reduced both cyclopiazonic acid (CPA) ‐induced SOCE and ATP‐induced Ca2+ entry in PAECs from control and CH rats. We next assessed the effect of EpiCh on ATP and CPA‐induced Ca2+ currents in PAEC from control rats using patch clamp. Both EpiCh and La3+ inhibited ATP‐induced current similarly. Although T‐type voltage gated Ca2+ channels contribute to ATP responses, mibefradil (T‐type inhibitor) had no further effect in EpiCh treated cells. Likewise, CPA‐induced current was reduced in cells treated with EpiCh. We conclude that membrane Chol is required for both agonist‐induced and store‐operated Ca2+ entry in PAEC and that reduced Ca2+ seen after CH is possibly due to loss of this key regulator.

Grant Funding Source: Supported by NIH grant HL95640
AntimiR‐145 therapy improves right ventricular structure in experimental pulmonary arterial hypertension (1090.1)
Authors: McLendon Jared , Joshi Sachindra , Fewell Jason , Oka Masahiko , McMurtry Ivan , Gerthoffer William
Published: June 2021

Our previous work shows miRNA‐145 is increased in pulmonary arterial hypertension (PAH) and knockdown of miR‐145 in rats exposed to Sugen‐5416, hypoxia, and normoxia, repairs pulmonary arteriopathy and improves RV function. One explanation for improvements is that partial repair of the arteriopathy reduces pulmonary arterial pressure (and/or increases vascular compliance) which reduces RV afterload. Normal repair mechanisms regulating cardiac structure may then result in reduced RV hypertrophy at the cellular and tissue levels. To test this we measured ventricular weights, RV myocyte cross‐sectional areas and collagen deposition. AntimiR‐145 produced a modest decrease in the RV/LV+S ratio as well as decreased RV weight/body weight ratio. Interestingly, the positive effect on tissue mass was not a result of normalizing RV cardiomyocyte hypertrophy because cellular cross‐sectional area was unaffected by antimiR‐145. These results are more consistent with the response to antimiR‐145 being a reduction in cell number and tissue mass rather than cell atrophy. The therapeutic response also includes beneficial effects on matrix remodeling as shown by reduced collagen deposition. Together the results demonstrate subchronic treatment with antimiR‐145 has beneficial cardiac effects in rats with PAH that result in increased cardiac output as well as partial regression of RV hypertrophy and matrix remodeling.

Grant Funding Source: Supported by NIH grant HL097220 to WTG and AHA fellowship 13PRE17070053 to JMM.
High expression of CD147 in endometriosis and its role in regulating apoptosis and migration in human endometrial cells (1093.3)
Authors: Chen Hao , Wang Chaoqun , Jing Aihong , Zhou Xiaping , Cai Zhiming , Chan Hsiaochang
Published: June 2021

Endometriosis is a common gynecological disease, which contributes to 40% of infertile women in reproductive age. CD147 has been found highly expressed in ectopic endometrium and stimulated the MMPs in human uterine fibroblasts. However, it is poorly understood if and how CD147 affects endometrium epithelial cell migration during the progression of endometriosis. We demonstrate here that the expression of human CD147 was significantly higher in ovarian ectopic endometrium than that of normal endometrium by real‐time PCR. Interfering CD147 function decreased cell migration and cell viability in the normal human uterine epithelial cells (HES). Surprisingly, MMP‐2 expression and activity were not changed when HES treated with anti‐CD147 antibody. Further studies revealed that immunodepletion of CD147 induced apoptosis in HES cells, which led to Caspase‐3 and poly ADP‐ribose polymerase (PARP) activation. The present study demonstrated that CD147 regulates apoptosis and the cell migration in a MMP‐2 independent manner in human endometrial epithelial cells and that abnormally high expression of CD147 in the ectopic endometriosis lesions may contribute to the pathogenesis of endometriosis.

