Downregulated bone morphogenetic protein signaling in nitrofen-induced congenital diaphragmatic hernia.

PubMed

Makanga, Martine; Dewachter, Céline; Maruyama, Hidekazu; Vuckovic, Aline; Rondelet, Benoit; Naeije, Robert; Dewachter, Laurence

2013-08-01

Bone morphogenetic proteins (BMP) have been shown to play crucial roles in not only lung and heart development, but also in the pathogenesis of pulmonary vascular remodeling in pulmonary hypertension (PH). We therefore hypothesized that BMP signaling could be altered in nitrofen-induced congenital diaphragmatic hernia (CDH) and associated PH. Pregnant rats were exposed to either 100 mg nitrofen or vehicle on embryonic day (E) 9.5. On E17 and E21, fetuses were delivered by cesarean section, killed and checked for left-sided CDH. The tissue was then harvested for pathobiological evaluation. In nitrofen-induced CDH, pulmonary expressions of BMP4, BMP receptor (BMPR) type 2 and Id1 decreased on E17 and E21. On E17, pulmonary gremlin-1 expression increased, while BMP7 decreased. In the lungs, Id1 expression was correlated to BMP4 and BMPR2 and inversely correlated to gremlin-1 expression. Myocardial expressions of BMPR2, BMPR1A, BMP7 and SERCA-2A decreased, while gremlin-1 and noggin expressions increased on E17. On E21, myocardial expressions of Id1 and SERCA-2A decreased, while gremlin-1 expression increased. Moreover, BMPR2 and BMPR1A expressions were correlated to SERCA-2A expression and inversely correlated to pro-apoptotic Bax/Bcl2 ratio within the myocardium. Downregulation of BMP signaling seems to contribute to pulmonary and myocardial anomalies observed in nitrofen-induced CDH.

Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) activity and impairs cardiac function in mice

PubMed Central

Asahi, Michio; Otsu, Kinya; Nakayama, Hiroyuki; Hikoso, Shungo; Takeda, Toshihiro; Gramolini, Anthony O.; Trivieri, Maria G.; Oudit, Gavin Y.; Morita, Takashi; Kusakari, Yoichiro; Hirano, Shuta; Hongo, Kenichi; Hirotani, Shinichi; Yamaguchi, Osamu; Peterson, Alan; Backx, Peter H.; Kurihara, Satoshi; Hori, Masatsugu; MacLennan, David H.

2004-01-01

Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) by direct binding and is superinhibitory if it binds through phospholamban (PLN). To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic (TG) mice were generated with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope). The level of NF-SLN expression (the NF-SLN/PLN expression ratio) was equivalent to that which induces profound superinhibition when coexpressed with PLN and SERCA2a in HEK-293 cells. In TG hearts, the apparent affinity of SERCA2a for Ca2+ was decreased compared with non-TG littermate control hearts. Invasive hemodynamic and echocardiographic analyses revealed impaired cardiac contractility and ventricular hypertrophy in TG mice. Basal PLN phosphorylation was reduced. In isolated papillary muscle subjected to isometric tension, peak amplitudes of Ca2+ transients and peak tensions were reduced, whereas decay times of Ca2+ transients and relaxation times of tension were increased in TG mice. Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts. No compensatory changes in expression of SERCA2a, PLN, ryanodine receptor, and calsequestrin were observed in TG hearts. Coimmunoprecipitation indicated that overexpressed NF-SLN was bound to both SERCA2a and PLN, forming a ternary complex. These data suggest that NF-SLN overexpression inhibits SERCA2a through stabilization of SERCA2a–PLN interaction in the absence of PLN phosphorylation and through the inhibition of PLN phosphorylation. Inhibition of SERCA2a impairs contractility and calcium cycling, but responsiveness to β-adrenergic agonists may prevent progression to heart failure. PMID:15201433

Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

PubMed

Tong, X; Kono, T; Evans-Molina, C

2015-06-18

The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered β-cell calcium homeostasis and reduced β-cell survival in diabetes.

Intramyocardial injection of SERCA2a-expressing lentivirus improves myocardial function in doxorubicin-induced heart failure.

PubMed

Mattila, Minttu; Koskenvuo, Juha; Söderström, Mirva; Eerola, Kim; Savontaus, Mikko

2016-07-01

Doxorubicin is an effective anticancer drug. The major limitation to its use is the induction of dose-dependent cardiomyopathy. No specific treatment exists for doxorubicin-induced cardiomyopathy and treatments used for other forms of heart failure have only limited beneficial effects. The contraction-relaxation cycle of the heart is controlled by cytosolic calcium concentrations, which, in turn, are critically regulated by the activity of the sarcoplasmic reticulum Ca(2) (+) ATPase (SERCA2a) pump. We hypothesized that SERCA2a gene transfer would ameliorate doxorubicin-induced cardiomyopathy. Lentiviral vectors LV-SERCA2a-GFP and LV-GFP were constructed and in vitro gene transfer of LV-SERCA2a-GFP confirmed SERCA2a expression by western blot analysis. Heart failure was induced by giving a single intraperitoneal injection of doxorubicin. LV-SERCA2a-GFP, LV-GFP vectors and phosphate-buffered saline (PBS) were injected under echocardiographic control to the anterior wall of the left ventricle. Echocardiography analyses were performed on the injection day and 28 days postinjection. On the injection day, there were no significant differences in the average ejection fractions (EFs) among SERCA2a (40.0%), GFP (41.1%) and PBS (39.4%) injected animals. On day 28, EF in the SERCA2a group had increased by 16.6 ± 6.7% to 46.4 ± 2.1%. By contrast, EFs in the GFP (40.2 ± 1.3%) and PBS (40.6 ± 1.4%) groups remained at pre-injection levels. In addition, end systolic and end diastolic left ventricle volumes were significantly smaller in the SERCA2a group compared to controls. SERCA2a gene transfer significantly improves left ventricle function and dimensions in doxorubicin-induced cardiomyopathy, thus making LV-SERCA2a gene transfer an attractive treatment modality for doxorubicin-induced heart failure. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Pancreatic and Duodenal Homeobox Protein 1 (Pdx-1) Maintains Endoplasmic Reticulum Calcium Levels through Transcriptional Regulation of Sarco-endoplasmic Reticulum Calcium ATPase 2b (SERCA2b) in the Islet β Cell*

PubMed Central

Johnson, Justin S.; Kono, Tatsuyoshi; Tong, Xin; Yamamoto, Wataru R.; Zarain-Herzberg, Angel; Merrins, Matthew J.; Satin, Leslie S.; Gilon, Patrick; Evans-Molina, Carmella

2014-01-01

Although the pancreatic duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in β cell development and secretory function, recent data also implicate Pdx-1 in the maintenance of endoplasmic reticulum (ER) health. The sarco-endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ gradient between the cytosol and ER lumen. In models of diabetes, our data demonstrated loss of β cell Pdx-1 that occurs in parallel with altered SERCA2b expression, whereas in silico analysis of the SERCA2b promoter revealed multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b levels and activity with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. The results revealed reduced SERCA2b expression and decreased ER Ca2+, which was measured using fluorescence lifetime imaging microscopy. Cotransfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity 3- to 4-fold relative to an empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1+/− mice were fed a high-fat diet. Isolated islets demonstrated an increased spliced-to-total Xbp1 ratio, whereas SERCA2b overexpression reduced the Xbp1 ratio to that of wild-type controls. Together, these results identify SERCA2b as a novel transcriptional target of Pdx-1 and define a role for altered ER Ca2+ regulation in Pdx-1-deficient states. PMID:25271154

Transcription of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase type 3 gene, ATP2A3, is regulated by the calcineurin/NFAT pathway in endothelial cells

PubMed Central

Hadri, Lahouaria; Pavoine, Catherine; Lipskaia, Larissa; Yacoubi, Sabrina; Lompré, Anne-Marie

2005-01-01

Histamine, known to induce Ca2+ oscillations in endothelial cells, was used to alter Ca2+ cycling. Treatment of HUVEC (human umbilical-vein endothelial cell)-derived EA.hy926 cells with histamine for 1–3 days increased the levels of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 3, but not of SERCA 2b, transcripts and proteins. Promoter-reporter gene assays demonstrated that this increase in expression was due to activation of SERCA 3 gene transcription. The effect of histamine was abolished by mepyramine, but not by cimetidine, indicating that the H1 receptor, but not the H2 receptor, was involved. The histamine-induced up-regulation of SERCA 3 was abolished by cyclosporin A and by VIVIT, a peptide that prevents calcineurin and NFAT (nuclear factor of activated T-cells) from interacting, indicating involvement of the calcineurin/NFAT pathway. Histamine also induced the nuclear translocation of NFAT. NFAT did not directly bind to the SERCA 3 promoter, but activated Ets-1 (E twenty-six-1), which drives the expression of the SERCA 3 gene. Finally, cells treated with histamine and loaded with fura 2 exhibited an improved capacity in eliminating high cytosolic Ca2+ concentrations, in accordance with an increase in activity of a low-affinity Ca2+-ATPase, like SERCA 3. Thus chronic treatment of endothelial cells with histamine up-regulates SERCA 3 transcription. The effect of histamine is mediated by the H1R (histamine 1 receptor) and involves activation of the calcineurin/NFAT pathway. By increasing the rate of Ca2+ sequestration, up-regulation of SERCA 3 counteracts the cytosolic increase in Ca2+ concentration. PMID:16250893

The CCAAT box in the proximal SERCA2 gene promoter regulates basal and stress-induced transcription in cardiomyocytes.

PubMed

Fragoso-Medina, Jorge; Rodriguez, Gabriela; Zarain-Herzberg, Angel

2018-05-01

The cardiac sarco/endoplasmic reticulum Ca 2+ -ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position -78 bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing -254 and -2579 bp of 5'-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPβ transcription factors to the SERCA2 gene proximal promoter.

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

PubMed

Lalli, M J; Yong, J; Prasad, V; Hashimoto, K; Plank, D; Babu, G J; Kirkpatrick, D; Walsh, R A; Sussman, M; Yatani, A; Marbán, E; Periasamy, M

2001-07-20

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

A protein interaction mechanism for suppressing the mechanosensitive Piezo channels.

PubMed

Zhang, Tingxin; Chi, Shaopeng; Jiang, Fan; Zhao, Qiancheng; Xiao, Bailong

2017-11-27

Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca 2+ ATPase (SERCA), including the widely expressed SERCA2, as Piezo interacting proteins. SERCA2 strategically suppresses Piezo1 via acting on a 14-residue-constituted intracellular linker connecting the pore-module and mechanotransduction-module. Mutating the linker impairs mechanogating and SERCA2-mediated modulation of Piezo1. Furthermore, the synthetic linker-peptide disrupts the modulatory effects of SERCA2, demonstrating the key role of the linker in mechanogating and regulation. Importantly, the SERCA2-mediated regulation affects Piezo1-dependent migration of endothelial cells. Collectively, we identify SERCA-mediated regulation of Piezos and the functional significance of the linker, providing important insights into the mechanogating and regulation mechanisms of Piezo channels.

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes.

PubMed

Meraviglia, Viviana; Bocchi, Leonardo; Sacchetto, Roberta; Florio, Maria Cristina; Motta, Benedetta M; Corti, Corrado; Weichenberger, Christian X; Savi, Monia; D'Elia, Yuri; Rosato-Siri, Marcelo D; Suffredini, Silvia; Piubelli, Chiara; Pompilio, Giulio; Pramstaller, Peter P; Domingues, Francisco S; Stilli, Donatella; Rossini, Alessandra

2018-01-31

SERCA2a is the Ca 2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.

Short-term effects of β2-AR blocker ICI 118,551 on sarcoplasmic reticulum SERCA2a and cardiac function of rats with heart failure.

PubMed

Gong, Haibin; Li, Yanfei; Wang, Lei; Lv, Qian; Wang, Xiuli

2016-09-01

The study was conducted to examine the effects of ICI 118,551 on the systolic function of cardiac muscle cells of rats in heart failure and determine the molecular mechanism of selective β2-adrenergic receptor (β2-AR) antagonist on these cells. The chronic heart failure model for rats was prepared through abdominal aortic constriction and separate cardiac muscle cells using the collagenase digestion method. The rats were then divided into Sham, HF and HF+ICI 50 nM goups and cultivated for 48 h. β2-AR, Gi/Gs and sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) protein expression levels in the cardiac muscle cells were evaluated by western blotting and changes in the systolic function of cardiac muscle cells based on the boundary detection system of contraction dynamics for individual cells was measured. The results showed that compared with the Sham group, the survival rate, percentage of basic contraction and maximum contraction amplitude percentage of cardiac muscle cells with heart failure decreased, Gi protein expression increased while Gs and SERCA2a protein expression decreased. Compared with the HF group, the maximum contraction amplitude percentage of cardiac muscle cells in group HF+ICI 50 nM decreased, the Gi protein expression level increased while the SERCA2a protein expression level decreased. Following the stimulation of Ca 2+ and ISO, the maximum contraction amplitude percentage of cardiac muscle cells in the HF+ICI 50 nM group was lower than that in group HF. This indicated that ICI 118,551 has negative inotropic effects on cardiac muscle cells with heart failure, which may be related to Gi protein. Systolic function of cardiac muscle cells with heart failure can therefore be reduced by increasing Gi protein expression and lowering SERCA2a protein expression.

Influence of infrasound exposure on the whole L-type calcium currents in rat ventricular myocytes.

PubMed

Pei, Zhaohui; Zhuang, Zhiqiang; Xiao, Pingxi; Chen, Jingzao; Sang, Hanfei; Ren, Jun; Wu, Zhenbiao; Yan, Guangmei

2009-06-01

This study was designed to examine the effect of infrasound exposure (5 Hz at 130 dB) on whole-cell L-type Ca2+ currents (WLCC) in rat ventricular myocytes and the underlying mechanism(s) involved. Thirty-two adult Sprague-Dawley rats were randomly assigned to infrasound exposure and control groups. [Ca2+](i), WLCC, mRNA expression of the a(1c) subunit of L-type Ca2+ channels (LCC), and SERCA2 protein were examined on day 1, 7, and 14 after initiation of infrasound exposure. Fluo-3/AM fluorescence and the laser scanning confocal microscope techniques were used to measure [Ca2+](i) in freshly isolated ventricular myocytes. The Ca2+ fluorescence intensity (FI), denoting [Ca2+](i) in cardiomyocytes, was significantly elevated in a time-dependent manner in the exposure groups. There was a significant increase in WLCC in the 1-day group and a further significant increase in the 7- and 14-day groups. LCC mRNA expression measured by RT-PCR revealed a significant rise in the 1-day group and a significant additional rise in the 7- and 14-day groups compared with control group. SERCA2 expression was significantly upregulated in the 1-day group followed by an overt decrease in the 7- and 14-day groups. Prolonged exposure of infrasound altered WLCC in rat cardiomyocytes by shifting the steady-state inactivation curves to the right (more depolarized direction) without altering the slope and biophysical properties of I (Ca,L). Taken together, our data suggest that changes in [Ca2+](I) levels as well as expression of LCC and SERCA2 may contribute to the infrasound exposure-elicited cardiac response.

Modulation of cardiac contractility by the phospholamban/SERCA2a regulatome.

PubMed

Kranias, Evangelia G; Hajjar, Roger J

2012-06-08

Heart disease remains the leading cause of death and disability in the Western world. Current therapies aim at treating the symptoms rather than the subcellular mechanisms, underlying the etiology and pathological remodeling in heart failure. A universal characteristic, contributing to the decreased contractile performance in human and experimental failing hearts, is impaired calcium sequestration into the sarcoplasmic reticulum (SR). SR calcium uptake is mediated by a Ca(2+)-ATPase (SERCA2), whose activity is reversibly regulated by phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA and phosphorylation of PLN relieves this inhibition. However, the initial simple view of a PLN/SERCA regulatory complex has been modified by our recent identification of SUMO, S100 and the histidine-rich Ca-binding protein as regulators of SERCA activity. In addition, PLN activity is regulated by 2 phosphoproteins, the inhibitor-1 of protein phosphatase 1 and the small heat shock protein 20, which affect the overall SERCA-mediated Ca-transport. This review will highlight the regulatory mechanisms of cardiac contractility by the multimeric SERCA/PLN-ensemble and the potential for new therapeutic avenues targeting this complex by using small molecules and gene transfer methods.

Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

PubMed

Kjelstrup, Signe; de Meis, Leopoldo; Bedeaux, Dick; Simon, Jean-Marc

2008-11-01

We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca(2+) in the Ca(2+)-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca(2+)-uptake, and heat up during ATP synthesis and Ca(2+)-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca(2+)-ATPases, not only act as Ca(2+)-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes

PubMed Central

Meraviglia, Viviana; Sacchetto, Roberta; Motta, Benedetta M.; Corti, Corrado; D’Elia, Yuri; Rosato-Siri, Marcelo D.; Suffredini, Silvia; Pompilio, Giulio; Pramstaller, Peter P.; Stilli, Donatella

2018-01-01

SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency. PMID:29385061

MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis.

PubMed

Boštjančič, Emanuela; Zidar, Nina; Glavač, Damjan

2012-10-15

Cardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3'-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI. The protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed. Combing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.

Basal and β-adrenergic cardiomyocytes contractility dysfunction induced by dietary protein restriction is associated with downregulation of SERCA2a expression and disturbance of endoplasmic reticulum Ca2+ regulation in rats.

PubMed

Penitente, Arlete R; Novaes, Rômulo D; Silva, Marcelo E; Silva, Márcia F; Quintão-Júnior, Judson F; Guatimosim, Silvia; Cruz, Jader S; Chianca, Deoclécio A; Natali, Antônio J; Neves, Clóvis A

2014-01-01

The mechanisms responsible for the cardiac dysfunction associated with dietary protein restriction (PR) are poorly understood. Thus, this study was designed to evaluate the effects of PR on calcium kinetics, basal and β-adrenergic contractility in murine ventricular cardiomyocytes. After breastfeeding male Fisher rats were distributed into a control group (CG, n = 20) and a protein-restricted group (PRG, n = 20), receiving isocaloric diets for 35 days containing 15% and 6% protein, respectively. Biometric and hemodynamic variables were measured. After euthanasia left ventricles (LV) were collected for histopathological evaluation, SERCA2a expression, cardiomyocytes contractility and Ca(2+)sparks analysis. PRG animals showed reduced general growth, increased heart rate and arterial pressure. These animals presented extracellular matrix expansion and disorganization, cardiomyocytes hypotrophy, reduced amplitudes of shortening and maximum velocity of contraction and relaxation at baseline and after β-adrenergic stimulation. Reduced SERCA2a expression as well as higher frequency and lower amplitude of Ca(2+)sparks were observed in PRG cardiomyocytes. The observations reveal that protein restriction induces marked myocardial morphofunctional damage. The pathological changes of cardiomyocyte mechanics suggest the potential involvement of the β-adrenergic system, which is possibly associated with changes in SERCA2a expression and disturbances in Ca(2+) intracellular kinetics. © 2014 S. Karger AG, Basel.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toki, Hideaki; Minowa, Osamu; Inoue, Maki

Dominant mutations in the Serca2 gene, which encodes sarco(endo)plasmic reticulum calcium-ATPase, predispose mice to gastrointestinal epithelial carcinoma [1–4] and humans to Darier disease (DD) [14–17]. In this study, we generated mice harboring N-ethyl-N-nitrosourea (ENU)-induced allelic mutations in Serca2: three missense mutations and one nonsense mutation. Mice harboring these Serca2 mutations developed tumors that were categorized as either early onset squamous cell tumors (SCT), with development similar to null-type knockout mice [2,4] (aggressive form; M682, M814), or late onset tumors (mild form; M1049, M1162). Molecular analysis showed no aberration in Serca2 mRNA or protein expression levels in normal esophageal cells ofmore » any of the four mutant heterozygotes. There was no loss of heterozygosity at the Serca2 locus in the squamous cell carcinomas in any of the four lines. The effect of each mutation on Ca{sup 2+}-ATPase activity was predicted using atomic-structure models and accumulated mutated protein studies, suggesting that putative complete loss of Serca2 enzymatic activity may lead to early tumor onset, whereas mutations in which Serca2 retains residual enzymatic activity result in late onset. We propose that impaired Serca2 gene product activity has a long-term effect on squamous cell carcinogenesis from onset to the final carcinoma stage through an as-yet unrecognized but common regulatory pathway. -- Highlights: •Novel mutations in murine Serca2 caused early onset or late onset of tumorigenesis. •They also caused higher or lower incidence of Darier Disease phenotype. •3D structure model suggested the former mutations led to severer defect on ATPase. •Driver gene mutations via long-range effect on Ca2+ distributions are suggested.« less

Recirculating cardiac delivery of AAV2/1SERCA2a improves myocardial function in an experimental model of heart failure in large animals.