Grant Funding Source: Supported by NSFC (31100841) and Shenzhen Projects of Science and Technology (JCYJ20120614085604107)
The promotion of osteogenic differentiation of human adipose‐derived mesenchymal stem cells induced by chitosan‐conjugated gold nanoparticles (1094.1)
Authors: Choi Seon Young , Song Min‐Seok , Ryu Pan‐Dong , Joo Sang‐Woo , Lee So Yeong
Published: June 2021

Gold nanoparticles are attractive materials for use in biomedicine due to their unique properties. Increasing evidence suggests that several nanoparticles induce differentiation of human mesenchymal stem cells (hMSCs) into osteoblast and adipocyte. In this study, we hypothesized that chitosan‐conjugated gold nanoparticles promote osteogenic differentiation of hMSCs. In order to investigate promoted osteogenic differentiation of hMSCs and related signaling pathway, chitosan‐conjugated gold nanoparticles were added to an osteogenic induction medium for 7, 10, 14 and 21 days. For evaluation of osteogenic differentiation, alizarin red staining, alamar blue assay and quantitative real time PCR were performed. In order to examine specific signaling pathways, immunofluorescence and western blotting assay were performed. Our results demonstrate that nanoparticles increase the deposit of calcium content and the expression of marker genes related to the early stage of osteogenic differentiation in hMSCs at non‐toxic concentrations. These results indicate that nanoparticles promote osteogenesis through Wnt/β‐catenin signaling pathway and may be used as a promoting reagent for bone formation.

Grant Funding Source: supported by the National Research Foundation of Korea (2012R1A2A2A01047151)
25‐Hydroxycholesterol inhibits adipogenic differentiation of C3H10T1/2 mouse stem cells through inhibition of PPARγ expression (1094.2)
Authors: Moseti Dorothy , Regassa Alemu , Kim Woo
Published: June 2021

Oxysterols have been shown to inhibit adipogenic differentiation of mesenchymal stem cells while inducing their osteogenic differentiation. However, the effects of oxysterols on the adipogenic differentiation of C3H10T1/2 mouse stem cells are poorly understood. This study was conducted to examine the effects of specific oxysterols on the expression of adipogenic transcripts and adipogenic differentiation of C3H10T1/2 mouse stem cells. Cells were treated with 10% Fetal Bovine Serum (control), 500nM Dexamethasone, 0.5mM 3‐Isobutyl‐1‐methlyxanthine, 20µg/ml Insulin (DMI) + 10µM PPARγ agonist Troglitazone (Tro) (DMITro) and DMITro + 10µM 20S hydroxycholesterol (20S), 25 hydroxycholesterol (25), 22R hydroxycholesterol (22R) or 22S hydroxycholesterol (22S) for six days. Gene expression was analysed by real‐time quantitative polymerase chain reaction and expression data analysed using the Statistical Analysis Software (SAS 9.2). Compared to the control, DMITro significantly increased the expression of peroxisome proliferator‐activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), fatty acid binding protein 2 (FABP2) and lipoprotein lipase (LPL). Compared to DMITro, treatment of cells with DMITro + 25 significantly decreased the expression of PPARγ, the master regulator of adipogenesis (P 蠄 0.05). Expression of C/EBPα was significantly decreased by DMITro + 25 and DMITro + 20S (P 蠄 0.05), while the expression of FABP2 was significantly decreased by DMITro + 25, DMITro + 20S and DMITro + 22R (P 蠄 0.05). Lastly, the expression of LPL was significantly decreased by DMITro + 25 and DMITro + 22R (P 蠄 0.05). In conclusion, the results showed that 25 hydroxycholesterol was the most potent in inhibiting the expression of key adipogenic transcripts and adipogenesis of C3H10T1/2 mouse stem cells suggesting its potential application in reducing adipogenesis and obesity.

Key words: Oxysterols, C3H10T1/2 stem cells, differentiation, PPARγ

Grant Funding Source: Supported by Canadian natural science and engineering research council.
Mapping the dynamic Interactomes of “druggable” membrane proteins: roles in human health and disease (1095.20)
Authors: Stagljar Igor
Published: June 2021

Despite extensive research in the past decade, there is a lack of in‐depth understanding of protein networks associated with integral membrane proteins because of their unique biochemical features, enormous complexity and multiplicity. This is a major obstacle to understanding the biology of deregulation of these integral membrane proteins which leads to numerous human diseases, and consequently hinders our development of improved and more targeted therapies to help treat these diseases. To address this challenge, we previously developed an in vivo genetic system, called the Membrane Yeast Two‐Hybrid (MYTH) assay, to identify and characterize protein interactors of all yeast ABC transporters and human receptor tyrosine kinases (RTKs), as well as selected cancer stem cell receptors (CSCRs)5 and G‐protein coupled receptors (GPCRs). However, despite MYTH being a robust technology suitable for mapping the PPIs of a wide‐range of membrane proteins, we have found that a significant percentage of mammalian integral membrane proteins cannot be properly analyzed using this system.To address this, we have recently developed a new variant of MYTH suitable for use in mammalian cells, which we have called the Mammalian Membrane Two‐Hybrid (MaMTH) system. During my talk, I will discuss exciting new findings indicating that MaMTH can detect stimuli (hormone/agonist)‐ and phosphorylation‐dependent PPIs. In addition, I will show that MaMTH allows for monitoring of the phosphorylation states of ErbB‐receptor mutants and drug‐induced activity changes of oncogenic variants of the Epidermal Growth Factor Receptor (EGFR). In conclusion, our study illustrates that MaMTH is a powerful tool for investigating dynamic interactomes of human integral membrane proteins and promises significant contributions to therapeutic research.
The role of p38, ERK1/2 and JNK mitogen activated protein kinase in the anti‐hypertrophic action of AMP‐kinase in cardiomyocytes (1097.13)
Authors: Lopez Pablo , Barreto Giselle , Soto Jessica , Javadov Sabzali
Published: June 2021

Mitogen activated protein kinases (MAPKs) play an important role in cell grow, proliferation and apoptosis in the heart. The MAPK family involves three major subgroups including extracellular signal‐regulated protein kinase (ERK1/2), p38, and c‐jun N‐terminal kinases (JNK). Despite many studies demonstrating anti‐hypetrophic action of AMP‐kinase (AMPK), a metabolic sensor regulating energy metabolism, the relationship between AMPK activation and MAPKs is not clear yet. We hypothesize that beneficial effects of AMPK are mediated, at least in part, through modification of MAPKs activation. Experiments were performed on H9c2 myoblastic rat cardiomyocytes incubated at 370C with 95% air and 5% CO2. Hypertrophy was induced by treatment of the cells with 200 nM angiotensin II (Ang II, a peptide hormone stimulating hypertrophy) or 10 μM phenylephrine (PE, the α1‐adrenergic receptor agonist). First, we determined time‐course activation (phosphorylation) of ERK1/2, p38 and JNK in response to Ang II and PE. Second, we evaluated, if activation/inhibition of AMPK by metformin/compound C influences activation of the MAPKs induced by AngII or PE. Results demonstrated that maximum activation of all three MAPKs occurs at 10 min after addition of the hypertrophic agents. A time point of 10 min was selected to determine whether AMPK affects MAPKs activation in AngII‐treated cells. Metformin (2 mM) or compound C (10 μM) was added to the culture medium 45 min before AngII or PE treatment. Metformin significantly abrogated the activation of all three MAPKs induced by AngII or PE. Inhibition of AMPK by compound C had no effect on MAPK phosphorylation suggesting that the effects are mediated via AMPK. Surprisingly, losartan had no significant effects on MAPK activation in AngII‐treated cells. In conclusion, our findings demonstrate that ERK1/2, p38 and JNK MAPKs play a crucial role in mediating anti‐hypertrophic effects of AMPK in cardiomyocyte hypertrophy.

Grant Funding Source: Supported by the NHLBI NIH grant SC1HL118669 (S.J.) and APS/NHLBI STRIDE Summer Research Fellowship
Dopamine D1‐like receptors differently regulate small G proteins in LRs in human embryonic kidney cells heterologously expressing human D1 or D5 receptors (1097.7)
Authors: Yu Peiying , Yang Yu , Jose Pedro
Published: June 2021