PubMed

Byrne, M J; Power, J M; Preovolos, A; Mariani, J A; Hajjar, R J; Kaye, D M

2008-12-01

Abnormal excitation-contraction coupling is a key pathophysiologic component of heart failure (HF), and at a molecular level reduced expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) is a major contributor. Previous studies in small animals have suggested that restoration of SERCA function is beneficial in HF. Despite this promise, the means by which this information might be translated into potential clinical application remains uncertain. Using a recently established cardiac-directed recirculating method of gene delivery, we administered adeno-associated virus 2 (AAV2)/1SERCA2a to sheep with pacing-induced HF. We explored the effects of differing doses of AAV2/1SERCA2a (low 1 x 10(10) d.r.p.; medium 1 x 10(12) d.r.p. and high 1 x 10(13) d.r.p.) in conjunction with an intra-coronary delivery group (2.5 x 10(13) d.r.p.). At the end of the study, haemodynamic, echocardiographic, histopathologic and molecular biologic assessments were performed. Cardiac recirculation delivery of AAV2/1SERCA2a elicited a dose-dependent improvement in cardiac performance determined by left ventricular pressure analysis, (+d P/d t(max); low dose -220+/-70, P>0.05; medium dose 125+/-53, P<0.05; high dose 287+/-104, P<0.05) and echocardiographically (fractional shortening: low dose -3+/-2, P>0.05; medium dose 1+/-2, P>0.05; high dose 6.5+/-3.9, P<0.05). In addition to favourable haemodynamic effects, brain natriuretic peptide expression was reduced consistent with reversal of the HF molecular phenotype. In contrast, direct intra-coronary infusion did not elicit any effect on ventricular function. As such, AAV2/1SERCA2a elicits favourable functional and molecular actions when delivered in a mechanically targeted manner in an experimental model of HF. These observations lay a platform for potential clinical translation.

Concomitant Intravenous Nitroglycerin With Intracoronary Delivery of AAV1.SERCA2a Enhances Gene Transfer in Porcine Hearts

PubMed Central

Karakikes, Ioannis; Hadri, Lahouaria; Rapti, Kleopatra; Ladage, Dennis; Ishikawa, Kiyotake; Tilemann, Lisa; Yi, Geng-Hua; Morel, Charlotte; Gwathmey, Judith K; Zsebo, Krisztina; Weber, Thomas; Kawase, Yoshiaki; Hajjar, Roger J

2012-01-01

SERCA2a gene therapy improves contractile and energetic function of failing hearts and has been shown to be associated with benefits in clinical outcomes, symptoms, functional status, biomarkers, and cardiac structure in a phase 2 clinical trial. In an effort to enhance the efficiency and homogeneity of gene uptake in cardiac tissue, we examined the effects of nitroglycerin (NTG) in a porcine model following AAV1.SERCA2a gene delivery. Three groups of Göttingen minipigs were assessed: (i) group A: control intracoronary (IC) AAV1.SERCA2a (n = 6); (ii) group B: a single bolus IC injection of NTG (50 µg) immediately before administration of intravenous (IV) AAV1.SERCA2a (n = 6); and (iii) group C: continuous IV NTG (1 µg/kg/minute) during the 10 minutes of AAV1.SERCA2a infusion (n = 6). We found that simultaneous IV infusion of NTG and AAV1.SERCA2a resulted in increased viral transduction efficiency, both in terms of messenger RNA (mRNA) as well as SERCA2a protein levels in the whole left ventricle (LV) compared to control animals. On the other hand, IC NTG pretreatment did not result in enhanced gene transfer efficiency, mRNA or protein levels when compared to control animals. Importantly, the transgene expression was restricted to the heart tissue. In conclusion, we have demonstrated that IV infusion of NTG significantly improves cardiac gene transfer efficiency in porcine hearts. PMID:22215018

SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

PubMed Central

Ahmad, Shama; Nichols, David P.; Strand, Matthew; Rancourt, Raymond C.; Randell, Scott H.; White, Carl W.; Ahmad, Aftab

2011-01-01

Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target. PMID:22096575

Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

PubMed Central

Cabello, Nuria; Llach, Anna; Vallmitjana, Alexander; Benítez, Raúl; Badimon, Lina; Cinca, Juan; Llorente-Cortés, Vicenta; Hove-Madsen, Leif

2013-01-01

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal. PMID:23516438

The consequences of long-term glycogen synthase kinase-3 inhibition on normal and insulin resistant rat hearts.

PubMed

Flepisi, T B; Lochner, Amanda; Huisamen, Barbara

2013-10-01

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase, discovered as a regulator of glycogen synthase. GSK-3 may regulate the expression of SERCA-2a potentially affecting myocardial contractility. It is known to phosphorylate and inhibit IRS-1, thus disrupting insulin signalling. This study aimed to determine whether myocardial GSK-3 protein and its substrate proteins are dysregulated in obesity and insulin resistance, and whether chronic GSK-3 inhibition can prevent or reverse this. Weight matched male Wistar rats were rendered obese by hyperphagia using a special diet (DIO) for 16 weeks and compared to chow fed controls. Half of each group was treated with the GSK-3 inhibitor CHIR118637 (30 mg/kg/day) from week 12 to16 of the diet period. Biometric and biochemical parameters were measured and protein expression determined by Western blotting and specific antibodies. Ca(2+)ATPase activity was determined spectrophotometrically. Cardiomyocytes were prepared by collagenase perfusion and insulin stimulated 2-deoxy-glucose uptake determined. DIO rats were significantly heavier than controls, associated with increased intra-peritoneal fat and insulin resistance. GSK-3 inhibition did not affect weight but improved insulin resistance, also on cellular level. It had no effect on GSK-3 expression but elevated its phospho/total ratio and elevated IRS-2 expression. Obesity lowered SERCA-2a expression and activity while GSK-3 inhibition alleviated this. The phospho/total ratio of phospholamban underscored inhibition of SERCA-2a in obesity. In addition, signs of myocardial hypertrophy were observed in treated control rats. GSK-3 inhibition could not reverse all the detrimental effects of obesity but may be harmful in normal rat hearts. It regulates IRS-2, SERCA-2a and phospholamban expression but not IRS-1.

Ca2+ homeostasis in microvascular endothelial cells from an insulin-dependent diabetic model: role of endosomes/lysosomes

NASA Astrophysics Data System (ADS)

Sanka, Shankar C.; Bennett, David C.; Rojas, Jose D.; Tasby, Geraldine B.; Meininger, Cynthia J.; Wu, Guoyao; Wesson, Donald E.; Pfarr, Curtis M.; Martinez-Zaguilan, Raul

2000-04-01

Cytosolic Ca2+ ([Ca2+]cyt) regulates several cellular functions, e.g. cell growth, contraction, secretion, etc. In many cell types, ion homeostasis appears to be coupled with glucose metabolism. In certain cell types, a strict coupling between glycolysis and the activity of Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPases (SERCA) has been suggested. Glucose metabolism is altered in diabetes. We hypothesize that: (1) Ca2+ homeostasis is altered in microvascular endothelial cells from diabetic animals due to the dysfunction of glycolysis coupling the activity of SERCA; (2) endosomal/lysosomal compartments expressing SERCA are involved in the dysfunction associated with diabetes.

Myocardium expression of connexin 43, SERCA2a, and myosin heavy chain isoforms are preserved in nitrofen-induced congenital diaphragmatic hernia rat model.

PubMed

Baptista, Maria João; Recamán, Mónica; Melo-Rocha, Gustavo; Nogueira-Silva, Cristina; Roriz, José-Mário; Soares-Fernandes, João; Gonzaga, Silvia; Santos, Marta; Leite-Moreira, Adelino; Areias, José Carlos; Correia-Pinto, Jorge

2006-09-01

Previous morphological studies had produced controversial results with regard to heart development in congenital diaphragmatic hernia (CDH), whereas a few publications investigated cardiac function and myocardial maturation. Myocardium maturation is associated with age-dependent increasing of gene expression of gap junction protein connexin 43 (Cx43), adenosine triphosphatase of the sarcoplasmic reticulum (SERCA2a), as well as switching of myosin heavy chains (MHCs) from beta to alpha isoforms. Our aim was to evaluate myocardium maturity in nitrofen-induced CDH rat model. Fetuses from dated pregnant Sprague-Dawley rats were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from left ventricle free wall were processed to (i) quantification of messenger RNA (mRNA) of Cx43, SERCA2a, alpha and beta MHC isoforms, as well as beta-actin (housekeeping gene); and (ii) separation of MHC isoforms (alpha and beta isoforms) by sodium dodecyl sulfate polyacrylamide gel electrophoresis silver stained. We demonstrated that there is no difference in myocardial gene expression of Cx43 (control, 1.0 +/- 0.1; nitrofen, 1.1 +/- 0.2; CDH, 1.3 +/- 0.2) and SERCA2a (control, 1.0 +/- 0.1; nitrofen, 0.9 +/- 0.1; CDH, 1.0 +/- 0.2). Myocardial gene expressions of alpha and beta mRNA of MHC isoforms were slightly decreased both in nitrofen and CDH fetuses when compared with control fetuses, but evaluation of the alpha-to-beta ratios of MHC isoforms at protein level revealed no significant differences between CDH and control (control, 16.9 +/- 2.5; CDH, 17.0 +/- 2.0). Myocardial quantification of Cx43 and SERCA2a mRNA, as well as the expression pattern of MHC isoforms at protein levels, was similar in all studied groups. We predict, therefore, that acute heart failure commonly observed in CDH infants might be attributed predominantly to cardiac overload secondary to severe pulmonary hypertension rather than to myocardial immaturity.

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase C674 promotes ischemia- and hypoxia-induced angiogenesis via coordinated endothelial cell and macrophage function.

PubMed

Mei, Yu; Thompson, Melissa D; Shiraishi, Yasunaga; Cohen, Richard A; Tong, Xiaoyong

2014-11-01

Ischemia is a complex phenomenon modulated by the concerted action of several cell types. We have identified that sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase 2 (SERCA 2) cysteine 674 (C674) S-glutathiolation is essential for ischemic angiogenesis, vascular endothelial growth factor (VEGF)-mediated endothelial cell (EC) migration and network formation. A heterozygote SERCA 2 C674S knockin (SKI) mouse shows impaired ischemic blood flow recovery after femoral artery ligation, and its ECs show depleted endoplasmic reticulum (ER) Ca(2+) stores and impaired angiogenic behavior. Here we studied the role of SERCA 2 C674 in the interaction between ECs and macrophages in the context of ischemia and discovered the involvement of the ER stress response protein, ER oxidoreductin-1α (ERO1). In wild type (WT) mice, expression of ERO1 was increased in the ischemic hind limb in vivo, as well as in ECs and macrophages exposed to hypoxia in vitro. The increase in ERO1 to ischemia/hypoxia was less in SKI mice. In WT ECs, both vascular cell adhesion molecule 1 (VCAM1) expression and bone marrow-derived macrophage adhesion to ECs were increased by hypoxia, and both were attenuated in SKI ECs. In WT ECs, ERO1 siRNA blocked hypoxia-induced VCAM1 expression and macrophage adhesion. In WT macrophages, hypoxia also stimulated both ERO1 and VEGF expression, and both were less in SKI macrophages. Compared with conditioned media of hypoxic SKI macrophages, conditioned media from WT macrophages had a greater effect on EC angiogenic behavior, and were blocked by VEGF neutralizing antibody. Taken together, under hypoxic conditions, SERCA 2 C674 and ERO1 enable increased VCAM1 expression and macrophage adhesion to ECs, as well as macrophage VEGF production that, in turn, promote angiogenesis. This study highlights the hitherto unrecognized interaction of two ER proteins, SERCA 2 C674 and ERO1, which mediate the EC and macrophage angiogenic response to ischemia/hypoxia. Copyright © 2014 Elsevier Ltd. All rights reserved.

MALONDIALDEHYDE AND 4-HYDROXYNONENAL ADDUCTS ARE NOT FORMED ON CARDIAC RYANODINE RECEPTOR (RyR2) AND SARCO(ENDO)PLASMIC RETICULUM Ca2+ ATPase (SERCA2) IN DIABETES

PubMed Central

Moore, Caronda J.; Shao, Chun Hong; Nagai, Ryoji; Kutty, Shelby; Singh, Jaipaul; Bidasee, Keshore R.

2013-01-01

Recently, we reported an elevated level of glucose-derived carbonyl adducts on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) in hearts of streptozotocin(STZ)-induced diabetic rats. We also showed these adduct impaired RyR2 and SERCA2 activities, and altered evoked Ca2+ transients. What is less clear is if lipid-derived malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) also chemically react with and impair RyR2 and SERCA2 activities in diabetes. This study used Western blot assays with adduct-specific antibodies and confocal microscopy to assess levels of MDA, 4-HNE, Nε-carboxy(methyl)lysine (CML), pentosidine and pyrraline adducts on RyR2 and SERCA2 and evoked intracellular transient Ca2+ kinetics in myocytes from control, diabetic and treated-diabetic rats. MDA and 4-HNE adducts were not detected on RyR2 and SERCA2 from control or 8 weeks diabetic rats with altered evoked Ca2+ transients. However, CML, pentosidine, and pyrraline adducts were elevated 3–5 fold (p<0.05). Treating diabetic rats with pyridoxamine (a scavenger of reactive carbonyl species, RCS) or aminoguanidine (a mixed reactive oxygen species-reactive carbonyl species scavenger) reduced CML, pentosidine and pyrraline adducts on RyR2 and SERCA2 and blunted SR Ca2+ cycling changes. Treating diabetic rats with the superoxide dismutase mimetic tempol had no impact on MDA and 4-HNE adducts on RyR2 and SERCA2, and on SR Ca2+ cycling. From these data we conclude that lipid-derived MDA and 4-HNE adducts are not formed on RyR2 and SERCA2 in this model of diabetes, and are therefore unlikely to be directly contributing to the SR Ca2+ dysregulation. PMID:23354458

LncRNA ZFAS1 as a SERCA2a Inhibitor to Cause Intracellular Ca2+ Overload and Contractile Dysfunction in a Mouse Model of Myocardial Infarction.

PubMed

Zhang, Ying; Jiao, Lei; Sun, Lihua; Li, Yanru; Gao, Yuqiu; Xu, Chaoqian; Shao, Yingchun; Li, Mengmeng; Li, Chunyan; Lu, Yanjie; Pan, Zhenwei; Xuan, Lina; Zhang, Yiyuan; Li, Qingqi; Yang, Rui; Zhuang, Yuting; Zhang, Yong; Yang, Baofeng

2018-05-11

Ca 2+ homeostasis-a critical determinant of cardiac contractile function-is critically regulated by SERCA2a (sarcoplasmic reticulum Ca 2+ -ATPase 2a). Our previous study has identified ZFAS1 as a new lncRNA biomarker of acute myocardial infarction (MI). To evaluate the effects of ZFAS1 on SERCA2a and the associated Ca 2+ homeostasis and cardiac contractile function in the setting of MI. ZFAS1 expression was robustly increased in cytoplasm and sarcoplasmic reticulum in a mouse model of MI and a cellular model of hypoxia. Knockdown of endogenous ZFAS1 by virus-mediated silencing shRNA partially abrogated the ischemia-induced contractile dysfunction. Overexpression of ZFAS1 in otherwise normal mice created similar impairment of cardiac function as that observed in MI mice. Moreover, at the cellular level, ZFAS1 overexpression weakened the contractility of cardiac muscles. At the subcellular level, ZFAS1 deleteriously altered the Ca 2+ transient leading to intracellular Ca 2+ overload in cardiomyocytes. At the molecular level, ZFAS1 was found to directly bind SERCA2a protein and to limit its activity, as well as to repress its expression. The effects of ZFAS1 were readily reversible on knockdown of this lncRNA. Notably, a sequence domain of ZFAS1 gene that is conserved across species mimicked the effects of the full-length ZFAS1 . Mutation of this domain or application of an antisense fragment to this conserved region efficiently canceled out the deleterious actions of ZFAS1 . ZFAS1 had no significant effects on other Ca 2+ -handling regulatory proteins. ZFAS1 is an endogenous SERCA2a inhibitor, acting by binding to SERCA2a protein to limit its intracellular level and inhibit its activity, and a contributor to the impairment of cardiac contractile function in MI. Therefore, anti- ZFAS1 might be considered as a new therapeutic strategy for preserving SERCA2a activity and cardiac function under pathological conditions of the heart. © 2018 The Authors.

Sarcoplasmic reticulum Ca2+ uptake and leak properties, and SERCA isoform expression, in type I and type II fibres of human skeletal muscle.

PubMed

Lamboley, C R; Murphy, R M; McKenna, M J; Lamb, G D

2014-03-15

The Ca(2+) uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca(2+)] heavily buffered in the pCa range (-log10[Ca(2+)]) 7.3-6.0 for set times and the amount of net SR Ca(2+) accumulation determined from the force response elicited upon emptying the SR of all Ca(2+). Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.6. Maximal Ca(2+) uptake rate was ∼0.18 and ∼0.21 mmol Ca(2+) (l fibre)(-1) s(-1) in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg(2+)] from 1 to 3 mM had no significant effect on the net Ca(2+) uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca(2+) release channels during uptake in either fibre type. Ca(2+) leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca(2+) leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca(2+) uptake in the type I human fibres can account for the higher relative level of SR Ca(2+) loading observed in type I compared to type II fibres, and the SR Ca(2+) leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate.

New insights into SERCA2a gene therapy in heart failure: pay attention to the negative effects of B-type natriuretic peptides.