Small G proteins,e.g.,Rabs (Rab4, Rab5, and Rab11), are involved in G protein‐coupled receptor trafficking, including recycling. We tested the hypothesis that D1‐like receptors (D1R and D5R) differentially regulate the small G proteins Rab4, Rab5, and Rab11 in lipid rafts (LRs) in HEK‐293 cells heterologously expressing either the human D1R or D5R (hD1R or hD5R cells, respectively). In the basal state the Rabs, which co‐fractionated with flotillin‐1, an LR marker protein, were distributed in LR fractions (fractions 2‐6) and nonLR fractions (fractions 7‐12). The D1‐like receptor agonist fenoldopam (1‐5μM/15min), increased Rab4 in LRs in hD1R cells (+38.9±14.8%, vs. control 0.0±4.8 %) and in hD5R cells (+26.9±8.7% vs. control +0.1±3.8 %) (P<0.05, n=4, ANOVA). The effect of fenoldopam on Rab5 was opposite that of Rab4: fenoldopam decreased Rab5 protein in LRs in both cell lines (‐27.4±4.9% hD1R) and (‐19.7±3.0% hD5R) (P<0.05, n=5, ANOVA). Fenoldopam also increased Rab11 protein in LRs in hD1R (+41.6±16.3%) compared to its control (+0.0±0.5%) (P<0.01, n=4, ANOVA) but not in hD5R cells. Moreover, fenoldopam increased Rab11 protein co‐immunoprecipitation with D1R in a time dependent manner which peaked at 1h (22.9±2.5 DU), relative to vehicle‐treatment (9.8±1.9 DU) (P<0.01, n=4, ANOVA). Rab11 protein also co‐immunoprecipitated with D5R but was not affected by fenoldopam. Confocal images corroborated the cell fractionation and co‐immunoprecipitation data: fenoldopam decreased Rab5 in LRs in hD1R and hD5R cells, and increased Rab4 in LRs in hD1R and hD5R cells. Fenoldopam also increased Rab11 protein co‐localization with LR marker protein in LRs in hD1R cells. However, in D5R cells, the D5R and Rab11 co‐localization in LRs was not affected by fenoldopam. We conclude that dopamine D1R and D5R co‐fractionates with Rab4, Rab5, and Rab11 in LRs. These small G proteins may play an important role in D1R and D5R trafficking. Specifically, Rab11 protein may play an important role in D1R recycling.

Grant Funding Source: HL023081, HL074940, DK039308
Fluctuations in surface pH of maturing rat incisor enamel are a result of cycles of H+‐secretion by ameloblasts and variations in enamel buffer characteristics (1098.1)
Authors: Frische Sebastian , Damkier Helle , Josephsen Kaj , Takano Yoshiro , Zahn Dirk , Fejerskov Ole
Published: June 2021

The large hydroxyapatite (HA) crystals in dental enamel results from a long lasting process called enamel maturation, which follows a stage of enamel matrix secretion. Thus, after the full thickness of enamel is reached, the enamel still requires about 60‐70 % mineral to be deposited by HA‐crystals growing in width and thickness. This is facilitated by the ameloblasts, which during maturation change morphology every 6‐8th hour. This study aims to test the role of the ameloblasts in maintaining enamel pH in the maturation zone in order to elucidate the role of the cyclic changes in ameloblast morphology for HA‐crystal growth.

Colorimetric pH‐indicators and ratiometric fluorometry were used to measure surface pH in maturation zone enamel of rat incisors. Alternating acidic (down to pH 6.24±0.06) and alkaline zones (up to pH 7.34±0.08) corresponding to ruffled‐ended and smooth‐ended ameloblasts, respectively, were found along the tooth thus coinciding with ameloblast morphological cycles. Underlying the cyclic pattern, a gradual decrease in pH towards the incisal edge was seen. Vinblastine or FR167356 (H+‐ATPase‐inhibitor) disturbed ameloblast acid‐secretion, especially in the early parts of acidic zones.

At the pH‐values observed, PO43‐ would be protonated (pKa >12) and HA dissolved. However, by molecular dynamics simulations we estimate the pKa of HPO42‐ at an ideal HA surface to be 4.3. The acidic pH measured at the enamel surface may thus only dissolve non‐perfect domains of HA crystals in which HPO42‐ is less electrostatically shielded. As a result of the repeated alkaline/acidic cycles induced by ameloblasts, near‐perfect domains will therefore gradually replace less perfect domains and large near‐perfect HA crystals will be produced.

In conclusion, fluctuations in surface pH of maturing rat incisor enamel are a result of cycles of H+‐secretion by ameloblasts and variations in enamel buffer characteristics.
Regulation of ENaC, AE1 and H+ ATPase in renal intercalated cell specific NBCe2 knockout mice (1098.5)
Authors: Pedersen Fredrik , Praetorius Jeppe , Damkier Helle
Published: June 2021

Regulation of ENaC, AE1 and H+ ATPase in renal intercalated cell specific NBCe2 knockout mice Fredrik D Pedersen, Jeppe Praetorius and Helle H Damkier, Department of Biomedicine, Health, Aarhus University, Denmark NBCe2 is an electrogenic Na+ HCO3‐ cotransporter expressed in the brain and kidney. In mouse, NBCe2 mRNA is localized in the collecting duct principal cells; however, attempts to locate the protein in mice have been unsuccessful. In humans, immunohistochemical studies localized NBCe2 to the apical membrane of apparent intercalated cells. Mice lacking NBCe2 are hypertensive and have lower urinary pH, but the mechanism behind and cellular origin of this phenotype is unknown.

The aim of the study is to define the cell type responsible for development of hypertension in NBCe2 knockout mice.

Intercalated cell specific NBCe2 knockout mice (IC‐KO) were generated using the loxP/cre system. Mice expressing cre recombinase driven by the promotor for the intercalated cell H+ ATPase, B1 were crossed with floxed NBCe2 mice.

Tail cuff measurements and urine electrolyte analyses revealed no differences in blood pressure or urine composition between the IC‐KO mice and wildtype littermates. However, immunoblot analysis revealed decreased abundance of the membrane transporters AE1, αENaC and H+ ATPase in the IC‐KO mice. Furthermore, immunohistochemical analysis showed increased γENaC abundance in the apical plasma membrane of the IC‐KO mice.

In conclusion, the IC‐KO mouse model does not display the hypertensive phenotype of the full NBCe2 knockout mouse. Nevertheless, the changes in abundance and subcellular localization of key renal membrane transporters indicate a significance of NBCe2 in intercalated cells in mice. However, the changes could also reflect the activity of cre recombinase in a subset of cortical principal cells that has been described for the cre mouse.
Aquaglyceroporin HC‐3 mediates hypotonicity‐induced cell volume and shape changes in cultured erythrocytes from Cope’s gray treefrog, Hyla chrysoscelis (1099.4)
Authors: Hawk Mark , Nickell Philip , Frisbie James , Goldstein David , Krane Carissa
Published: June 2021

Freeze tolerant Cope’s gray treefrog, Hyla chrysoscelis, accumulates glycerol during a process of cold‐acclimation in anticipation of freezing. Glycerol acts as a cryoprotectant to control osmotic gradients formed by extracellular ice crystals during freezing. The aquaglyceroporin, HC‐3, is abundantly expressed in the membrane of erythrocytes from H. chrysoscelis, where it facilitates osmotically driven transmembrane water flux and glycerol diffusion, both important in freeze tolerance. We hypothesize that HC‐3 functions to moderate the dynamic cell shape changes that occur during the freeze/thaw process as a result of hypotonicity induced cell volume expansion. Cultured erythrocytes subjected to moderate hypotonic stress (70 mOsM) in the absence of 0.3 mM HgCl2, underwent a series of shape changes: they swelled, initially elongated, then became swollen and round, before returning to an elongated state. Erythrocytes treated with an HC‐3 morpholino to knockdown HC‐3 expression become spherical and remained spherical in response to hypotonic challenge. Within 600 seconds of the initiation of the hypotonic challenge, 80% of control cells succumbed to hypotonicity‐induced cell lysis, whereas 70% of the morpholino‐treated cells remained intact. These data indicate that HC‐3 functions in regulating erythrocyte cell volume in response to hypotonic challenge, implicating a role for HC‐3 in cellular freeze tolerance.