PubMed

Zhai, Yuting; Luo, Yuanyuan; Wu, Pei; Li, Dongye

2018-05-01

Sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) is a target of interest in gene therapy for heart failure with reduced ejection fraction (HFrEF). However, the results of an important clinical study, the Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial, were controversial. Promising results were observed in the CUPID 1 trial, but the results of the CUPID 2 trial were negative. The factors that caused the controversial results remain unclear. Importantly, enrolled patients were required to have a higher plasma level of B-type natriuretic peptide (BNP) in the CUPID 2 trial. Moreover, BNP was shown to inhibit SERCA2a expression. Therefore, it is possible that high BNP levels interact with treatment effects of SERCA2a gene transfer and accordingly lead to negative results of CUPID 2 trial. From this point of view, effects of SERCA2a gene therapy should be explored in heart failure with preserved ejection fraction, which is characterised by lower BNP levels compared with HFrEF. In this review, we summarise the current knowledge of SERCA2a gene therapy for heart failure, analyse potential interaction between BNP levels and therapeutic effects of SERCA2a gene transfer and provide directions for future research to solve the identified problems. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression

PubMed Central

Zatyka, Malgorzata; Da Silva Xavier, Gabriela; Bellomo, Elisa A.; Leadbeater, Wendy; Astuti, Dewi; Smith, Joel; Michelangeli, Frank; Rutter, Guy A.; Barrett, Timothy G.

2015-01-01

Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca2+ imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca2+ concentration ([Ca2+]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis. PMID:25274773

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy.

PubMed

Gehrig, Stefan M; van der Poel, Chris; Sayer, Timothy A; Schertzer, Jonathan D; Henstridge, Darren C; Church, Jarrod E; Lamon, Severine; Russell, Aaron P; Davies, Kay E; Febbraio, Mark A; Lynch, Gordon S

2012-04-04

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

Resistance Training Regulates Cardiac Function through Modulation of miRNA-214

PubMed Central

Melo, Stéphano Freitas Soares; Barauna, Valério Garrone; Júnior, Miguel Araújo Carneiro; Bozi, Luiz Henrique Marchesi; Drummond, Lucas Rios; Natali, Antônio José; de Oliveira, Edilamar Menezes

2015-01-01

Aims: To determine the effects of resistance training (RT) on the expression of microRNA (miRNA)-214 and its target in sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), and on the morphological and mechanical properties of isolated left ventricular myocytes. Main methods: Male Wistar rats were divided into two groups (n = 7/group): Control (CO) or trained (TR). The exercise-training protocol consisted of: 4 × 12 bouts, 5×/week during 8 weeks, with 80% of one repetition maximum. Key findings: RT increased the left ventricular myocyte width by 15% and volume by 12%, compared with control animals (p < 0.05). The time to half relaxation and time to peak were 8.4% and 4.4% lower, respectively, in cells from TR group as compared to CO group (p < 0.05). RT decreased miRNA-214 level by 18.5% while its target SERCA2a expression were 18.5% higher (p < 0.05). Significance: Our findings showed that RT increases single left ventricular myocyte dimensions and also leads to faster cell contraction and relaxation. These mechanical adaptations may be related to the augmented expression of SERCA2a which, in turn, may be associated with the epigenetic modification of decreased miRNA-214 expression. PMID:25822872

Exposure to low mercury concentration in vivo impairs myocardial contractile function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furieri, Lorena Barros; Fioresi, Mirian; Junior, Rogerio Faustino Ribeiro

2011-09-01

Increased cardiovascular risk after mercury exposure has been described but cardiac effects resulting from controlled chronic treatment are not yet well explored. We analyzed the effects of chronic exposure to low mercury concentrations on hemodynamic and ventricular function of isolated hearts. Wistar rats were treated with HgCl{sub 2} (1st dose 4.6 {mu}g/kg, subsequent dose 0.07 {mu}g/kg/day, im, 30 days) or vehicle. Mercury treatment did not affect blood pressure (BP) nor produced cardiac hypertrophy or changes of myocyte morphometry and collagen content. This treatment: 1) in vivo increased left ventricle end diastolic pressure (LVEDP) without changing left ventricular systolic pressure (LVSP)more » and heart rate; 2) in isolated hearts reduced LV isovolumic systolic pressure and time derivatives, and {beta}-adrenergic response; 3) increased myosin ATPase activity; 4) reduced Na{sup +}-K{sup +} ATPase (NKA) activity; 5) reduced protein expression of SERCA and phosphorylated phospholamban on serine 16 while phospholamban expression increased; as a consequence SERCA/phospholamban ratio reduced; 6) reduced sodium/calcium exchanger (NCX) protein expression and {alpha}-1 isoform of NKA, whereas {alpha}-2 isoform of NKA did not change. Chronic exposure for 30 days to low concentrations of mercury does not change BP, heart rate or LVSP but produces small but significant increase of LVEDP. However, in isolated hearts mercury treatment promoted contractility dysfunction as a result of the decreased NKA activity, reduction of NCX and SERCA and increased PLB protein expression. These findings offer further evidence that mercury chronic exposure, even at small concentrations, is an environmental risk factor affecting heart function. - Highlights: > Unchanges blood pressure, heart rate, systolic pressure. > Increases end diastolic pressure. > Promotes cardiac contractility dysfunction. > Decreases NKA activity, NCX and SERCA, increases PLB protein expression. > Small concentrations constitutes environmental cardiovascular risk factor.« less

Transmembrane helix M6 in sarco(endo)plasmic reticulum Ca(2+)-ATPase forms a functional interaction site with phospholamban. Evidence for physical interactions at other sites.

PubMed

Asahi, M; Kimura, Y; Kurzydlowski, K; Tada, M; MacLennan, D H

1999-11-12

In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), mutation of amino acids on one face of the phospholamban (PLN) transmembrane helix led to loss of PLN inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) molecules. This helical face was proposed to form a site of PLN interaction with a transmembrane helix in SERCA molecules. To determine whether predicted transmembrane helices M4, M5, M6, or M8 in SERCA1a interact with PLN, SERCA1a mutants were co-expressed with wild-type PLN and effects on Ca(2+) dependence of Ca(2+) transport were measured. Wild-type inhibitory interactions shifted apparent Ca(2+) affinity of SERCA1a by an average of -0.34 pCa units, but four of the seven mutations in M4 led to a more inhibitory shift in apparent Ca(2+) affinity, averaging -0.53 pCa units. Seven mutations in M5 led to an average shift of -0.32 pCa units and seven mutations in M8 led to an average shift of -0.30 pCa units. Among 11 mutations in M6, 1, Q791A, increased the inhibitory shift (-0.59 pCa units) and 5, V795A (-0.11), L802A (-0.07), L802V (-0.04), T805A (-0.11), and F809A (-0.12), reduced the inhibitory shift, consistent with the view that Val(795), Leu(802), Thr(805), and Phe(809), located on one face of a predicted M6 helix, form a site in SERCA1a for interaction with PLN. Those mutations in M4, M6, or M8 of SERCA1a that enhanced PLN inhibitory function did not enhance PLN physical association with SERCA1a, but mutants V795A and L802A in M6, which decreased PLN inhibitory function, decreased physical association, as measured by co-immunoprecipitation. In related studies, those PLN mutants that gained inhibitory function also increased levels of co-immunoprecipitation of wild-type SERCA1a and those that lost inhibitory function also reduced association, correlating functional interaction sites with physical interaction sites. Thus, both functional and physical data confirm that PLN interacts with M6 SERCA1a.

Insulin receptor substrate proteins create a link between the tyrosine phosphorylation cascade and the Ca2+-ATPases in muscle and heart.

PubMed

Algenstaedt, P; Antonetti, D A; Yaffe, M B; Kahn, C R

1997-09-19

Following phosphorylation by the insulin receptor kinase, the insulin receptor substrates (IRS)-1 and IRS-2 bind to and activate several Src homology 2 (SH2) domain proteins. To identify novel proteins that interact with IRS proteins in muscle, a human skeletal muscle cDNA expression library was created in the lambdaEXlox system and probed with baculovirus-produced and tyrosine-phosphorylated human IRS-1. One clone of the 10 clones which was positive through three rounds of screening represented the C terminus of the human homologue of the adult fast twitch skeletal muscle Ca2+-ATPase (SERCA1) including the cytoplasmic tail and part of transmembrane region 10. Western blot analysis of extracts of rat muscle demonstrated co-immunoprecipitation of both IRS-1 and IRS-2 with the skeletal muscle Ca2+-ATPase (SERCA1) and the cardiac muscle isoform (SERCA2). In both cases, injection of insulin stimulated a 2- to 6-fold increase in association of which was maximal within 5 min. In primary cultures of aortic smooth muscle cells and C2C12 cells, the insulin-stimulated interaction between IRS proteins and SERCA1 and -2 was dose-dependent with a maximum induction at 100 nM insulin. This interaction was confirmed in a "pull down" experiment using a glutathione S-transferase fusion protein containing the C terminus of the human SERCA isoform and phosphorylated IRS-1 in vitro and could be blocked by a FLVRES-like domain peptide present in the human SERCA sequence. Affinity chromatography of phosphopeptide libraries using the glutathione S-transferase fusion protein of the C terminus of SERCA1 indicated a consensus sequence for binding of XpYGSS; this is identical to potential tyrosine phosphorylation sites at position 431 of human IRS-1 and at position 500 of human IRS-2. In streptozotocin diabetic rats the interaction between IRS proteins and SERCA1 in skeletal muscle and SERCA2 in cardiac muscle was significantly reduced. Taken together, these results indicate that the IRS proteins bind to the Ca2+-ATPase of the sarcoplasmic reticulum in an insulin-regulated fashion, thus creating a potential link between the tyrosine phosphorylation cascade and effects of insulin on calcium.

Mechanisms of Intracellular Calcium Homeostasis in MC3T3-E1 Cells and Bone Tissues of Sprague-Dawley Rats Exposed to Fluoride.

PubMed

Duan, Xiao-qin; Li, Yan-hui; Zhang, Xiu-yun; Zhao, Zhi-tao; Wang, Ying; Wang, Huan; Li, Guang-sheng; Jing, Ling

2016-04-01

Calcium homeostasis of osteoblasts (OBs) has an important role in the physiology and pathology of bone tissue. In order to study the mechanisms of intracellular calcium homeostasis, MC3T3-E1 cells and Sprague-Dawley rats were treated with different concentrations of fluoride. Then, we examined intracellular-free calcium ion ([Ca(2+)]i) in MC3T3-E1 cells as well as mRNA and protein levels of Cav1.2, the main subunit of L-type voltage-dependent calcium channels (VDCCs), Na(+)/Ca(2+) exchange carriers (NCS), and plasma membrane Ca(2+)-ATPase (PMCA), inositol 1,4,5-trisphosphate receptor (IP3R) channels, sarco/endoplasmic reticulum calcium ATPase 2b (SERCA2b)/ATP2A2 in vitro, and rat bone tissues in vivo. Our results showed that [Ca(2+)]i of fluoride-treated OBs increased in a concentration-dependent manner with an increase in the concentration of fluoride. We also found that the low dose of fluoride led to high expression levels of Cav1.2, NCS-1, and PMCA and low expression levels of IP3R and SERCA2b/ATP2A2, while the high dose of fluoride induced an increase in SERCA2b/ATP2A2 levels and decrease in Cav1.2, PMCA, NCS-1, and IP3R levels. These results demonstrate that calcium channels and calcium pumps of plasma and endoplasmic reticulum (ER) membranes keep intracellular calcium homeostasis by regulating Cav1.2, NCS-1, PMCA, IP3R, and SERCA2b/ATP2A2 expression.

The modified Yi qi decoction protects cardiac ischemia-reperfusion induced injury in rats.

PubMed

Yu, Xiao; Zhao, Xiao-Dong; Bao, Rong-Qi; Yu, Jia-Yu; Zhang, Guo-Xing; Chen, Jing-Wei

2017-06-21

To investigate the effects and involved mechanisms of the modified Yi Qi decoction (MYQ) in cardiac ischemia-reperfusion (IR) induced injury. Male Sprague-Dawley rats were subjected to a 30-min coronary arterial occlusion followed by reperfusion, low or high dose decoction of MYQ was administrated orally for 1 week or 1 month. Both in 1 week and 1 month IR rat groups, cardiac function indexes were significantly impaired compared with sham group rats, accompanied with higher ratio of infarct size to risk size, decreased expressions of sodium calcium exchanger (NCX1) and sarcoplasmic reticulum Ca 2+ -ATPase (Serca2a), and different expressions of autophagic proteins, Beclin-1 and LC3. Treatment with MYQ (low or high dose) for 1 week showed no marked beneficial effects on cardiac function and cardiac injury (ratio of infarct size to risk size), although expressions of anti-apoptotic protein, Bcl-2, NCX1 and Serca2a were increased. Treatment with MYQ (low or high dose) for 1 month showed significantly improved effects on cardiac function and cardiac injury (ratio of infarct size to risk size), accompanied with increase of Bcl-2, NCX1 and Serca2a expressions, and decrease of Bax (a pro-apoptotic protein) and Beclin-1 expressions. The results show that MYQ have potential therapeutic effects on IR-induced cardiac injury, which may be through regulation of apoptotic proteins, cytosolic Ca 2+ handling proteins and autophagic proteins signal pathways.

Thyroid hormone regulates muscle function during cold acclimation in zebrafish (Danio rerio).

PubMed

Little, Alexander G; Seebacher, Frank

2013-09-15

Thyroid hormone (TH) is a universal regulator of growth, development and metabolism during cold exposure in mammals. In zebrafish (Danio rerio), TH regulates locomotor performance and metabolism during cold acclimation. The influence of TH on locomotor performance may be via its effect on metabolism or, as has been shown in mammals, by modulating muscle phenotypes. Our aim was to determine whether TH influences muscle phenotypes in zebrafish, and whether this could explain changes in swimming capacity in response to thermal acclimation. We used propylthiouracil and iopanoic acid to induce hypothyroidism in zebrafish over a 3-week acclimation period to either 18 or 28°C. To verify that physiological changes following hypothyroid treatment were in fact due to the action of TH, we supplemented hypothyroid fish with 3,5-diiodothryronine (T2) or 3,5,3'-triiodothyronine (T3). Cold-acclimated fish had significantly greater sustained swimming performance (Ucrit) but not burst speed. Greater Ucrit was accompanied by increased tail beat frequency, but there was no change in tail beat amplitude. Hypothyroidism significantly decreased Ucrit and burst performance, as well as tail beat frequency and SERCA activity in cold-acclimated fish. However, myofibrillar ATPase activity increased in cold-acclimated hypothyroid fish. Hypothyroid treatment also decreased mRNA concentrations of myosin heavy chain fast isoforms and SERCA 1 isoform in cold-acclimated fish. SERCA 1 mRNA increased in warm-acclimated hypothyroid fish, and SERCA 3 mRNA decreased in both cold- and warm-acclimated hypothyroid fish. Supplementation with either T2 or T3 restored Ucrit, burst speed, tail beat frequency, SERCA activity and myosin heavy chain and SERCA 1 and 3 mRNA levels of hypothyroid fish back to control levels. We show that in addition to regulating development and metabolism in vertebrates, TH also regulates muscle physiology in ways that affect locomotor performance in fish. We suggest that the role of TH in modulating SERCA1 expression during cold exposure may have predisposed it to regulate endothermic thermogenesis.

Glutathione adducts on sarcoplasmic/endoplasmic reticulum Ca2+ ATPase Cys-674 regulate endothelial cell calcium stores and angiogenic function as well as promote ischemic blood flow recovery.

PubMed

Thompson, Melissa D; Mei, Yu; Weisbrod, Robert M; Silver, Marcy; Shukla, Praphulla C; Bolotina, Victoria M; Cohen, Richard A; Tong, Xiaoyong

2014-07-18

The sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is key to Ca(2+) homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca(2+) uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca(2+) studies showed decreased nitric oxide (·NO)-induced (45)Ca(2+) uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca(2+) stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca(2+) influx. Adenoviral overexpression of calreticulin, an ER Ca(2+) binding protein, increased ionomycin-releasable stores, VEGF-induced Ca(2+) influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca(2+) homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca(2+) stores. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer.

PubMed

Blackwell, Daniel J; Zak, Taylor J; Robia, Seth L

2016-09-20

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase.

PubMed

Yamamoto, Sachiko; Takehara, Munenori; Ushimaru, Makoto

2017-01-01

SERCA maintains intracellular Ca 2+ homeostasis by sequestering cytosolic Ca 2+ into SR/ER stores. Two primary fatty acid amides (PFAAs), oleamide (18:1 9-cis ) and linoleamide (18:2 9,12-cis ), induce an increase in intracellular Ca 2+ levels, which might be caused by their inhibition of SERCA. Three major SERCA isoforms, rSERCA1a, hSERCA2b, and hSERCA3a, were individually overexpressed in COS-1 cells, and the inhibitory action of PFAAs on Ca 2+ -ATPase activity of SERCA was examined. The Ca 2+ -ATPase activity of each SERCA was inhibited in a concentration-dependent manner strongly by linoleamide (IC 50 15-53μM) and partially by oleamide (IC 50 8.3-34μM). Inhibition by other PFAAs, such as stearamide (18:0) and elaidamide (18:1 9-trans ), was hardly or slightly observed. With increasing dose, linoleamide decreased the apparent affinity for Ca 2+ and the apparent maximum velocity of Ca 2+ -ATPase activity of all SERCAs tested. Oleamide also lowered these values for hSERCA3a. Meanwhile, oleamide uniquely reduced the apparent Ca 2+ affinity of rSERCA1a and hSERCA2b: the reduction was considerably attenuated above certain concentrations of oleamide. The dissociation constants for SERCA interaction varied from 6 to 45μM in linoleamide and from 1.6 to 55μM in oleamide depending on the isoform. Linoleamide and oleamide inhibit SERCA activity in the micromolar concentration range, and in a different manner. Both amides mainly suppress SERCA activity by lowering the Ca 2+ affinity of the enzyme. Our findings imply a novel role of these PFAAs as modulators of intracellular Ca 2+ homeostasis via regulation of SERCA activity. Copyright © 2016 Elsevier B.V. All rights reserved.

Saturation of SERCA's lipid annulus may protect against its thermal inactivation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fajardo, Val Andrew; Center for Bone and Muscle Health, Brock University, St. Catharines, ON; Department of Health Sciences, Brock University, St. Catharines, ON

The sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA) pumps are integral membrane proteins that catalyze the active transport of Ca{sup 2+} into the sarcoplasmic reticulum, thereby eliciting muscle relaxation. SERCA pumps are highly susceptible to oxidative damage, and cytoprotection of SERCA dampens thermal inactivation and is a viable therapeutic strategy in combating diseases where SERCA activity is impaired, such as muscular dystrophy. Here, we sought to determine whether increasing the percent of saturated fatty acids (SFA) within SERCA's lipid annulus through diet could protect SERCA pumps from thermal inactivation. Female Wistar rats were fed either a semi-purified control diet (AIN93G, 7% soybeanmore » oil by weight) or a modified AIN93G diet containing high SFA (20% lard by weight) for 17 weeks. Soleus muscles were extracted and SERCA lipid annulus and activity under thermal stress were analyzed. Our results show that SERCA's lipid annulus is abundant with short-chain (12–14 carbon) fatty acids, which corresponds well with SERCA's predicted bilayer thickness of 21 Å. Under control-fed conditions, SERCA's lipid annulus was already highly saturated (79%), and high-fat feeding did not increase this any further. High-fat feeding did not mitigate the reductions in SERCA activity seen with thermal stress; however, correlational analyses revealed significant and strong associations between % SFA and thermal stability of SERCA activity with greater %SFA being associated with lower thermal inactivation and greater % polyunsaturation and unsaturation index being associated with increased thermal inactivation. Altogether, these findings show that SERCA's lipid annulus may influence its susceptibility to oxidative damage, which could have implications in muscular dystrophy and age-related muscle wasting. - Highlights: • SERCA's lipid annulus in rat soleus was measured after immunoconcentration. • Short fatty acid chains surround SERCA and may ensure optimal hydrophobic matching. • SERCA's annulus is highly saturated in control-fed and high-fat fed rats. • Greater saturation associates with small levels of thermal inactivation. • Greater unsaturation associates with large levels of thermal inactivation.« less

Underlying mechanism of the contractile dysfunction in atrophied ventricular myocytes from a murine model of hypothyroidism.