Grant Funding Source: This research was supported by NSF Research Grant IOS‐1121457.
Impact of hypercapnic incubation on hatchling common snapping turtle (Chelydra serpentina) growth and metabolism (1101.3)
Authors: Wearing Oliver , Eme John , Kemp Adam , Crossley Dane
Published: June 2021

Some reptilian embryos are naturally exposed to low levels of O2 and high levels of CO2. This phenomenon has provoked much research on the physiological effects of developmental hypoxia in reptiles and associated implications on survival. However, notably less attention has been given to the effects of hypercapnia. In this study, we investigated the growth trajectories and metabolic rates (resting and postprandial) of common snapping turtle (Chelydra serpentina) hatchlings that were chronically incubated as embryos in one of three environmental conditions: Atmospheric CO2 (control); 3.5% CO2 (3.5%); and 7% CO2 (7%). Control and 7% hatchlings had similar growth trajectories and mean masses. However, the 3.5% animals had both a higher mean mass and a steeper trajectory compared to the other conditions. VO2 experiments revealed that resting metabolic rate was significantly elevated in the hypercapnic conditions compared to the control condition. Moreover, the postprandial metabolic rate was 1.6‐ to 1.7‐fold higher than resting in the hypercapnic, compared to a 2‐fold increase in the normal incubated turtles. This study not only demonstrates that snapping turtles can survive considerable hypercapnia during embryonic development, but also that incubation condition has lasting effects on physiology post‐hatching.

Grant Funding Source: Supported by NSF CAREER IBN IOS‐0845741 to DAC
Role of synemin, an intermediate filament protein, in adult skeletal muscle (1102.25)
Authors: Garcia‐Pelagio Karla , Muriel Joaquin , Lund Linda , Bond Meredith , Bloch Robert
Published: June 2021

Intermediate filament (IF) related diseases are linked to defects in proteins that are associated with the contractile apparatus of skeletal and cardiac muscle, and with costameres. Synemin is a large IF protein that associates with dystrobrevin, vinculin, and talin at costameres at the sarcolemma of striated muscle, as well as with α‐actinin and desmin at Z disks. Synemin can be expressed in either 210 kDa α‐ or 180 kDa β‐ alternatively spliced forms. We generated mice null for synemin by homologous recombination to study synemin’s function in skeletal muscle. Skeletal muscle in the knock out (syn KO) mouse does not make synemin mRNA or protein. Preliminary characterization of the syn KO mouse suggests that it has a mild skeletal muscle phenotype. The organization of costameres appears to be normal, but the fibers appear smaller than controls. Treadmill running uphill test results was not significantly affected when compared to controls. More notably, we have observed differences in the biomechanical properties of the sarcolemma. Stabilization time of blebs generated by applying fixed suction pressure through a pipette to the surface membrane of single Extensor digitorum longus fibers, were slower in the syn KO than in controls but faster than in desmin KO and mdx fibers, which lack dystrophin. Furthermore, the pressure at which the blebs separated from underlying contractile structures were lower than in controls. These results suggest that the viscoelastic properties of the sarcolemma‐costamere‐myofibril complex are significantly influenced by synemin, but less than by desmin and dystrophin.

Grant Funding Source: Supported by APS Postdoctoral Fellowship Physiological Genomics and CONACyT to KGP. By NIH to RJB
Radiation induced inhibition of myogenesis following muscle damage from cardiotoxin injection (1102.37)
Authors: Cotter Joshua , Baker Michael , Acharya Munjal , Limoli Charles , Caiozzo Vincent
Published: June 2021

Skeletal muscle is routinely exposed to radiation during diagnostic screening and therapeutic management of disease which may inhibit myogenesis. Previously it has been demonstrated in tissue culture that clinical doses of radiation blunt the activation of myogenic stem cells, referred to as “satellite” cells, which are important for the regeneration of skeletal muscle from injury/disease. The objective of the current study was to investigate the effects of radiation on myogenesis in the tibialis anterior (TA) following muscle damage from cardiotoxin (CTX). Adult male mice (C57BL/J6) were assigned to one of four groups: 1) CTX only ‐ 4 days, 2) CTX + irradiation (IRR) ‐ 4 days, 3) CTX only ‐ 14 days, or 4) CTX + IRR ‐ 14 days. Following injection, mice in the irradiation groups were exposed to a dose of 7 Gy of gamma irradiation; a clinically relevant dose of radiation. Following each time period, the TA was harvested and prepared for histological analysis by hematoxylin and eosin staining. The number of regenerative fibers was examined within the zone of damage. Regenerative fibers were identified by those fibers with centralized nuclei. CTX injection resulted in significant damage and subsequent fiber regeneration in all groups. Muscle regeneration was blunted in the CTX+IRR group as fibers with centralized nuclei were not observed until 14 days following CTX injection. Extensive regeneration was evident at 4 days and nearly complete at 14 days in the CTX group. The results indicate that a single, clinically relevant dose of radiation resulted in significant inhibition of myogenesis following muscle degeneration in an in vivo mouse model.
Regional differences in the cutaneous thermal sensitivity to wetness across the torso (1104.10)
Authors: Filingeri Davide , Redortier Bernard , Hodder Simon , Havenith George
Published: June 2021

Humans are not provided with humidity receptors and the ability to perceive skin wetness seems to rely on the sensory inputs arising from thermal cold afferents. Given the regional variability in thermal sensitivity across the torso, we examined whether regional differences in the sensitivity to wetness exist. Sixteen male participants were tested at rest. Twelve regions of the torso were stimulated with a dry or wet thermal probe (25cm²) set at a temperature of 15°C below the local skin temperature, which was measured pre and post stimulation. Thermal and wetness sensations were recorded using likert scales. As a result of the same relative cold stimuli, the skin cooling response varied by location and regional differences were found in thermal and wetness sensations. The lateral chest showed the greater drop in local skin temperature. Colder sensations were reported on the lateral abdomen and lower back. Greater wetness was reported on the lower back. No differences were perceived between wet and dry stimuli, the dry ones perceived as wet as the wet ones. The regional variability in wetness sensitivity showed a pattern similar to the thermal sensitivity to cold. We conclude that, as thermal cold afferents seem to have a critical role in driving the perception of wetness, based on the regional variability in thermal sensitivity, humans present regional differences in the cutaneous sensitivity to wetness across the torso.

Grant Funding Source: Supported by Loughborough University and Oxylane Research
Impaired enteroendocrine cells differentiation signaling pathways through microbiota transfer (1107.12)
Authors: Sakar Yassine , Langelier Benedicte , Devime Fabienne , Covasa Mihai
Published: June 2021

Recently, we showed that susceptibility to obesity is characterized by an unfavorable microbiome and that the gut chemosensory signature can be transferred via microbiota. To examine whether changes in the signaling pathways controlling differentiation of enteroendocrine cells (EEC) are transmitted through gut microbiota, we assessed gene and protein expression of intestinal bHLH transcription factors, in germ free mice conventionalized with microbiota from obese prone and resistant rats. Gut permeability, intestinal lipid and carbohydrates transporters as well as circulating levels and intestinal protein expression of gut peptides were determined. We showed that obese animals exhibited decreased expression of bHLH transcription factors controlling EECs differentiation (MATH1, NGN3, NEUROD1), increased expression of bHLH factors modulating absorptive enterocyte expression. and decreased number of total EECs L‐, I‐ and S‐cells. This was accompanied by increased gut permeability and expression of lipid and carbohydrates transporters. Transfer of obese gut microbiota replicated obesity‐associated decreased in bHLH transcription factors in germ free mice. These findings demonstrate that gut microbiota modulates complex molecular signaling machinery responsible for EECs differentiation pathways. It shows that susceptibility to obesity and associated microbiome impacts EEC differentiation through downregulation of bHLH transcription factors resulting in reduced EEC number and satiety gut hormone levels.
Efficacy of a novel green coffee bean extract (GCB‐70) in weight management (1107.2)
Authors: Swaroop Anand , Bagchi Manashi , Kumar Pawan , Bagchi Debasis
Published: June 2021

Green coffee beans (GCB) are the unroasted beans or raw seeds from the Coffee fruits, which are novel, natural source of a powerful antioxidant, chlorogenic acids, as well as caffeine, caffeic acid, vanillic acid, quinides, lignans and trigonelline. Chlorogenic acids enhance the taste and caffeine solubility. Chlorogenic acid has been demonstrated to reduce the absorption of fat from food intake and improves glucose metabolism, and promotes anti‐aging pathophysiology. Our laboratories have developed a novel, patented GCB extract GCB‐70 enriched with 70% total chlorogenic acids (tCGA) which includes 35% of 5‐caffeoylquinic acid (5‐CQA). The presently available GCB extracts contain only 45% tCGA including 15% 5‐CQA. Studies demonstrated that GCB‐70 is safe, potent, which potentiates antioxidant efficacy against oxidative stress, regulates blood pressure and improve cognitive functions. Furthermore, GCB‐70 accelerates glucose and fat metabolism, and may be an effective and inexpensive means for weight management. These results are very encouraging. Further research studies are in progress.