PubMed

Montalvo, Dolores; Pérez-Treviño, Perla; Madrazo-Aguirre, Katheryne; González-Mondellini, Fabio A; Miranda-Roblero, Hipólito O; Ramonfaur-Gracia, Diego; Jacobo-Antonio, Mariana; Mayorga-Luna, Maritza; Gómez-Víquez, Norma L; García, Noemí; Altamirano, Julio

2018-06-01

Hypothyroidism (Hypo) is a risk factor for cardiovascular diseases, including heart failure. Hypo rapidly induces Ca 2+ mishandling and contractile dysfunction (CD), as well as atrophy and ventricular myocytes (VM) remodeling. Hypo decreases SERCA-to-phospholamban ratio (SERCA/PLB), and thereby contributes to CD. Nevertheless, detailed spatial and temporal Ca 2+ cycling characterization in VM is missing, and contribution of other structural and functional changes to the mechanism underlying Ca 2+ mishandling and CD, as transverse tubules (T-T) remodeling, mitochondrial density (D mit ) and energy availability, is unclear. Therefore, in a rat model of Hypo, we aimed to characterize systolic and diastolic Ca 2+ signaling, T-T remodeling, D mit , citrate synthase (CS) activity and high-energy phosphate metabolites (ATP and phosphocreatine). We confirmed a decrease in SERCA/PLB (59%), which slowed SERCA activity (48%), reduced SR Ca 2+ (19%) and blunted Ca 2+ transient amplitude (41%). Moreover, assessing the rate of SR Ca 2+ release (dRel/dt), we found that early and maximum dRel/dt decreased, and this correlated with staggered Ca 2+ transients. However, dRel/dt persisted during Ca 2+ transient relaxation due to abundant late Ca 2+ sparks. Isoproterenol significantly up-regulated systolic Ca 2+ cycling. T-T were unchanged, hence, cannot explain staggered Ca 2+ transients and altered dRel/dt. Therefore, we suggest that these might be caused by RyR2 clusters desynchronization, due to diminished Ca 2+ -dependent sensitivity of RyR2, which also caused a decrease in diastolic SR Ca 2+ leak. Furthermore, D mit was unchanged and CS activity slightly decreased (14%), however, the ratio phosphocreatine/ATP did not change, therefore, energy deficiency cannot account for Ca 2+ and contractility dysregulation. We conclude that decreased SR Ca 2+ , due to slower SERCA, disrupts systolic RyR2 synchronization, and this underlies CD. Copyright © 2018 Elsevier Ltd. All rights reserved.

CGP-37157 inhibits the sarcoplasmic reticulum Ca²+ ATPase and activates ryanodine receptor channels in striated muscle.

PubMed

Neumann, Jake T; Diaz-Sylvester, Paula L; Fleischer, Sidney; Copello, Julio A

2011-01-01

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca²+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca²+ channels and plasmalemma Na+/Ca²+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca²+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca²+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC₅₀ values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased V(max) of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥ 5 μM) also increased RyR-mediated Ca²+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC(50) values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca²+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca²+ transporters.

CGP-37157 Inhibits the Sarcoplasmic Reticulum Ca2+ ATPase and Activates Ryanodine Receptor Channels in Striated Muscle

PubMed Central

Neumann, Jake T.; Diaz-Sylvester, Paula L.; Fleischer, Sidney

2011-01-01

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca2+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca2+ channels and plasmalemma Na+/Ca2+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca2+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca2+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC50 values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased Vmax of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥5 μM) also increased RyR-mediated Ca2+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC50 values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca2+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca2+ transporters. PMID:20923851

Heat stress responses modulate calcium regulations and electrophysiological characteristics in atrial myocytes.

PubMed

Chen, Yao-Chang; Kao, Yu-Hsun; Huang, Chun-Feng; Cheng, Chen-Chuan; Chen, Yi-Jen; Chen, Shih-Ann

2010-04-01

Heat stress-induced responses change the ionic currents and calcium homeostasis. However, the molecular insights into the heat stress responses on calcium homeostasis remain unclear. The purposes of this study were to examine the mechanisms of heat stress responses on calcium handling and electrophysiological characteristics in atrial myocytes. We used indo-1 fluorimetric ratio technique and whole-cell patch clamp to investigate the intracellular calcium, action potentials, and ionic currents in isolated rabbit single atrial cardiomyocytes with or without (control) exposure to heat stress (43 degrees C, 15 min) 5+/-1 h before experiments. The expressions of sarcoplasmic reticulum ATPase (SERCA2a), and Na(+)-Ca(2+) exchanger (NCX) in the control and heat stress-treated atrial myocytes were evaluated by Western blot and real-time PCR. As compared with control myocytes, the heat stress-treated myocytes had larger sarcoplasmic reticulum calcium content and larger intracellular calcium transient with a shorter decay portion. Heat stress-treated myocytes also had larger L-type calcium currents, transient outward potassium currents, but smaller NCX currents. Heat stress responses increased the protein expressions, SERCA2a, NCX, and heat shock protein. However, heat stress responses did not change the RNA expression of SERCA2a and NCX. In conclusion, heat stress responses change calcium handling through protein but not RNA regulation. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Preferential cytotoxicity of bortezomib toward highly malignant human liposarcoma cells via suppression of MDR1 expression and function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Yamei; Wang, Lingxian; Wang, Lu

2015-02-15

Liposarcoma is the most common soft tissue sarcoma with a high risk of relapse. Few therapeutic options are available for the aggressive local or metastatic disease. Here, we report that the clinically used proteasome inhibitor bortezomib exhibits significantly stronger cytotoxicity toward highly malignant human liposarcoma SW872-S cells compared with its parental SW872 cells, which is accompanied by enhanced activation of apoptotic signaling both in vitro and in vivo. Treatment of cells with Jun-N-terminal kinase (JNK) inhibitor SP60015 or the translation inhibitor cycloheximide ameliorated this enhanced apoptosis. Bortezomib inhibited MDR1 expression and function more effectively in SW872-S cells than in SW872more » cells, indicating that the increased cytotoxicity relies on the degree of proteasome inhibition. Furthermore, the pharmacological or genetic inhibition of sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2, which is highly expressed in SW872-S cells, resulted in partial reversal of cell growth inhibition and increase of MDR1 expression in bortezomib-treated SW872-S cells. These results show that bortezomib exhibits preferential cytotoxicity toward SW872-S cells possibly via highly expressed SERCA2-associated MDR1 suppression and suggest that bortezomib may serve as a potent agent for treating advanced liposarcoma. - Highlights: • We compare the cytotoxicity of different drugs between SW872-S and SW872 cells. • Highly malignant liposarcoma cells SW872-S show hypersensitivity to bortezomib. • Apoptotic signaling is robustly enhanced in bortezomib-treated SW872-S cells. • Bortezomib has strong suppression on MDR1 expression and function in SW872-S cells. • Inhibition of SERCA2 protects SW872-S cells from bortezomib.« less

Atrial SERCA2a Overexpression Has No Affect on Cardiac Alternans but Promotes Arrhythmogenic SR Ca2+ Triggers

PubMed Central

Nassal, Michelle M. J.; Wan, Xiaoping; Laurita, Kenneth R.; Cutler, Michael J.

2015-01-01

Background Atrial fibrillation (AF) is the most common arrhythmia in humans, yet; treatment has remained sub-optimal due to poor understanding of the underlying mechanisms. Cardiac alternans precede AF episodes, suggesting an important arrhythmia substrate. Recently, we demonstrated ventricular SERCA2a overexpression suppresses cardiac alternans and arrhythmias. Therefore, we hypothesized that atrial SERCA2a overexpression will decrease cardiac alternans and arrhythmias. Methods Adult rat isolated atrial myocytes where divided into three treatment groups 1) Control, 2) SERCA2a overexpression (Ad.SERCA2a) and 3) SERCA2a inhibition (Thapsigargin, 1μm). Intracellular Ca2+ was measured using Indo-1AM and Ca2+ alternans (Ca-ALT) was induced with a standard ramp pacing protocol. Results As predicted, SR Ca2+ reuptake was enhanced with SERCA2a overexpression (p< 0.05) and reduced with SERCA2a inhibition (p<0.05). Surprisingly, there was no difference in susceptibility to Ca-ALT with either SERCA2a overexpression or inhibition when compared to controls (p = 0.73). In contrast, SERCA2a overexpression resulted in increased premature SR Ca2+ (SCR) release compared to control myocytes (28% and 0%, p < 0.05) and concomitant increase in SR Ca2+ load (p<0.05). Based on these observations we tested in-vivo atrial arrhythmia inducibility in control and Ad.SERCA2a animals using an esophageal atrial burst pacing protocol. There were no inducible atrial arrhythmias in Ad.GFP (n = 4) animals though 20% of Ad.SERCA2a (n = 5) animals had inducible atrial arrhythmias (p = 0.20). Conclusions Our findings suggest that unlike the ventricle, SERCA2a is not a key regulator of cardiac alternans in the atrium. Importantly, SERCA2a overexpression in atrial myocytes can increase SCR, which may be arrhythmogenic. PMID:26352986

Synergistic interactions of biotic and abiotic environmental stressors on gene expression.

PubMed

Altshuler, Ianina; McLeod, Anne M; Colbourne, John K; Yan, Norman D; Cristescu, Melania E

2015-03-01

Understanding the response of organisms to multiple stressors is critical for predicting if populations can adapt to rapid environmental change. Natural and anthropogenic stressors often interact, complicating general predictions. In this study, we examined the interactive and cumulative effects of two common environmental stressors, lowered calcium concentration, an anthropogenic stressor, and predator presence, a natural stressor, on the water flea Daphnia pulex. We analyzed expression changes of five genes involved in calcium homeostasis - cuticle proteins (Cutie, Icp2), calbindin (Calb), and calcium pump and channel (Serca and Ip3R) - using real-time quantitative PCR (RT-qPCR) in a full factorial experiment. We observed strong synergistic interactions between low calcium concentration and predator presence. While the Ip3R gene was not affected by the stressors, the other four genes were affected in their transcriptional levels by the combination of the stressors. Transcriptional patterns of genes that code for cuticle proteins (Cutie and Icp2) and a sarcoplasmic calcium pump (Serca) only responded to the combination of stressors, changing their relative expression levels in a synergistic response, while a calcium-binding protein (Calb) responded to low calcium stress and the combination of both stressors. The expression pattern of these genes (Cutie, Icp2, and Serca) were nonlinear, yet they were dose dependent across the calcium gradient. Multiple stressors can have complex, often unexpected effects on ecosystems. This study demonstrates that the dominant interaction for the set of tested genes appears to be synergism. We argue that gene expression patterns can be used to understand and predict the type of interaction expected when organisms are exposed simultaneously to natural and anthropogenic stressors.

Different oxidative status and expression of calcium channel components in stress-induced dysfunctional chicken muscle.

PubMed

Xing, T; Zhao, X; Wang, P; Chen, H; Xu, X; Zhou, G

2017-04-01

The objective of this study was to assess the effects of transport stress at high ambient temperatures on the oxidation status and the expression of essential elements responsible for the Ca transport (sarco- (endo-) plasmic reticulum Ca-ATPase (SERCA1) and the ryanodine receptor (RyR) in (PM) muscles of broilers. Briefly, Arbor Acres broiler chickens ( = 112) were randomly categorized into 2 treatments: unstressed control (C) and 0.5 h transport (T). Each treatment consisted of 8 replicates of 7 birds each. Birds were transported according to a designed protocol. PM muscle samples in T group were collected and classified as normal (T-NOR) or pale, soft, and exudative-like (T-PSE) using meat quality parameters. The results indicated that production of corticosterone (CORT) and reactive oxygen species (ROS) increased significantly after transportation ( < 0.05). Thiobarbituric acid reactive substance values and carbonyl contents increased significantly in the T group ( < 0.05). Moreover, the extent of lipid peroxidation and protein oxidation was more severe in the T-PSE group compared to the T-NOR group ( < 0.05). The mRNA and protein expression of SERCA1 and αRyR increased in the T-NOR group but decreased significantly in the T-PSE group compared to the CON group ( < 0.05). The mRNA expression of βRyR was found to be enhanced in the T-NOR group compared to the CON group, whereas there was no difference in the T-PSE group ( < 0.05). The results indicate that short-distance transport of broilers affects their physiological responses and biochemical changes which may lead to different oxidative states and, importantly, to different expressions of SERCA and RyR. These induced changes in abnormal sarcoplasmic Ca homeostasis have significant implications for the development of PSE-like meat.

A transcriptomic approach to search for novel phenotypic regulators in McArdle disease.

PubMed

Nogales-Gadea, Gisela; Consuegra-García, Inés; Rubio, Juan C; Arenas, Joaquin; Cuadros, Marc; Camara, Yolanda; Torres-Torronteras, Javier; Fiuza-Luces, Carmen; Lucia, Alejandro; Martín, Miguel A; García-Arumí, Elena; Andreu, Antoni L

2012-01-01

McArdle disease is caused by lack of glycogen phosphorylase (GP) activity in skeletal muscle. Patients experience exercise intolerance, presenting as early fatigue and contractures. In this study, we investigated the effects produced by a lack of GP on several genes and proteins of skeletal muscle in McArdle patients. Muscle tissue of 35 patients and 7 healthy controls were used to identify abnormalities in the patients' transcriptomic profile using low-density arrays. Gene expression was analyzed for the influence of variables such as sex and clinical severity. Differences in protein expression were studied by immunoblotting and 2D electrophoresis analysis, and protein complexes were examined by two-dimensional, blue native gel electrophoresis (BN-PAGE). A number of genes including those encoding acetyl-coA carboxylase beta, m-cadherin, calpain III, creatine kinase, glycogen synthase (GS), and sarcoplasmic reticulum calcium ATPase 1 (SERCA1), were found to be downregulated in patients. Specifically, compared to controls, GS and SERCA1 proteins were reduced by 50% and 75% respectively; also, unphosphorylated GS and SERCA1 were highly downregulated. On BN-PAGE analysis, GP was present with GS in two muscle protein complexes. Our findings revealed some issues that could be important in understanding the physiological consequences of McArdle disease: (i) SERCA1 downregulation in patients could result in impaired calcium transport in type II (fast-twitch) muscle fibers, leading to early fatigability during exercise tasks involving type II fibers (which mostly use glycolytic metabolism), i.e. isometric exercise, lifting weights or intense dynamic exercise (stair climbing, bicycling, walking at a very brisk pace), (ii) GP and GS were found together in two protein complexes, which suggests a new regulatory mechanism in the activity of these glycogen enzymes.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a).

PubMed

Chen, Jialin; De Raeymaecker, Joren; Hovgaard, Jannik Brøndsted; Smaardijk, Susanne; Vandecaetsbeek, Ilse; Wuytack, Frank; Møller, Jesper Vuust; Eggermont, Jan; De Maeyer, Marc; Christensen, Søren Brøgger; Vangheluwe, Peter

2017-04-28

The Golgi/secretory pathway Ca 2+ /Mn 2+ -transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca 2+ -dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca 2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di- tert -butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a)

PubMed Central

Chen, Jialin; De Raeymaecker, Joren; Hovgaard, Jannik Brøndsted; Smaardijk, Susanne; Vandecaetsbeek, Ilse; Wuytack, Frank; Møller, Jesper Vuust; Eggermont, Jan; De Maeyer, Marc; Christensen, Søren Brøgger; Vangheluwe, Peter

2017-01-01

The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed. PMID:28264934

Muscle RANK is a key regulator of Ca2+ storage, SERCA activity, and function of fast-twitch skeletal muscles.

PubMed

Dufresne, Sébastien S; Dumont, Nicolas A; Boulanger-Piette, Antoine; Fajardo, Val A; Gamu, Daniel; Kake-Guena, Sandrine-Aurélie; David, Rares Ovidiu; Bouchard, Patrice; Lavergne, Éliane; Penninger, Josef M; Pape, Paul C; Tupling, A Russell; Frenette, Jérôme

2016-04-15

Receptor-activator of nuclear factor-κB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Here we show that RANK is also expressed in fully differentiated myotubes and skeletal muscle. Muscle RANK deletion has inotropic effects in denervated, but not in sham, extensor digitorum longus (EDL) muscles preventing the loss of maximum specific force while promoting muscle atrophy, fatigability, and increased proportion of fast-twitch fibers. In denervated EDL muscles, RANK deletion markedly increased stromal interaction molecule 1 content, a Ca(2+)sensor, and altered activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) modulating Ca(2+)storage. Muscle RANK deletion had no significant effects on the sham or denervated slow-twitch soleus muscles. These data identify a novel role for RANK as a key regulator of Ca(2+)storage and SERCA activity, ultimately affecting denervated skeletal muscle function. Copyright © 2016 the American Physiological Society.

Muscle RANK is a key regulator of Ca2+ storage, SERCA activity, and function of fast-twitch skeletal muscles

PubMed Central

Dufresne, Sébastien S.; Dumont, Nicolas A.; Boulanger-Piette, Antoine; Fajardo, Val A.; Gamu, Daniel; Kake-Guena, Sandrine-Aurélie; David, Rares Ovidiu; Bouchard, Patrice; Lavergne, Éliane; Penninger, Josef M.; Pape, Paul C.; Tupling, A. Russell

2016-01-01

Receptor-activator of nuclear factor-κB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Here we show that RANK is also expressed in fully differentiated myotubes and skeletal muscle. Muscle RANK deletion has inotropic effects in denervated, but not in sham, extensor digitorum longus (EDL) muscles preventing the loss of maximum specific force while promoting muscle atrophy, fatigability, and increased proportion of fast-twitch fibers. In denervated EDL muscles, RANK deletion markedly increased stromal interaction molecule 1 content, a Ca2+ sensor, and altered activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) modulating Ca2+ storage. Muscle RANK deletion had no significant effects on the sham or denervated slow-twitch soleus muscles. These data identify a novel role for RANK as a key regulator of Ca2+ storage and SERCA activity, ultimately affecting denervated skeletal muscle function. PMID:26825123

Effect of Phosphorylation on Interactions between Transmembrane Domains of SERCA and Phospholamban.

PubMed

Martin, Peter D; James, Zachary M; Thomas, David D

2018-06-05

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB's TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However-based on these results-at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the previously observed structural shift in the cytoplasmic domain. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Serca2a and Na(+)/Ca(2+) exchanger are involved in left ventricular function following cardiac remodelling of female rats treated with anabolic androgenic steroid.