Grant Funding Source: Company
Compound 21 ameliorates type 2 diabetes in Zucker diabetic fatty rats by islet preservation (1108.13)
Authors: Shao Chunhong , Yu Li , Gao Lie
Published: June 2021

This study aimed to evaluate the therapeutic potential of Compound 21 (C21), a nonpeptide AT2R agonist, on type 2 diabetes in Zucker Diabetic Fatty (ZDF) rats. Rats received C21 infusion (0.3 mg/rat/day; saline as the control) from the age of 4 weeks to 16 weeks. The blood glucose, water intake, and urine excretion were monitored weekly. At the end of C21 treatment, the islet function, morphology, endoplasmic reticulum (ER) stress markers, beta‐cell proliferation markers, and apoptosis were examined by oral glucose tolerance test, immunofluorescence, western blotting, and TUNEL staining. The serum insulin concentration was measured by ELISA. We found that, compared with control ZDF rats, C21‐treated ZDF rats displayed significantly lower blood glucose, water intake, and urine excretion from the day 7 post C21 treatment. These rats also exhibited significantly less islet destruction, elevated serum insulin level (2.45 ± 0.21 vs 1.24 ± 0.04 ng/ml; P < 0.05), and improved glucose tolerance (peak glucose level: 131.5 ± 1.7 vs 219.0 ± 17.0 mg/dl; P < 0.05). In the islets of these rats, we found lower Bip (17.1 ± 1.24 vs 54.2 ± 3.6 %; P < 0.05) and Chop signals, less apoptosis (21.3 ± 1.07 vs 61.0 ± 5.34 %; P < 0.05), and up‐regulated Ki67 expressions. Our data suggested that C21 treatment ameliorated type 2 diabetes by preserving islets through suppressing ER stress, attenuating apoptosis, and promoting proliferation of beta‐cells.

Grant Funding Source: Supported by NIH
AMPK regulates the vacuolar proton‐ATPase via 14‐3‐3 proteins (1109.11)
Authors: Al‐bataineh Mohammad , Gong Fan , Li Hui , Marciszyn Allison , Bhalla Vivek , Hallows Kenneth , Pastor‐Soler Núria
Published: June 2021

The vacuolar proton‐ATPase (V‐ATPase) mediates proton transport across membranes. The V‐ATPase is expressed at the apical membrane of kidney type A collecting duct intercalated cells (ICs). Defects in the V‐ATPase may cause renal tubular acidosis with kidney and systemic complications. In a cell line of IC origin, Clone C cells, we showed that the V‐ATPase A subunit is phosphorylated directly by AMPK at Ser‐384. In addition, AMPK activators induced an acute cytoplasmic redistribution of the V‐ATPase in ICs. AMPK regulates transport proteins by promoting their ubiquitination and degradation, mechanisms that are likely important for inhibition of membrane transport during cellular metabolic stress. We have shown that Ser‐384 is necessary for the AMPK‐mediated cytoplasmic redistribution of the pump. It has been previously shown that the V‐ATPase A subunit binds to 14‐3‐3 proteins (14‐3‐3s), and we noticed that Ser‐384 exists within a ‘mode 1’ 14‐3‐3 binding motif. Dimeric 14‐3‐3s bind to phosphorylated sites on target proteins and thereby modulate protein‐protein interactions. We thus hypothesized that AMPK may promote V‐ATPase A subunit ubiquitination as well as increased binding to 14‐3‐3s. We detected increased co­immunoprecipitation of 14‐3‐3s to FLAG‐tagged A subunit expressed in Clone C ICs treated with the AMPK activator AICAR as compared with untreated cells. Furthermore, AICAR‐treated Clone C ICs had greater A subunit ubiquitination than untreated cells. We propose that A subunit 14‐3‐3 binding and ubiquitination are mediated by AMPK‐dependent Ser‐384 phosphorylation, thus linking regulation of the V­ATPase to AMPK during metabolic depletion in ICs.

Grant Funding Source: Supported by NIH/NIDDK