PubMed

Nascimento, Andrews Marques do; Lima, Ewelyne Miranda de; Brasil, Girlandia Alexandre; Caliman, Izabela Facco; Silva, Josiane Fernandes da; Lemos, Virgínia Soares; Andrade, Tadeu Uggere de; Bissoli, Nazaré Souza

2016-06-15

Anabolic-androgenic steroids are misused, including by women, but little is known about the cardiovascular effects of these drugs on women. To evaluated the effects of nandrolone decanoate (ND) and resistive physical exercise on cardiac contractility in young female rats. Female Wistar rats were separated into 4 groups: C (untrained animals); E (animals were submitted to resistance exercise by jumping in water 5 times per week); ND (animals were treated with ND, 20mg/kg/week for 4weeks); and NDE (trained and treated). The haemodynamic parameters (+dP/dtmax, -dP/dtmin and Tau) were assessed in the left ventricle. The heart was collected for histological analyses and collagen deposition. The gastrocnemius muscle was weighed, and hypertrophy was assessed by the ratio of their weights to gastrocnemius/tibia length. The expression of calcium handling proteins was measured by western blot analysis. ND treatment and physical exercise increased cardiac contractility and relaxation. In addition, ND promoted increases in phospholamban phosphorylated (p-PLB) and isoforms of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a) expression, while resistance exercise increased the phosphorylation of PLB and expression of Na(+)/Ca(2+) exchangers (NCX). Cardiac hypertrophy and collagen deposition were observed after ND treatment. Regulatory components of cytosolic calcium, such as SERCA2a and p-PLB, play important roles in modulating the contractility and relaxation effects of ND in females. Copyright © 2016 Elsevier Inc. All rights reserved.

T-tubule Disruption Promotes Calcium Alternans in Failing Ventricular Myocytes: Mechanistic Insights from Computational Modeling

PubMed Central

Nivala, Michael; Song, Zhen; Weiss, James N.; Qu, Zhilin

2015-01-01

In heart failure (HF), T-tubule (TT) disruption contributes to dyssynchronous calcium (Ca) release and impaired contraction, but its role in arrhythmogenesis remains unclear. In this study, we investigate the mechanisms of TT disruption and other HF remodeling factors on Ca alternans in ventricular myocytes using computer modeling. A ventricular myocyte model with detailed spatiotemporal Ca cycling modeled by a coupled Ca release unit (CRU) network was used, in which the L-type Ca channels and the ryanodine receptor (RyR) channels were simulated by random Markov transitions. TT disruption, which removes the L-type Ca channels from the associated CRUs, results in “orphaned” RyR clusters and thus provides increased opportunity for spark-induced Ca sparks to occur. This effect combined with other HF remodeling factors promoted alternans by two distinct mechanisms: 1) for normal sarco-endoplasmic reticulum Ca ATPase (SERCA) activity, alternans was caused by both CRU refractoriness and coupling. The increased opportunity for spark-induced sparks by TT disruption combined with the enhanced CRU coupling by Ca elevation in the presence or absence of increased RyR leakiness facilitated spark synchronization on alternate beats to promote Ca alternans; 2) for down-regulated SERCA, alternans was caused by the sarcoplasmic reticulum (SR) Ca load-dependent mechanism, independent of CRU refractoriness. TT disruption and increased RyR leakiness shifted and steepened the SR Ca release-load relationship, which combines with down-regulated SERCA to promote Ca alternans. In conclusion, the mechanisms of Ca alternans for normal and down-regulated SERCA are different, and TT disruption promotes Ca alternans by both mechanisms, which may contribute to alternans at different stages of HF. PMID:25450613

T-tubule disruption promotes calcium alternans in failing ventricular myocytes: mechanistic insights from computational modeling.

PubMed

Nivala, Michael; Song, Zhen; Weiss, James N; Qu, Zhilin

2015-02-01

In heart failure (HF), T-tubule (TT) disruption contributes to dyssynchronous calcium (Ca) release and impaired contraction, but its role in arrhythmogenesis remains unclear. In this study, we investigate the effects of TT disruption and other HF remodeling factors on Ca alternans in ventricular myocytes using computer modeling. A ventricular myocyte model with detailed spatiotemporal Ca cycling modeled by a coupled Ca release unit (CRU) network was used, in which the L-type Ca channels and the ryanodine receptor (RyR) channels were simulated by random Markov transitions. TT disruption, which removes the L-type Ca channels from the associated CRUs, results in "orphaned" RyR clusters and thus provides increased opportunity for spark-induced Ca sparks to occur. This effect combined with other HF remodeling factors promoted alternans by two distinct mechanisms: 1) for normal sarco-endoplasmic reticulum Ca ATPase (SERCA) activity, alternans was caused by both CRU refractoriness and coupling. The increased opportunity for spark-induced sparks by TT disruption combined with the enhanced CRU coupling by Ca elevation in the presence or absence of increased RyR leakiness facilitated spark synchronization on alternate beats to promote Ca alternans; 2) for down-regulated SERCA, alternans was caused by the sarcoplasmic reticulum (SR) Ca load-dependent mechanism, independent of CRU refractoriness. TT disruption and increased RyR leakiness shifted and steepened the SR Ca release-load relationship, which combines with down-regulated SERCA to promote Ca alternans. In conclusion, the mechanisms of Ca alternans for normal and down-regulated SERCA are different, and TT disruption promotes Ca alternans by both mechanisms, which may contribute to alternans at different stages of HF. Copyright © 2014 Elsevier Ltd. All rights reserved.

AFos Dissociates Cardiac Myocyte Hypertrophy and Expression of the Pathological Gene Program

PubMed Central

Jeong, Mark Y.; Kinugawa, Koichiro; Vinson, Charles; Long, Carlin S.

2005-01-01

Background Although induction of activator protein-1 (AP-1) transcription factor activity has been observed in cardiac hypertrophy, a direct role for AP-1 in myocardial growth and gene expression remains obscure. Methods and Results Hypertrophy was induced in cultured neonatal rat cardiomyocytes with phenylephrine or overexpression of a constitutively active MAP3K, MKK6. In both treatment groups, induction of the pathological gene profile was observed, ie, expression of β-myosin heavy chain (βMHC), atrial/brain natriuretic peptides (ANP/BNP), and skeletal α-actin (sACT) was increased, whereas expression for α-myosin heavy chain (αMHC) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA) genes was repressed. The role of AP-1 in the hypertrophic phenotype was evaluated with the use of an adenoviral construct expressing a dominant negative mutant of the c-Fos proto-oncogene (AdAFos). Although AFos did not change the myocyte growth response, it abrogated the gene profile to both agonists, including the upregulation of both αMHC and SERCA expression. Conclusions Although c-Fos/AP-1 is necessary for induction of the pathological/fetal gene program, it does not appear to be critical for cardiomyocyte hypertrophy. PMID:15795322

Right ventricular beneficial effects of beta adrenergic receptor kinase inhibitor (betaARKct) gene transfer in a rat model of severe pressure overload.

PubMed

Molina, Ezequiel J; Gupta, Dipin; Palma, Jon; Gaughan, John P; Macha, Mahender

2009-06-01

Heart failure is associated with abnormalities in betaAR cascade regulation, calcium cycling, expression of inflammatory mediators and apoptosis. Adenoviral mediated gene transfer of betaARKct has beneficial indirect effects on these pathologic processes upon the left ventricular myocardium. The concomitant biochemical changes that occur in the right ventricle have not been well characterized. Sprague-Dawley rats underwent aortic banding and were followed by echocardiography. After a decrease in fractional shortening of 25% from baseline, intracoronary injection of adenoviral-betaARKct (n=14) or adenoviral-beta-galactosidase (control, n=13) was performed. Rats were randomly euthanized on post-operative day 7, 14 or 21. Protein analysis including RV myocardial levels of betaARKct, betaARK1, SERCA(2a), inflammatory tissue mediators (IL-1, IL-6 and TNF-alpha), apoptotic markers (bax and bak), and MAP kinases (jnk, p38 and erk) was performed. ANOVA was employed for group comparison. Adenoviral-betaARKct treated animals showed increased expression of betaARKct and decreased levels of betaARK1 compared with controls. This treatment group also demonstrated normalization of SERCA(2a) expression and decreased levels of the inflammatory markers IL-1, IL-6 and TNF-alpha. The pro-apoptotic markers bax and bak were similarly improved. Ventricular levels of the MAP kinase jnk were increased. Differences were most significant 7 days after gene transfer, but the majority of these changes persisted at 21 days. These results suggest that attenuation of the pathologic mechanisms of beta adrenergic receptor desensitization, SERCA(2a) expression, inflammation and apoptosis, not only occur in the left ventricle but also in the right ventricular myocardium after intracoronary gene transfer of betaARKct during heart failure.

Serca2a and Na{sup +}/Ca{sup 2+} exchanger are involved in left ventricular function following cardiac remodelling of female rats treated with anabolic androgenic steroid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nascimento, Andrews Marques do; Lima, Ewelyne Mira

Anabolic-androgenic steroids are misused, including by women, but little is known about the cardiovascular effects of these drugs on women. Aim: To evaluated the effects of nandrolone decanoate (ND) and resistive physical exercise on cardiac contractility in young female rats. Main methods: Female Wistar rats were separated into 4 groups: C (untrained animals); E (animals were submitted to resistance exercise by jumping in water 5 times per week); ND (animals were treated with ND, 20 mg/kg/week for 4 weeks); and NDE (trained and treated). The haemodynamic parameters (+ dP/dt{sub max}, − dP/dt{sub min} and Tau) were assessed in the leftmore » ventricle. The heart was collected for histological analyses and collagen deposition. The gastrocnemius muscle was weighed, and hypertrophy was assessed by the ratio of their weights to gastrocnemius/tibia length. The expression of calcium handling proteins was measured by western blot analysis. Results: ND treatment and physical exercise increased cardiac contractility and relaxation. In addition, ND promoted increases in phospholamban phosphorylated (p-PLB) and isoforms of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a) expression, while resistance exercise increased the phosphorylation of PLB and expression of Na{sup +}/Ca{sup 2+} exchangers (NCX). Cardiac hypertrophy and collagen deposition were observed after ND treatment. Conclusion: Regulatory components of cytosolic calcium, such as SERCA2a and p-PLB, play important roles in modulating the contractility and relaxation effects of ND in females. - Highlights: • ND and resistive exercise enhanced the cardiac function and increased expression of cytosolic calcium regulatory components.« less

PGC-1{alpha} accelerates cytosolic Ca{sup 2+} clearance without disturbing Ca{sup 2+} homeostasis in cardiac myocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Min, E-mail: chenminyx@gmail.com; Yunnan Centers for Diseases Prevention and Control, Kunming 650022; Wang, Yanru

2010-06-11

Energy metabolism and Ca{sup 2+} handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1{alpha}) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1{alpha} in cardiac Ca{sup 2+} signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1{alpha} via adenoviral transduction. Our data shows that overexpressing PGC-1{alpha} improved myocyte contractility without increasing the amplitude of Ca{sup 2+}more » transients, suggesting that myofilament sensitivity to Ca{sup 2+} increased. Interestingly, the decay kinetics of global Ca{sup 2+} transients and Ca{sup 2+} waves accelerated in PGC-1{alpha}-expressing cells, but the decay rate of caffeine-elicited Ca{sup 2+} transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca{sup 2+}-ATPase (SERCA2a), but not Na{sup +}/Ca{sup 2+} exchange (NCX) contribute to PGC-1{alpha}-induced cytosolic Ca{sup 2+} clearance. Furthermore, PGC-1{alpha} induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1{alpha} did not disturb cardiac Ca{sup 2+} homeostasis, because SR Ca{sup 2+} load and the propensity for Ca{sup 2+} waves remained unchanged. These data suggest that PGC-1{alpha} can ameliorate cardiac Ca{sup 2+} cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1{alpha}-calcium handing pathway sheds new light on the role of PGC-1{alpha} in the therapy of cardiac diseases.« less

Contributions of SERCA pump and ryanodine-sensitive stores to presynaptic residual Ca2+

PubMed Central

Scullin, Chessa S.; Partridge, L. Donald

2010-01-01

The presynaptic Ca2+ signal, which triggers vesicle release, disperses to a broadly distributed residual [Ca2+] ([Ca2+]res) that plays an important role in synaptic plasticity. We have previously reported a slowing in the decay timecourse of [Ca2+]res during the second of paired pulses. In this study, we investigated the contributions of organelle and plasma membrane Ca2+ flux pathways to the reduction of effectiveness of [Ca2+]res clearance during short-term plasticity in Schaffer collateral terminals in the CA1 field of the hippocampus. We show that the slowed decay timecourse is mainly the result of a transport-dependent Ca2+ clearance process; that presynaptic caffeine-sensitive Ca2+ stores are not functionally loaded in the unstimulated terminal, but that these stores can effectively take up Ca2+ even during high frequency trains of stimuli; and that a rate limiting step of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) kinetics following the first pulse is responsible for a large portion of the observed slowing of [Ca2+]res clearance during the second pulse. We were able to accurately fit our [Ca2+]res data with a kinetic model based on these observations and this model predicted a reduction in availability of unbound SERCA during paired pulses, but no saturation of Ca2+ buffer in the endoplasmic reticulum. PMID:20153896

SERCA directs cell migration and branching across species and germ layers

PubMed Central

Lansdale, Nick; Navarro, Sonia; Truong, Thai V.; Bower, Dan J.; Featherstone, Neil C.; Connell, Marilyn G.; Al Alam, Denise; Frey, Mark R.; Trinh, Le A.; Fernandez, G. Esteban; Warburton, David; Fraser, Scott E.; Bennett, Daimark; Jesudason, Edwin C.

2017-01-01

ABSTRACT Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding. PMID:28821490

Gestational exposure to diethylstilbestrol alters cardiac structure/function, protein expression and DNA methylation in adult male mice progeny

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haddad, Rami, E-mail: rami.haddad@mail.mcgill.ca; Division of Experimental Medicine, Department of Medicine, McGill University, 850 Sherbrooke Street, Montréal, Québec, Canada H3A 1A2; Kasneci, Amanda, E-mail: amanda.kasneci@mail.mcgill.ca

2013-01-01

Pregnant women, and thus their fetuses, are exposed to many endocrine disruptor compounds (EDCs). Fetal cardiomyocytes express sex hormone receptors making them potentially susceptible to re-programming by estrogenizing EDCs. Diethylstilbestrol (DES) is a proto-typical, non-steroidal estrogen. We hypothesized that changes in adult cardiac structure/function after gestational exposure to the test compound DES would be a proof in principle for the possibility of estrogenizing environmental EDCs to also alter the fetal heart. Vehicle (peanut oil) or DES (0.1, 1.0 and 10.0 μg/kg/da.) was orally delivered to pregnant C57bl/6n dams on gestation days 11.5–14.5. At 3 months, male progeny were left sedentarymore » or were swim trained for 4 weeks. Echocardiography of isoflurane anesthetized mice revealed similar cardiac structure/function in all sedentary mice, but evidence of systolic dysfunction and increased diastolic relaxation after swim training at higher DES doses. The calcium homeostasis proteins, SERCA2a, phospholamban, phospho-serine 16 phospholamban and calsequestrin 2, are important for cardiac contraction and relaxation. Immunoblot analyses of ventricle homogenates showed increased expression of SERCA2a and calsequestrin 2 in DES mice and greater molecular remodeling of these proteins and phospho-serine 16 phospholamban in swim trained DES mice. DES increased cardiac DNA methyltransferase 3a expression and DNA methylation in the CpG island within the calsequestrin 2 promoter in heart. Thus, gestational DES epigenetically altered ventricular DNA, altered cardiac function and expression, and reduced the ability of adult progeny to cardiac remodel when physically challenged. We conclude that gestational exposure to estrogenizing EDCs may impact cardiac structure/function in adult males. -- Highlights: ► Gestational DES changes cardiac SERCA2a and CASQ2 expression. ► Echocardiography identified systolic dysfunction and increased diastolic relaxation. ► DES increased DNMT3a expression and increased CpG DNA methylation. ► DES impacts fetal heart reducing cardiac reserve on challenge in adulthood. ► Fetal heart can be re-programmed by a non-steroidal estrogen.« less

Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation.

PubMed

Betzer, Cristine; Lassen, Louise Berkhoudt; Olsen, Anders; Kofoed, Rikke Hahn; Reimer, Lasse; Gregersen, Emil; Zheng, Jin; Calì, Tito; Gai, Wei-Ping; Chen, Tong; Moeller, Arne; Brini, Marisa; Fu, Yuhong; Halliday, Glenda; Brudek, Tomasz; Aznar, Susana; Pakkenberg, Bente; Andersen, Jens Peter; Jensen, Poul Henning

2018-05-01

Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca 2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca 2+ levels followed by a later Ca 2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro , and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca 2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

From contraction to gene expression: nanojunctions of the sarco/endoplasmic reticulum deliver site- and function-specific calcium signals.

PubMed

Evans, A Mark; Fameli, Nicola; Ogunbayo, Oluseye A; Duan, Jingxian; Navarro-Dorado, Jorge

2016-08-01

Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum" hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2b and RyR1 are preferentially targeted to the sarcoplasmic reticulum (SR) proximal to the plasma membrane (PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. RyR3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of RyR2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear RyR3s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via RyR2 in order to elicit contraction. Data also suggest that unique SERCA and RyR are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.

Improved exercise capacity and reduced systemic inflammation after adenoviral-mediated SERCA-2a gene transfer.

PubMed

Gupta, Dipin; Palma, Jon; Molina, Ezequiel; Gaughan, John P; Long, Walter; Houser, Steven; Macha, Mahender

2008-04-01

We hypothesized that sarcoplasmic reticulum Ca2+ ATPase pump (SERCA-2a) gene delivery would have beneficial effects upon exercise capacity and markers of inflammation in the setting of heart failure. A pressure-overload model of experimental heart failure was used in rats. Following a decrease in fractional shortening of >or=25%, animals underwent intracoronary adenoviral-mediated gene transfection using SERCA-2a. Heart failure animals were randomized to receive the SERCA-2a gene, the beta galactosidase (control) gene, or followed without any further intervention. Exercise and hemodynamic testing were performed, and myocardial and systemic markers of inflammation were assayed after 7 and 21 d. Animals receiving Ad.SERCA-2a showed an increase in exercise tolerance (499.0 +/- 14.9 versus 312.8 +/- 10.5 s, P < 0.0001) relative to Ad.Gal group. Groups treated with Ad.SERCA-2a had significantly decreased serum levels of the inflammatory markers interleukin-1, interleukin-6, and tumor necrosis factor-alpha compared with Ad.Gal-treated animals. Serum levels of atrial natriuretic peptide were decreased in animals receiving Ad.SERCA-2a compared with animals receiving Ad.Gal at day 7 (0.35 +/- 0.03 versus 0.52 +/- 0.11 pg/mL, P = 0.001). Myocardial levels of the proapoptotic protein bax were reduced in Ad.SERCA-2a -treated animals compared with those receiving Ad.Gal at day 7 (protein level/actin: 0.24 +/- 0.05 versus 0.33 +/- 0.04, P = 0.04) and day 21 (protein level/actin: 0.61 +/- 0.04 versus 0.69 +/- 0.01, P = 0.001). Genetic modulation of heart failure using the SERCA-2a gene was associated with improvement in cardiac function and exercise capacity as well as improvements in heart-failure associated inflammatory markers.

Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy.

PubMed

Denmeade, Samuel R; Mhaka, Annastasiah M; Rosen, D Marc; Brennen, W Nathaniel; Dalrymple, Susan; Dach, Ingrid; Olesen, Claus; Gurel, Bora; Demarzo, Angelo M; Wilding, George; Carducci, Michael A; Dionne, Craig A; Møller, Jesper V; Nissen, Poul; Christensen, S Brøgger; Isaacs, John T

2012-06-27

Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP.

PubMed

Henderson, Mark J; Wires, Emily S; Trychta, Kathleen A; Yan, Xiaokang; Harvey, Brandon K

2015-09-06

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Highly efficient and specific modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban.

PubMed

Fechner, H; Suckau, L; Kurreck, J; Sipo, I; Wang, X; Pinkert, S; Loschen, S; Rekittke, J; Weger, S; Dekkers, D; Vetter, R; Erdmann, V A; Schultheiss, H-P; Paul, M; Lamers, J; Poller, W

2007-02-01

Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum Ca(2+) pump (SERCA2a) contributes to cardiac dysfunction in heart failure (HF). PLB downregulation may increase SERCA2a activity and improve cardiac function. Small interfering (si)RNAs mediate efficient gene silencing by RNA interference (RNAi). However, their use for in vivo gene therapy is limited by siRNA instability in plasma and tissues, and by low siRNA transfer rates into target cells. To address these problems, we developed an adenoviral vector (AdV) transcribing short hairpin (sh)RNAs against rat PLB and evaluated its potential to silence the PLB gene and to modulate SERCA2a-mediated Ca(2+) sequestration in primary neonatal rat cardiomyocytes (PNCMs). Over a period of 13 days, vector transduction resulted in stable > 99.9% ablation of PLB-mRNA at a multiplicity of infection of 100. PLB protein gradually decreased until day 7 (7+/-2% left), whereas SERCA, Na(+)/Ca(2+) exchanger (NCX1), calsequestrin and troponin I protein remained unchanged. PLB silencing was associated with a marked increase in ATP-dependent oxalate-supported Ca(2+) uptake at 0.34 microM of free Ca(2+), and rapid loss of responsiveness to protein kinase A-dependent stimulation of Ca(2+) uptake was maintained until day 7. In summary, these results indicate that AdV-derived PLB-shRNA mediates highly efficient, specific and stable PLB gene silencing and modulation of active Ca(2+) sequestration in PNCMs. The availability of the new vector now enables employment of RNAi for the treatment of HF in vivo.

Sarcoendoplasmic reticulum Ca(2+) ATPase. A critical target in chlorine inhalation-induced cardiotoxicity.

PubMed

Ahmad, Shama; Ahmad, Aftab; Hendry-Hofer, Tara B; Loader, Joan E; Claycomb, William C; Mozziconacci, Olivier; Schöneich, Christian; Reisdorph, Nichole; Powell, Roger L; Chandler, Joshua D; Day, Brian J; Veress, Livia A; White, Carl W

2015-04-01

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.

Sarcoendoplasmic Reticulum Ca2+ ATPase. A Critical Target in Chlorine Inhalation–Induced Cardiotoxicity

PubMed Central

Ahmad, Aftab; Hendry-Hofer, Tara B.; Loader, Joan E.; Claycomb, William C.; Mozziconacci, Olivier; Schöneich, Christian; Reisdorph, Nichole; Powell, Roger L.; Chandler, Joshua D.; Day, Brian J.; Veress, Livia A.; White, Carl W.

2015-01-01

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation–induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration–approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia–reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure. PMID:25188881

Coordinate downregulation of CaM kinase II and phospholamban accompanies contractile phenotype transition in the hyperthyroid rabbit soleus.

PubMed

Jiang, M; Xu, A; Jones, D L; Narayanan, N

2004-09-01

This study investigated the effects of l-thyroxine-induced hyperthyroidism on Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaM kinase II)-mediated sarcoplasmic reticulum (SR) protein phosphorylation, SR Ca(2+) pump (Ca(2+)-ATPase) activity, and contraction duration in slow-twitch soleus muscle of the rabbit. Phosphorylation of Ca(2+)-ATPase and phospholamban (PLN) by endogenous CaM kinase II was found to be significantly lower (30-50%) in soleus of the hyperthyroid compared with euthyroid rabbit. Western blotting analysis revealed higher levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) 1 ( approximately 150%) Ca(2+) pump isoform, unaltered levels of SERCA2 Ca(2+) pump isoform, and lower levels of PLN ( approximately 50%) and delta-, beta-, and gamma-CaM kinase II (40 approximately 70%) in soleus of the hyperthyroid rabbit. SR vesicles from hyperthyroid rabbit soleus displayed approximately twofold higher ATP-energized Ca(2+) uptake and Ca(2+)-stimulated ATPase activities compared with that from euthyroid control. The V(max) of Ca(2+) uptake (in nmol Ca(2+).mg SR protein(-1).min(-1): euthyroid, 818 +/- 73; hyperthyroid, 1,649 +/- 90) but not the apparent affinity of the Ca(2+)-ATPase for Ca(2+) (euthyroid, 0.97 +/- 0.02 microM, hyperthyroid, 1.09 +/- 0.04 microM) differed significantly between the two groups. CaM kinase II-mediated stimulation of Ca(2+) uptake by soleus muscle SR was approximately 60% lower in the hyperthyroid compared with euthyroid. Isometric twitch force of soleus measured in situ was significantly greater ( approximately 36%), and the time to peak force and relaxation time were significantly lower ( approximately 30-40%), in the hyperthyroid. These results demonstrate that thyroid hormone-induced transition in contractile properties of the rabbit soleus is associated with coordinate downregulation of the expression and function of PLN and CaM kinase II and selective upregulation of the expression and function of SERCA1, but not SERCA2, isoform of the SR Ca(2+) pump.

Discovery of novel SERCA inhibitors by virtual screening of a large compound library.

PubMed

Elam, Christopher; Lape, Michael; Deye, Joel; Zultowsky, Jodie; Stanton, David T; Paula, Stefan

2011-05-01

Two screening protocols based on recursive partitioning and computational ligand docking methodologies, respectively, were employed for virtual screens of a compound library with 345,000 entries for novel inhibitors of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA), a potential target for cancer chemotherapy. A total of 72 compounds that were predicted to be potential inhibitors of SERCA were tested in bioassays and 17 displayed inhibitory potencies at concentrations below 100 μM. The majority of these inhibitors were composed of two phenyl rings tethered to each other by a short link of one to three atoms. Putative interactions between SERCA and the inhibitors were identified by inspection of docking-predicted poses and some of the structural features required for effective SERCA inhibition were determined by analysis of the classification pattern employed by the recursive partitioning models. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Structural requirements for inhibitory effects of bisphenols on the activity of the sarco/endoplasmic reticulum calcium ATPase

PubMed Central

Woeste, Matthew; Steller, Jeffrey; Hofmann, Emily; Kidd, Taylor; Patel, Rahul; Connolly, Kevin; Jayasinghe, Manori; Paula, Stefan

2013-01-01

Bisphenols (BPs) are a class of small organic compounds with widespread industrial applications. Previous studies have identified several BPs that interfere with the activity of the ion-translocating enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). In order to define the molecular determinants of BP-mediated SERCA inhibition, we conducted enzyme activity assays with rabbit SERCA to determine the inhibitory potencies of 27 commercially available BPs, which were the basis for structure-activity relationships. The most potent BPs inhibited SERCA at low micromolar concentrations and carried at their two phenyl rings multiple non-polar substituents, such as small alkyl groups or halides. Furthermore, the presence of methyl groups or a cyclohexyl group at the central carbon atom connecting the two phenyl moieties correlated with good potencies. For a characterization and visualization of inhibitor/enzyme interactions, molecular docking was performed, which suggested that hydrogen bonding with Asp254 and hydrophobic interactions were the major driving forces for BP binding to SERCA. Calcium imaging studies with a selection of BPs showed that these inhibitors were able to increase intracellular calcium levels in living human cells, a behavior consistent with that of a SERCA inhibitor. PMID:23643898

Sodium Accumulation in SERCA Knockout-Induced Heart Failure

PubMed Central

Li, Liren; Louch, William E.; Niederer, Steven A.; Aronsen, Jan M.; Christensen, Geir; Sejersted, Ole M.; Smith, Nicolas P.

2012-01-01

In cardiomyocytes, a major decrease in the level of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) can severely impair systolic and diastolic functions. In mice with cardiomyocyte-specific conditional excision of the Serca2 gene (SERCA2 KO), end-stage heart failure developed between four and seven weeks after gene deletion combined with [Na+]i elevation and intracellular acidosis. In this study, to investigate the underpinning changes in Ca2+ dynamics and metabolic homeostasis, we developed data-driven mathematical models of Ca2+ dynamics in the ventricular myocytes of the control, four-week, and seven-week SERCA2 knockout (KO) mice. The seven-week KO model showed that elevated [Na+]i was due to increased Na+ influxes through the Na+/Ca2+ exchanger (NCX) and the Na+/H+ exchanger, with the latter exacerbated by intracellular acidosis. Furthermore, NCX upregulation in the seven-week KO model resulted in increased ATP consumption for ion transport. Na+ accumulation in the SERCA KO due to NCX upregulation and intracellular acidosis potentially play a role in the development of heart failure, by initiating a reinforcing cycle involving: a mismatch between ATP demand and supply; an increasingly compromised metabolism; a decreased pHi; and, finally, an even greater [Na+]i elevation. PMID:22824267

Enhanced adsorption of Ca-ATPase containing vesicles on a negatively charged solid-supported-membrane for the investigation of membrane transporters.

PubMed

Sacconi, Alessio; Moncelli, Maria Rosa; Margheri, Giancarlo; Tadini-Buoninsegni, Francesco

2013-11-12

A convenient model system for a biological membrane is a solid-supported membrane (SSM), which consists of a gold-supported alkanethiol|phospholipid bilayer. In combination with a concentration jump method, SSMs have been used for the investigation of several membrane transporters. Vesicles incorporating sarcoplasmic reticulum Ca-ATPase (SERCA) were adsorbed on a negatively charged SSM (octadecanethiol|phosphatidylserine bilayer). The current signal generated by the adsorbed vesicles following an ATP concentration jump was compared to that produced by SERCA-containing vesicles adsorbed on a conventional SSM (octadecanethiol|phosphatidylcholine bilayer). A significantly higher current amplitude was recorded on the serine-based SSM. The adsorption of SERCA-incorporating vesicles on the SSM was then characterized by surface plasmon resonance (SPR). The SPR measurements clearly indicate that in the presence of Ca(2+) and Mg(2+), the amount of adsorbed vesicles on the serine-based SSM is about twice that obtained using the conventional SSM, thereby demonstrating that the higher current amplitude recorded on the negatively charged SSM is correlated with a greater quantity of adsorbed vesicles. The enhanced adsorption of membrane vesicles on the PS-based SSM may be useful to study membrane preparations with a low concentration of transport protein generating small current signals, as in the case of various recombinantly expressed proteins.

Presence of a thapsigargin-sensitive calcium pump in Trypanosoma evansi: Immunological, physiological, molecular and structural evidences.

PubMed

Pérez-Gordones, M C; Serrano, M L; Rojas, H; Martínez, J C; Uzcanga, G; Mendoza, M

2015-12-01

In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca(2+)-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 μM TG or 25 μM 2',5'-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca(2+) and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG. Copyright © 2015 Elsevier Inc. All rights reserved.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats

PubMed Central

Silveira, C.F.S.M.P.; Campos, D.H.S.; Freire, P.P.; Deus, A.F.; Okoshi, K.; Padovani, C.R.; Cicogna, A.C.

2017-01-01

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group. PMID:28423119

Characterization of sarcoplasmic reticulum Ca{sup 2+} ATPase nucleotide binding domain mutants using NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myint, Wazo; Gong, Qingguo; Ahn, Jinwoo

2011-02-04

Research highlights: {yields} Structural consequence by substitution mutations on the isolated SERCA-nucleotide binding (SERCA-N) domain was studied. {yields} The study fills a gap between the previous clinical, physiological, and biochemical data and the molecular basis of SERCA-N. {yields} The E412G mutation, known to be seen in patients with Darier's disease, was found to maintain the active conformation but exhibit reduced protein stability. -- Abstract: Sarcoplasmic reticulum Ca{sup 2+} ATPase (SERCA) is essential for muscle function by transporting Ca{sup 2+} from the cytosol into the sarcoplasmic reticulum through ATP hydrolysis. In this report, the effects of substitution mutations on the isolatedmore » SERCA-nucleotide binding domain (SERCA-N) were studied using NMR. {sup 15}N-{sup 1}H HSQC spectra of substitution mutants at the nucleotide binding site, T441A, R560V, and C561A, showed chemical shift changes, primarily in residues adjacent to the mutation sites, indicating only local effects. Further, the patterns of chemical shift changes upon AMP-PNP binding to these mutants were similar to that of the wild type SERCA-N (WT). In contrast to these nucleotide binding site mutants, a mutant found in patients with Darier's disease, E412G, showed small but significant chemical shift changes throughout the protein and rapid precipitation. However, the AMP-PNP dissociation constant ({approx}2.5 mM) was similar to that of WT ({approx}3.8 mM). These results indicate that the E412G mutant retains its catalytic activity but most likely reduces its stability. Our findings provide molecular insight into previous clinical, physiological, and biochemical observations.« less

High-throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

PubMed Central

Schaaf, Tory M.; Peterson, Kurt C.; Grant, Benjamin D.; Bawaskar, Prachi; Yuen, Samantha; Li, Ji; Muretta, Joseph M.; Gillispie, Gregory D.; Thomas, David D.

2017-01-01

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS. PMID:27899691

Overexpression of calreticulin sensitizes SERCA2a to oxidative stress.

PubMed

Ihara, Yoshito; Kageyama, Kan; Kondo, Takahito

2005-04-22

Calreticulin (CRT), a Ca(2+)-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac disorder in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In this study, the effect of overexpression of CRT on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. The in vitro activity of SERCA2a and uptake of (45)Ca(2+) into isolated microsomes were suppressed by H(2)O(2) in CRT-overexpressing cells compared with controls. Moreover, SERCA2a protein was degraded via a proteasome-dependent pathway following the formation of a complex with CRT under the stress with H(2)O(2). Thus, we conclude that overexpression of CRT enhances the inactivation and degradation of SERCA2a in the cells under oxidative stress, suggesting some pathophysiological functions of CRT in Ca(2+) homeostasis of myocardiac disease.

One Dimensional Finite Element Method Approach to Study Effect of Ryanodine Receptor and Serca Pump on Calcium Distribution in Oocytes

NASA Astrophysics Data System (ADS)

Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2013-11-01

Oocyte is a female gametocyte or germ cell involved in reproduction. Calcium ions (Ca2+) impact nearly all aspects of cellular life as they play an important role in a variety of cellular functions. Calcium ions contributes to egg activation upon fertilization. Since it is the internal stores which provide most of the calcium signal, much attention has been focused on the intracellular channels. There are mainly two types of calcium channels which release calcium from the internal stores to the cytoplasm in many cell types. These channels are IP3-Receptor and Ryanodine Receptor (RyR). Further it is essential to maintain low cytosolic calcium concentration, the cell engages the Serco/Endoplasmic reticulum Ca2+ ATPases (SERCA) present on the ER or SR membrane for the re-uptake of cytosolic calcium at the expense of ATP hydrolysis. In view of above an attempt has been made to study the effect of the Ryanodine receptor (RyR) and the SERCA pump on the calcium distribution in oocytes. The main aim of this paper is to study the calcium concentration in absence and presence of these parameters. The FEM is used to solve the proposed Mathematical model under appreciate initial and boundary conditions. The program has been developed in MATLAB 7.10 for the entire problem to get numerical results.

REACTIVE CARBONYL SPECIES AND THEIR ROLES IN SARCOPLASMIC RETICULUM Ca2+ CYCLING DEFECT IN THE DIABETIC HEART

PubMed Central

Tian, Chengju; Alomar, Fadhel; Moore, Caronda J; Shao, Chun Hong; Kutty, Shelby; Singh, Jaipaul; Bidasee, Keshore R.

2016-01-01

Efficient and rhythmic cardiac contractions depend critically on the adequate and synchronized release of Ca2+ from the sarcoplasmic reticulum (SR) via ryanodine receptor Ca2+ release channels (RyR2) and its reuptake via sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a). It is well established that this orchestrated process becomes compromised in diabetes. What remain incompletely defined are the molecular mechanisms responsible for the dysregulation of RyR2 and SERCA2a in diabetes. Earlier, found elevated levels of carbonyl adducts on RyR2 and SERCA2a isolated from hearts of type 1 diabetic rats and showed the presence of these post-translational modifications compromised their functions. We also showed that these mono- and di-carbonyl reactive carbonyl species (RCS) do not indiscriminately react with all basic amino acid residues on RyR2 and SERCA2a; some residues are more susceptible to carbonylation (modification by RCS) than others. A key unresolved question in the field is which of the many RCS that are upregulated in the heart in diabetes chemically react with RyR2 and SERCA2a? This brief review introduces readers to the field of RCS and their roles in perturbing SR Ca2+ cycling in diabetes. It also provides new experimental evidence that not all RCS that are upregulated in the heart in diabetes chemically react with RyR2 and SERCA2a, methylglyoxal and glyoxal preferentially do. PMID:23430128

Rosiglitazone induces the unfolded protein response, but has no significant effect on cell viability, in monocytic and vascular smooth muscle cells.

PubMed

Caddy, J; Isa, S; Mainwaring, L S; Adam, E; Roberts, A; Lang, D; Morris, R H K; Thomas, A W; Webb, R

2010-10-01

Given the safety concerns expressed over negative cardiovascular outcomes resulting from the clinical use of rosiglitazone, and the view that rosiglitazone exerts PPARγ-independent effects alongside its insulin-sensitising PPARγ-dependent effects, we hypothesised that rosiglitazone may trigger Unfolded Protein Responses (UPRs) due to disruptions in [Ca(2+)](i) homeostasis within two cardiovascular cell types: monocytic (MM6) and vascular smooth muscle (A7r5) cells. In microsomal samples derived from both cell types, pre-incubation with rosiglitazone rapidly (30min) brought about concentration-dependent PPARγ-independent inhibition of Ca(2+)ATPase activity (IC(50) ∼2μM). Fluo-3 fluorimetric data demonstrated in intact cells that 1h treatment with 1 or 10μM rosiglitazone caused Ca(2+) ions to leak into the cytoplasm. Gene expression analysis showed that within 4h of rosiglitazone exposure, the UPR transcription factor XBP-1 was activated (likely due to corresponding ER Ca(2+) depletion), and the UPR target genes BiP and SERCA2b were subsequently upregulated within 24-72h. After 72h 1 or 10μM rosiglitazone treatment, microsomal Ca(2+)ATPase activity increased to >2-fold of that seen in control microsomes, while [Ca(2+)](i) returned to basal, indicating that UPR-triggered SERCA2b upregulation was responsible for enhanced enzymatic Ca(2+) sequestration within the ER. This appeared to be sufficient to replenish ER Ca(2+) stores and restore normal cell physiology, as cell viability levels were not decreased due to rosiglitazone treatment throughout a 2-week study. Thus, incubation with 1-10μM rosiglitazone triggers the UPR, but does not prove cytotoxic, in cells of the cardiovascular system. This observation provides an important contribution to the current debate over the use of rosiglitazone in the clinical treatment of Type-2 Diabetes. Copyright © 2010 Elsevier Inc. All rights reserved.

SERCA2a upregulation ameliorates cellular alternans induced by metabolic inhibition

PubMed Central

Stary, Victoria; Puppala, Dheeraj; Scherrer-Crosbie, Marielle; Dillmann, Wolfgang H.

2016-01-01

Cardiac alternans has been associated with the incidence of ventricular tachyarrhythmias and sudden cardiac death. The aim of this study was to investigate the effect of impaired mitochondrial function in the genesis of cellular alternans and to examine whether modulating the sarcoplasmic reticulum (SR) Ca2+ ameliorates the level of alternans. Cardiomyocytes isolated from control and doxycyline-induced sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a)-upregulated mice were loaded with two different Ca2+ indicators to selectively measure mitochondrial and cytosolic Ca2+ using a custom-made fluorescence photometry system. The degree of alternans was defined as the alternans ratio (AR) [1 − (small Ca2+ intensity)/(large Ca2+ intensity)]. Blocking of complex I and II, cytochrome-c oxidase, F0F1 synthase, α-ketoglutarate dehydrogenase of the electron transport chain, increased alternans in both control and SERCA2a mice (P < 0.01). Changes in AR in SERCA2a-upregulated mice were significantly less pronounced than those observed in control in seven of nine tested conditions (P < 0.04). N-acetyl-l-cysteine (NAC), rescued alternans in myocytes that were previously exposed to an oxidizing agent (P < 0.001). CGP, an antagonist of the mitochondrial Na+-Ca2+ exchanger, had the most severe effect on AR. Exposure to cyclosporin A, a blocker of the mitochondrial permeability transition pore reduced CGP-induced alternans (P < 0.0001). The major findings of this study are that impairment of mitochondrial Ca2+ cycling and energy production leads to a higher amplitude of alternans in both control and SERCA2a-upregulated mice, but changes in SERCA2a-upregulated mice are less severe, indicating that SERCA2a mice are more capable of sustaining electrical stability during stress. This suggests a relationship between sarcoplasmic Ca2+ content and mitochondrial dysfunction during alternans, which may potentially help to understand changes in Ca2+ signaling in myocytes from diseased hearts, leading to new therapeutic targets. PMID:26846549

The Change of Left Ventricular Function in Rats with Subclinical Hypothyroid and the Effects of Thyroxine Replacement.

PubMed

Chen, Xuedi; Gao, Cuixia; Gong, Ningning; Wang, Yu; Tian, Limin

2018-01-01

The main purpose of this study was to explore the relationships between serca2a, Ryr2, adipokines, and the left ventricular function in the subclinical hypothyroidism with different TSH levels and to determine the impact of L-T4 treatment on these indexes. Sixty-five male Wistar rats were randomly divided into five groups: control group; sHT A, B, and C group; and sHT + T4 group. The sHT rats were induced by methimazole (MMI), and the sHT + T4 rats were administered with L-T4 treatment after 8 weeks of MMI administration. Serum TT4, TSH, APN, chemerin, and TNF- α were detected by radioimmunoassay kits and ELISA kits; left ventricular function was measured by PowerLab system via subclavian artery catheter. The expression of Serca2a, Ryr2, APN, chemerin, and TNF- α were detected by RT-PCR, Western blot, and immunohistochemistry. The sHT groups had significantly higher TSH, chemerin, and TNF- α and lower Serca2a, Ryr2, and APN. The left ventricular pressure and heart rate in sHT groups were significantly lower in control and sHT + T4 group. Histopathological examination revealed the pathological changes in the sHT rats' heart. L-T4 administration reduced TSH level and improved left ventricular function. TSH can impair left ventricular function by regulating several factors, and L-T4 treatment ameliorates it in sHT rats.

Artemisinin dimer anti-cancer activity correlates with heme-catalyzed ROS generation and ER stress induction

PubMed Central

Stockwin, Luke H.; Han, Bingnan; Yu, Sherry X.; Hollingshead, Melinda G.; ElSohly, Mahmoud A.; Gul, Waseem; Slade, Desmond; Galal, Ahmed M.; Newton, Dianne L.

2009-01-01

Analogs of the malaria therapeutic, artemisinin, possess in vitro and in vivo anti-cancer activity. In this study, two dimeric artemisinins (NSC724910 and 735847) were studied to determine their mechanism of action. Dimers were >1000 fold more active than monomer and treatment was associated with increased reactive oxygen species (ROS) and apoptosis induction. Dimer activity was inhibited by the anti-oxidant L-NAC, the iron chelator desferroxamine, and exogenous hemin. Similarly, induction of heme oxygenase (HMOX) with CoPPIX inhibited activity while inhibition of HMOX with SnPPIX enhanced it. These results emphasize the importance of iron, heme and ROS in activity. Microarray analysis of dimer treated cells identified DNA damage; iron/heme and cysteine/methionine metabolism, antioxidant response, and endoplasmic reticulum (ER) stress as affected pathways. Detection of an ER-stress response was relevant because in malaria, artemisinin inhibits pfATP6, the plasmodium orthologue of mammalian ER-resident SERCA Ca2+-ATPases. A comparative study of NSC735847 with thapsigargin, a specific SERCA inhibitor and ER-stress inducer showed similar behavior in terms of transcriptomic changes, induction of endogenous SERCA and ER calcium mobilization. However, thapsigargin had little effect on ROS production, modulated different ER-stress proteins and had greater potency against purified SERCA1. Furthermore, an inactive derivative of NSC735847 that lacked the endoperoxide had identical inhibitory activity against purified SERCA1, suggesting that direct inhibition of SERCA has little inference on overall cytotoxicity. In summary, these data implicate indirect ER-stress induction as a central mechanism of artemisinin dimer activity. PMID:19533749

Memantine can improve chronic ethanol exposure-induced spatial memory impairment in male C57BL/6 mice by reducing hippocampal apoptosis.

PubMed

Wang, Xiaolong; Yu, Hao; You, Jiabin; Wang, Changliang; Feng, Chunmei; Liu, Zhaodi; Li, Ya; Wei, Rucheng; Xu, Siqi; Zhao, Rui; Wu, Xu; Zhang, Guohua

2018-05-22

Chronic ethanol intake can induce neuronal apoptosis, leading to dementia. We investigated the protective effects of memantine on spatial memory impairment induced by chronic ethanol exposure in mice. Male C57BL/6 mice were administered 10% (m/V) or 20% (m/V) ethanol as the only choice of drinking water. Mice were treated for 60 d, 90 d, or 180 d. Mice were treated with memantine for the same duration (daily 10 mg/kg oral). The Morris water maze and radial arm maze test were used to measure spatial memory. Mice were sacrificed after the behavioral tests. Brains were removed to prepare for paraffin sections, and hippocampi were isolated for protein and RNA extraction. 4',6-diamidino-2-phenylindole (DAPI) staining and immunohistochemical staining of cleaved caspase-3 were performed. Western blot analysis was used to detect the expression of cleaved caspase-3 and calcium-related proteins, including N-methyl-d-aspartic acid receptor 1 (NR1), 1,4,5-trisphosphate receptor 1 (IP3R1), and sarco/endoplasmic reticulum calcium adenosine triphosphatase 1 (SERCA1). The changes of NR1, IP3R1 and SERCA1 mRNA were detected using quantitative polymerase chain reaction (qPCR). The results revealed that chronic ethanol exposure induced spatial memory impairment in mice, as well as increasing the expression of NR1, IP3R1 and SERCA1, the activation of caspase-3 and apoptosis in hippocampus. The effect was particularly prominent in the 20% ethanol group after 180 d exposure. Memantine decreased ethanol-induced spatial memory impairment, caspase-3 activation and apoptosis in the mouse hippocampus. These results suggest that disruption of intracellular calcium balance by ethanol can induce caspase-3 activation and apoptosis, which underlies subsequent spatial memory impairment in mice. Copyright © 2018 Elsevier B.V. All rights reserved.

Tracing Cytoplasmic Ca2+ Ion and Water Access Points in the Ca2+-ATPase

PubMed Central

Musgaard, Maria; Thøgersen, Lea; Schiøtt, Birgit; Tajkhorshid, Emad

2012-01-01

Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) transports two Ca2+ ions across the membrane of the sarco(endo)plasmic reticulum against the concentration gradient, harvesting the required energy by hydrolyzing one ATP molecule during each transport cycle. Although SERCA is one of the best structurally characterized membrane transporters, it is still largely unknown how the transported Ca2+ ions reach their transmembrane binding sites in SERCA from the cytoplasmic side. Here, we performed extended all-atom molecular dynamics simulations of SERCA. The calculated electrostatic potential of the protein reveals a putative mechanism by which cations may be attracted to and bind to the Ca2+-free state of the transporter. Additional molecular dynamics simulations performed on a Ca2+-bound state of SERCA reveal a water-filled pathway that may be used by the Ca2+ ions to reach their buried binding sites from the cytoplasm. Finally, several residues that are involved in attracting and guiding the cations toward the possible entry channel are identified. The results point to a single Ca2+ entry site close to the kinked part of the first transmembrane helix, in a region loaded with negatively charged residues. From this point, a water pathway outlines a putative Ca2+ translocation pathway toward the transmembrane ion-binding sites. PMID:22339863

The Ca2+-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum.

PubMed

Espinoza-Fonseca, L Michel

2017-03-28

Ca 2+ transport across the sarco/endoplasmic reticulum (SR) plays an essential role in intracellular Ca 2+ homeostasis, signalling, cell differentiation and muscle contractility. During SR Ca 2+ uptake and release, proton fluxes are required to balance the charge deficit generated by the exchange of Ca 2+ and other ions across the SR. During Ca 2+ uptake by the SR Ca 2+ -ATPase (SERCA), two protons are countertransported from the SR lumen to the cytosol, thus partially compensating for the charge moved by Ca 2+ transport. Studies have shown that protons are also transported from the cytosol to the lumen during Ca 2+ release, but a transporter that facilitates proton transport into the SR lumen has not been described. In this article we propose that SERCA forms pores that facilitate bidirectional proton transport across the SR. We describe the location and structure of water-filled pores in SERCA that form cytosolic and luminal pathways for protons to cross the SR membrane. Based on this structural information, we suggest mechanistic models for proton translocation to the cytosol during active Ca 2+ transport, and into the SR lumen during SERCA inhibition by endogenous regulatory proteins. Finally, we discuss the physiological consequences of SERCA-mediated bidirectional proton transport across the SR membrane of muscle and non-muscle cells.

Protective effects of circulating microvesicles derived from myocardial ischemic rats on apoptosis of cardiomyocytes in myocardial ischemia/reperfusion injury.

PubMed

Wang, Yao; Wei, Su; Wang, Yi-Lu; Liu, Miao; Shang, Man; Zhang, Qi; Wu, Yan-Na; Liu, Ming-Lin; Song, Jun-Qiu; Liu, Yan-Xia

2017-08-15

To investigate the effects of circulating microvesicles derived from myocardial ischemia (I-MVs) on apoptosis in myocardial ischemia/reperfusion (I/R) injury in rats. I-MVs from rats undergoing myocardial left anterior descending (LAD) coronary artery ligation were isolated by ultracentrifugation from circulating blood and characterized by flow cytometry. I-MVs were administered intravenously (4.8 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min of ischemia and 120-min of reperfusion of LAD in rats. Treatment with I-MVssignificantly reduced the size of myocardial infarction, the activities of serum CK-MB and LDH, and the number of apoptotic cardiomyocytes. The activities of caspase 3, caspase 9 and caspase 12 in myocardium were also decreased significantly with I-MVs treatment. Moreover, the expression of Bax was decreased but Bcl-2 was increased. The expression of glucose regulated protein 78 (GRP78), sarco/endoplasmic reticulum Ca 2+ -ATPase 2 (SERCA2) and phosphorylated phospholamban (p-PLB) were increased after being treated with I-MVs. I-MVs could protect hearts from I/R injury in rats through SERCA2 and p-PLB of calcium regulatory proteins to alleviate intrinsic myocardial apoptosis including mitochondrial and endoplasmic reticulum pathways.

SERCA2a upregulation ameliorates cellular alternans induced by metabolic inhibition.

PubMed

Stary, Victoria; Puppala, Dheeraj; Scherrer-Crosbie, Marielle; Dillmann, Wolfgang H; Armoundas, Antonis A

2016-04-15

Cardiac alternans has been associated with the incidence of ventricular tachyarrhythmias and sudden cardiac death. The aim of this study was to investigate the effect of impaired mitochondrial function in the genesis of cellular alternans and to examine whether modulating the sarcoplasmic reticulum (SR) Ca(2+)ameliorates the level of alternans. Cardiomyocytes isolated from control and doxycyline-induced sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a)-upregulated mice were loaded with two different Ca(2+)indicators to selectively measure mitochondrial and cytosolic Ca(2+)using a custom-made fluorescence photometry system. The degree of alternans was defined as the alternans ratio (AR) [1 - (small Ca(2+)intensity)/(large Ca(2+)intensity)]. Blocking of complex I and II, cytochrome-coxidase, F0F1synthase, α-ketoglutarate dehydrogenase of the electron transport chain, increased alternans in both control and SERCA2a mice (P< 0.01). Changes in AR in SERCA2a-upregulated mice were significantly less pronounced than those observed in control in seven of nine tested conditions (P< 0.04).N-acetyl-l-cysteine (NAC), rescued alternans in myocytes that were previously exposed to an oxidizing agent (P< 0.001). CGP, an antagonist of the mitochondrial Na(+)-Ca(2+)exchanger, had the most severe effect on AR. Exposure to cyclosporin A, a blocker of the mitochondrial permeability transition pore reduced CGP-induced alternans (P< 0.0001). The major findings of this study are that impairment of mitochondrial Ca(2+)cycling and energy production leads to a higher amplitude of alternans in both control and SERCA2a-upregulated mice, but changes in SERCA2a-upregulated mice are less severe, indicating that SERCA2a mice are more capable of sustaining electrical stability during stress. This suggests a relationship between sarcoplasmic Ca(2+)content and mitochondrial dysfunction during alternans, which may potentially help to understand changes in Ca(2+)signaling in myocytes from diseased hearts, leading to new therapeutic targets. Copyright © 2016 the American Physiological Society.

3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

PubMed

Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

2012-05-01

3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA. Copyright © 2012 Elsevier Ltd. All rights reserved.

Hydrophobic imbalance in the cytoplasmic domain of phospholamban is a determinant for lethal dilated cardiomyopathy.

PubMed

Ceholski, Delaine K; Trieber, Catharine A; Young, Howard S

2012-05-11

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) and its regulatory partner phospholamban (PLN) are essential for myocardial contractility. Arg(9) → Cys (R9C) and Arg(14) deletion (R14del) mutations in PLN are associated with lethal dilated cardiomyopathy in humans. To better understand these mutations, we made a series of amino acid substitutions in the cytoplasmic domain of PLN and tested their ability to inhibit SERCA. R9C is a complete loss-of-function mutant of PLN, whereas R14del is a mild loss-of-function mutant. When combined with wild-type PLN to simulate heterozygous conditions, the mutants had a dominant negative effect on SERCA function. A series of targeted mutations in this region of the PLN cytoplasmic domain ((8)TRSAIRR(14)) demonstrated the importance of hydrophobic balance in proper PLN regulation of SERCA. We found that Arg(9) → Leu and Thr(8) → Cys substitutions mimicked the behavior of the R9C mutant, and an Arg(14) → Ala substitution mimicked the behavior of the R14del mutant. The results reveal that the change in hydrophobicity resulting from the R9C and R14del mutations is sufficient to explain the loss of function and persistent interaction with SERCA. Hydrophobic imbalance in the cytoplasmic domain of PLN appears to be a predictor for the development and progression of dilated cardiomyopathy.

Increased thrombospondin-4 after nerve injury mediates disruption of intracellular calcium signaling in primary sensory neurons

PubMed Central

Guo, Yuan; Zhang, Zhiyong; Wu, Hsiang-en; Luo, Z. David; Hogan, Quinn H.; Pan, Bin

2017-01-01

Painful nerve injury disrupts Ca2+ signaling in primary sensory neurons by elevating plasma membrane Ca2+-ATPase (PMCA) function and depressing sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) function, which decreases endoplasmic reticulum (ER) Ca2+ stores and stimulates store-operated Ca2+ entry (SOCE). The extracellular matrix glycoprotein thrombospondin-4 (TSP4), which is increased after painful nerve injury, decreases Ca2+ current (ICa) through high-voltage–activated Ca2+ channels and increases ICa through low-voltage–activated Ca2+ channels in dorsal root ganglion neurons, which are events similar to the effect of nerve injury. We therefore examined whether TSP4 plays a critical role in injury-induced disruption of intracellular Ca2+ signaling. We found that TSP4 increases PMCA activity, inhibits SERCA, depletes ER Ca2+ stores, and enhances store-operated Ca2+ influx. Injury-induced changes of SERCA and PMCA function are attenuated in TSP4 knock-out mice. Effects of TSP4 on intracellular Ca2+ signaling are attenuated in voltage-gated Ca2+ channel α2δ1 subunit (Cavα2δ1) conditional knock-out mice and are also Protein Kinase C (PKC) signaling dependent. These findings suggest that TSP4 elevation may contribute to the pathogenesis of chronic pain following nerve injury by disrupting intracellular Ca2+ signaling via interacting with the Cavα2δ1 and the subsequent PKC signaling pathway. Controlling TSP4 mediated intracellular Ca2+ signaling in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain. PMID:28232180

Urotensin II induction of neonatal cardiomyocyte hypertrophy involves the CaMKII/PLN/SERCA 2a signaling pathway.

PubMed

Shi, Hongtao; Han, Qinghua; Xu, Jianrong; Liu, Wenyuan; Chu, Tingting; Zhao, Li

2016-05-25

Although studies have shown that Urotensin II (UII) can induce cardiomyocyte hypertrophy and UII-induced cardiomyocyte hypertrophy model has been widely used for hypertrophy research, but its precise mechanism remains unknown. Recent researches have demonstrated that UII-induced cardiomyocyte hypertrophy has a relationship with the changes of intracellular Ca(2+) concentration. Therefore, the aim of this study was to investigate the mechanisms of cardiomyocyte hypertrophy induced by UII and to explore whether the calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulating of phospholamban (PLN) Thr17-phosphorylation signaling pathway contributed to UII-induced cardiomyocyte hypertrophy. Primary cultures of neonatal rat cardiomyocytes were stimulated for 48h with UII. Cell size, protein/DNA contents and intracellular Ca(2+) were determined. Phosphorylated and total forms of CaMKII, PLN and the total amount of serco/endo-plasmic reticulum ATPases (SERCA 2a) were quantified by western blot. The responses of cardiomyocytes to UII were also evaluated after pretreatment with the CaMKII inhibitor, KN-93. These results showed that UII increased cell size, protein/DNA ratio and intracellular Ca(2+), consistent with a hypertrophic response. Furthermore, the phosphorylation of CaMKII and its downstream target PLN (Thr17), SERCA 2a levels were up-regulated by UII treatment. Conversely, treatment with KN-93 reversed all those effects of UII. Taken together, the results suggest that UII can induce cardiomyocyte hypertrophy through CaMKII-mediated up-regulating of PLN Thr17-phosphorylation signaling pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats

PubMed Central

Linde, Cristina I.; Karashima, Eiji; Raina, Hema; Zulian, Alessandra; Wier, Withrow G.; Hamlyn, John M.; Ferrari, Patrizia; Blaustein, Mordecai P.

2012-01-01

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na+ reabsorption. Recently we demonstrated that Ca2+ signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na+/Ca2+ exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca2+ signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1–100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca2+ signals in response to 5 μM PE or ATP in the absence and presence of extracellular Ca2+. These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca2+ release and increased Ca2+ entry, respectively. The increased SR Ca2+ release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca2+ signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca2+ signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension. PMID:22140038

Cardiac Overexpression of Antioxidant Catalase Attenuates Aging-Induced Cardiomyocyte Relaxation Dysfunction

PubMed Central

Ren, Jun; Li, Qun; Wu, Shan; Li, Shi-Yan; Babcock, Sara A.

2007-01-01

Catalase, an enzyme which detoxifies H2O2, may interfere with cardiac aging. To test this hypothesis, contractile and intracellular Ca2+ properties were evaluated in cardiomyocytes from young (3–4 mo) and old (26–28 mo) FVB and transgenic mice with cardiac overexpression of catalase. Contractile indices analyzed included peak shortening (PS), time-to-90% PS (TPS90), time-to-90% relengthening (TR90), half-width duration (HWD), maximal velocity of shortening/relengthening (± dL/dt) and intracellular Ca2+ levels or decay rate. Levels of advanced glycation endproduct (AGE), Na+/Ca2+ exchanger (NCX), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), phospholamban (PLB), myosin heavy chain (MHC), membrane Ca2+ and K+ channels were measured by western blot. Catalase transgene prolonged survival while did not alter myocyte function by itself. Aging depressed ± dL/dt, prolonged HWD, TR90 and intracellular Ca2+ decay without affecting other indices in FVB myocytes. Aged FVB myocytes exhibited a stepper decline in PS in response to elevated stimulus or a dampened rise in PS in response to elevated extracellular Ca2+ levels. Interestingly, aging-induced defects were nullified or significantly attenuated by catalase. AGE level was elevated by 5-fold in aged FVB compared with young FVB mice, which was reduced by catalase. Expression of SERCA2a, NCX and Kv1.2 K+ channel was significantly reduced although levels of PLB, L-type Ca2+ channel dihydropyridine receptor and β-MHC isozyme remained unchanged in aged FVB hearts. Catalase restored NCX and Kv1.2 K+ channel but not SERCA2a level in aged mice. In summary, our data suggested that catalase protects cardiomyocytes from aging-induced contractile defect possibly via improved intracellular Ca2+ handling. PMID:17250874

Tight coupling of Na+/K+-ATPase with glycolysis demonstrated in permeabilized rat cardiomyocytes.

PubMed

Sepp, Mervi; Sokolova, Niina; Jugai, Svetlana; Mandel, Merle; Peterson, Pearu; Vendelin, Marko

2014-01-01

The effective integrated organization of processes in cardiac cells is achieved, in part, by the functional compartmentation of energy transfer processes. Earlier, using permeabilized cardiomyocytes, we demonstrated the existence of tight coupling between some of cardiomyocyte ATPases and glycolysis in rat. In this work, we studied contribution of two membrane ATPases and whether they are coupled to glycolysis--sarcoplasmic reticulum Ca2+ ATPase (SERCA) and plasmalemma Na+/K+-ATPase (NKA). While SERCA activity was minor in this preparation in the absence of calcium, major role of NKA was revealed accounting to ∼30% of the total ATPase activity which demonstrates that permeabilized cell preparation can be used to study this pump. To elucidate the contribution of NKA in the pool of ATPases, a series of kinetic measurements was performed in cells where NKA had been inhibited by 2 mM ouabain. In these cells, we recorded: ADP- and ATP-kinetics of respiration, competition for ADP between mitochondria and pyruvate kinase (PK), ADP-kinetics of endogenous PK, and ATP-kinetics of total ATPases. The experimental data was analyzed using a series of mathematical models with varying compartmentation levels. The results show that NKA is tightly coupled to glycolysis with undetectable flux of ATP between mitochondria and NKA. Such tight coupling of NKA to PK is in line with its increased importance in the pathological states of the heart when the substrate preference shifts to glucose.

The Evolutionary History of Sarco(endo)plasmic Calcium ATPase (SERCA)

PubMed Central

Altshuler, Ianina; Vaillant, James J.; Xu, Sen; Cristescu, Melania E.

2012-01-01

Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na+/K+ transporters, H+/K+ transporters, and plasma membrane Ca2+ pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endo)plasmic reticulum calcium ATPase (SERCA), which maintains calcium homeostasis in the cell by actively pumping Ca2+ into the sarco(endo)plasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain. PMID:23285113

The evolutionary history of sarco(endo)plasmic calcium ATPase (SERCA).

PubMed

Altshuler, Ianina; Vaillant, James J; Xu, Sen; Cristescu, Melania E

2012-01-01

Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na(+)/K(+) transporters, H(+)/K(+) transporters, and plasma membrane Ca(2+) pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endo)plasmic reticulum calcium ATPase (SERCA), which maintains calcium homeostasis in the cell by actively pumping Ca(2+) into the sarco(endo)plasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain.

Arterial α2-Na+ pump expression influences blood pressure: lessons from novel, genetically engineered smooth muscle-specific α2 mice.

PubMed

Chen, Ling; Song, Hong; Wang, Youhua; Lee, Jane C; Kotlikoff, Michael I; Pritchard, Tracy J; Paul, Richard J; Zhang, Jin; Blaustein, Mordecai P

2015-09-01

Arterial myocytes express α1-catalytic subunit isoform Na(+) pumps (75-80% of total), which are ouabain resistant in rodents, and high ouabain affinity α2-Na(+) pumps. Mice with globally reduced α2-pumps (but not α1-pumps), mice with mutant ouabain-resistant α2-pumps, and mice with a smooth muscle (SM)-specific α2-transgene (α2 (SM-Tg)) that induces overexpression all have altered blood pressure (BP) phenotypes. We generated α2 (SM-DN) mice with SM-specific α2 (not α1) reduction (>50%) using nonfunctional dominant negative (DN) α2. We compared α2 (SM-DN) and α2 (SM-Tg) mice to controls to determine how arterial SM α2-pumps affect vasoconstriction and BP. α2 (SM-DN) mice had elevated basal mean BP (mean BP by telemetry: 117 ± 4 vs. 106 ± 1 mmHg, n = 7/7, P < 0.01) and enhanced BP responses to chronic ANG II infusion (240 ng·kg(-1)·min(-1)) and high (6%) NaCl. Several arterial Ca(2+) transporters, including Na(+)/Ca(2+) exchanger 1 (NCX1) and sarcoplasmic reticulum and plasma membrane Ca(2+) pumps [sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 (SERCA2) and plasma membrane Ca(2+)-ATPase 1 (PMCA1)], were also reduced (>50%). α2 (SM-DN) mouse isolated small arteries had reduced myogenic reactivity, perhaps because of reduced Ca(2+) transporter expression. In contrast, α2 (SM-Tg) mouse aortas overexpressed α2 (>2-fold), NCX1, SERCA2, and PMCA1 (43). α2 (SM-Tg) mice had reduced basal mean BP (104 ± 1 vs. 109 ± 2 mmHg, n = 15/9, P < 0.02) and attenuated BP responses to chronic ANG II (300-400 ng·kg(-1)·min(-1)) with or without 2% NaCl but normal myogenic reactivity. NCX1 expression was inversely related to basal BP in SM-α2 engineered mice but was directly related in SM-NCX1 engineered mice. NCX1, which usually mediates arterial Ca(2+) entry, and α2-Na(+) pumps colocalize at plasma membrane-sarcoplasmic reticulum junctions and functionally couple via the local Na(+) gradient to help regulate cell Ca(2+). Altered Ca(2+) transporter expression in SM-α2 engineered mice apparently compensates to minimize Ca(2+) overload (α2 (SM-DN)) or depletion (α2 (SM-Tg)) and attenuate BP changes. In contrast, Ca(2+) transporter upregulation, observed in many rodent hypertension models, should enhance Ca(2+) entry and signaling and contribute significantly to BP elevation. Copyright © 2015 the American Physiological Society.

Presenilin 1 mutation decreases both calcium and contractile responses in cerebral arteries.

PubMed

Toussay, Xavier; Morel, Jean-Luc; Biendon, Nathalie; Rotureau, Lolita; Legeron, François-Pierre; Boutonnet, Marie-Charlotte; Cho, Yoon H; Macrez, Nathalie

2017-10-01

Mutations or upregulation in presenilin 1 (PS1) gene are found in familial early-onset Alzheimer's disease or sporadic late-onset Alzheimer's disease, respectively. PS1 has been essentially studied in neurons and its mutation was shown to alter intracellular calcium (Ca 2+ ) signals. Here, we showed that PS1 is expressed in smooth muscle cells (SMCs) of mouse cerebral arteries, and we assessed the effects of the deletion of exon 9 of PS1 (PS1dE9) on Ca 2+ signals and contractile responses of vascular SMC. Agonist-induced contraction of cerebral vessels was significantly decreased in PS1dE9 both in vivo and ex vivo. Spontaneous activity of Ca 2+ sparks through ryanodine-sensitive channels (RyR) was unchanged, whereas the RyR-mediated Ca 2+ -release activated by caffeine was shorter in PS1dE9 SMC when compared with control. Moreover, PS1dE9 mutation decreased the caffeine-activated capacitive Ca 2+ entry, and inhibitors of SERCA pumps reversed the effects of PS1dE9 on Ca 2+ signals. PS1dE9 mutation also leads to the increased expression of SERCA3, phospholamban, and RyR3. These results show that PS1 plays a crucial role in the cerebrovascular system and the vascular reactivity is decreased through altered Ca 2+ signals in PS1dE9 mutant mice. Copyright © 2017 Elsevier Inc. All rights reserved.

Cardioprotective effects of baicalein on heart failure via modulation of Ca(2+) handling proteins in vivo and in vitro.

PubMed

Zhao, Fali; Fu, Lu; Yang, Wei; Dong, Yuhui; Yang, Jing; Sun, Shoubin; Hou, Yuling

2016-01-15

Baicalein is a widely used Chinese herbal medicine extracted from Labiatae plants Scutellaria baicalensis Georgi's dry root, which has multiple pharmacological activities. However, the precise mechanism of baicalein against myocardial remodeling remains poorly understood. The aim of our study was to investigate the underlying mechanism of baicalein treatment in rats model of heart failure (HF) and rat myocardial cells (H9C2). HF model was established by abdominal aorta constriction in rats and incubation with 50μM isoproterenol for 48h in H9C2 cells. Various molecular biological experiments were performed to assess the effects of baicalein on cardiac function, myocardial remodeling, apoptosis and Ca(2+) handling proteins. In the present study, first we found that baicalein alleviated HF in vivo. Additionally, treatment with baicalein inhibited the myocardial fibrosis, restrained the expression and activity of MMP2 and MMP9, and suppressed apoptosis in heart tissue. Moreover, baicalein could inhibit the cardiac myocyte hypertrophy and apoptosis induced by isoproterenol in vitro. Finally we found that baicalein could modulate the expressions and activities of Ca(2+) handling proteins, including downregulation of phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and expression of Na(+)/Ca(2+)-exchangers (NCX1), upregulation of sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and ryanodine receptor 2 (RYR2). Baicalein also restrained the decreased SERCA activity induced by aortic banding. Our studies suggested that baicalein alleviated myocardial remodeling and improved cardiac function via modulation of Ca(2+) handling proteins, which may be a potential phytochemical flavonoid for therapeutics of HF. Copyright © 2015 Elsevier Inc. All rights reserved.

Disbalance of calcium regulation-related genes in broiler hearts induced by selenium deficiency.

PubMed

Zhang, Ziwei; Liu, Man; Guan, Zhenqiong; Yang, Jie; Liu, Zhonghua; Xu, Shiwen

2017-06-01

Dietary selenium (Se) deficiency may influence the calcium (Ca) homeostasis in broilers. Our objective was to investigate the effects of Se deficiency on Ca regulation-related genes in broiler hearts. In the present study, 1-day-old broilers were fed either a commercial diet (as control group) with 0.15 mg/kg Se or a Se-deficient diet (as L group) with 0.033 mg/kg Se for 35 days. We examined the mRNA expression levels of 15 Ca regulation-related genes (ITPR 1, ITPR 2, ITPR3, RyR2, RyR3, SERCA1s, SLC8A1, PMCA1, CACNA1S, TRPC1, TRPC3, stromal interacting molecule 1, ORAI1, calmodulin (CaLM) and calreticulin (CRT) in broiler hearts. Then, Kyoto Encyclopedia of Genes and Genomes analysis, protein-protein interactions (PPI) analysis and correlation analysis were performed to analyse the relationships between these genes. The results showed that the mRNA expression levels of ITPR 1, ITPR 2, RyR2, RyR3, SERCA1s, SLC8A1, PMCA1, CACNA1S, CaLM and CRT were generally decreased by Se deficiency, while mRNA expression levels of TRPC1, TRPC3, stromal interacting molecule 1, ORAI1 and ITPR3 were increased by Se deficiency. Kyoto Encyclopedia of Genes and Genomes and PPI analysis showed that these Ca regulation-related genes are involved in the Ca signalling pathway and a total of 15 PPIs with a combined score of >0.4 were obtained. In conclusion, the results demonstrated that Se deficiency might cause heart injury via modulating the Ca-related pathway genes, and then induce Ca 2+ overload in the heart of broilers.

Mutual adaptation of a membrane protein and its lipid bilayer during conformational changes.

PubMed

Sonntag, Yonathan; Musgaard, Maria; Olesen, Claus; Schiøtt, Birgit; Møller, Jesper Vuust; Nissen, Poul; Thøgersen, Lea

2011-01-01

The structural elucidation of membrane proteins continues to gather pace, but we know little about their molecular interactions with the lipid environment or how they interact with the surrounding bilayer. Here, with the aid of low-resolution X-ray crystallography, we present direct structural information on membrane interfaces as delineated by lipid phosphate groups surrounding the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in its phosphorylated and dephosphorylated Ca(2+)-free forms. The protein-lipid interactions are further analysed using molecular dynamics simulations. We find that SERCA adapts to membranes of different hydrophobic thicknesses by inducing local deformations in the lipid bilayers and by undergoing small rearrangements of the amino-acid side chains and helix tilts. These mutually adaptive interactions allow smooth transitions through large conformational changes associated with the transport cycle of SERCA, a strategy that may be of general nature for many membrane proteins.

Sphingosine inhibits the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA) activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benaim, Gustavo, E-mail: gbenaim@idea.gob.ve; Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela; Pimentel, Adriana A., E-mail: adriana.pimentel@ucv.ve

2016-04-29

The increase in the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca{sup 2+}]{sub i} by inhibiting the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca{sup 2+} pump. The results showed that addition of sphingosine produced a release of Ca{sup 2+} from the endoplasmic reticulum followed by a Ca{sup 2+} entrance from the outside mileu. The results presented in this work support thatmore » this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca{sup 2+}]{sub I} in mammalian cells.« less

Calcitriol Prevents Cardiovascular Repercussions in Puromycin Aminonucleoside-Induced Nephrotic Syndrome

PubMed Central

Roberto, Roncon-Albuquerque

2018-01-01

Puromycin aminonucleoside-induced nephrotic syndrome (PAN-NS) is characterized by cardiac remodeling and increased local inflammatory activity. Patients with NS and animal models of NS have vitamin D3 deficiency. The aim of the present study was to evaluate the influence of calcitriol on cardiac remodeling and local inflammatory state in PAN-NS rat model. Male Sprague-Dawley rats were injected with PAN or vehicle on day 0. PAN and control rats were divided into two subgroups for the administration of calcitriol (PAN-D and Ct-D groups) or the vehicle (PAN-V and Ct-V groups) during 21 days. On day 21, the renal function, metabolic balance, calcitriol and FGF-23 plasma levels, prohypertrophy and proinflammatory markers (ET-1, TGF-β1, TNF-α, and IL-1β), and calcium signaling molecules (PLB and SERCA-2a) were evaluated. Twenty-one days after injection, PAN-V group presented cardiac hypertrophy and a modulation of proinflammatory markers local expression. Calcitriol treatment of PAN rats prevented cardiac hypertrophy and was associated with marked reduction in the cardiac expression levels of proinflammatory markers. Our results suggest that vitamin D3 deficiency in PAN-NS may contribute to cardiac remodeling and to the increase in local inflammatory activity. Calcitriol treatment prevents both cardiac repercussions and local inflammatory processes in PAN-NS. PMID:29607318

Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca2+ signaling in ventricular myocytes.

PubMed

Park, Seon-Ah; Hong, Bing-Zhe; Ha, Ki-Chan; Kim, Uh-Hyun; Han, Myung-Kwan; Kwak, Yong-Geun

2017-06-02

Cyclic ADP-ribose (cADPR) releases Ca 2+ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca 2+ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca 2+ ([Ca 2+ ] i ) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca 2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca 2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling

PubMed Central

De Ford, C; Heidersdorf, B; Haun, F; Murillo, R; Friedrich, T; Borner, C; Merfort, I

2016-01-01

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that preferentially affects children and adolescents. Over 50% of human T-ALLs possess activating mutations of Notch1. The clerodane diterpene casearin J (CJ) is a natural product that inhibits the sarcoendoplasmatic reticulum calcium ATPase (SERCA) pump and induces cell death in leukemia cells, but the molecular mechanism of cytotoxicity remains poorly understood. Here we show that owing to SERCA pump inhibition, CJ induces depletion of the endoplasmic reticulum calcium pools, oxidative stress, and apoptosis via the intrinsic signaling pathway. Moreover, Notch1 signaling is reduced in T-ALL cells with auto-activating mutations in the HD-domain of Notch1, but not in cells that do not depend on Notch1 signaling. CJ also provoked a slight activation of NF-κB, and consistent with this notion a combined treatment of CJ and the NF-κB inhibitor parthenolide (Pt) led to a remarkable synergistic cell death in T-ALL cells. Altogether, our data support the concept that inhibition of the SERCA pump may be a novel strategy for the treatment of T-ALL with HD-domain-mutant Notch1 receptors and that additional treatment with the NF-κB inhibitor parthenolide may have further therapeutic benefits. PMID:26821066

Spontaneous Ca(2+) transients in interstitial cells of Cajal located within the deep muscular plexus of the murine small intestine.

PubMed

Baker, Salah A; Drumm, Bernard T; Saur, Dieter; Hennig, Grant W; Ward, Sean M; Sanders, Kenton M

2016-06-15

Interstitial cells of Cajal at the level of the deep muscular plexus (ICC-DMP) in the small intestine generate spontaneous Ca(2+) transients that consist of localized Ca(2+) events and limited propagating Ca(2+) waves. Ca(2+) transients in ICC-DMP display variable characteristics: from discrete, highly localized Ca(2+) transients to regionalized Ca(2+) waves with variable rates of occurrence, amplitude, duration and spatial spread. Ca(2+) transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells. Ca(2+) transients in ICC-DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s). Functional intracellular Ca(2+) stores are essential for spontaneous Ca(2+) transients, and the sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump is necessary for maintenance of spontaneity. Ca(2+) release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3 Rs). Release from these channels is interdependent. ICC express transcripts of multiple RyRs and InsP3 Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression. Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC-DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC-DMP are mediated by activation of Ca(2+) -activated Cl(-) channels; thus, Ca(2+) signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca(2+) transients in ICC-DMP within intact jejunal muscles expressing a genetically encoded Ca(2+) indicator (GCaMP3) selectively in ICC. ICC-DMP displayed spontaneous Ca(2+) transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca(2+) transients was highly variable, and it was determined that firing was stochastic in nature. Ca(2+) transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 μm) significantly increased the occurrence of Ca(2+) transients, suggesting that ICC-DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca(2+) transients were minimally affected after 12 min in Ca(2+) free solution, indicating these events do not depend immediately upon Ca(2+) influx. However, inhibitors of sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3 R) and ryanodine receptor (RyR) channels blocked ICC Ca(2+) transients. These data suggest an interdependence between RyR and InsP3 R in the generation of Ca(2+) transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high-resolution recording of the subcellular Ca(2+) dynamics that control the behaviour of ICC-DMP in situ. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Spontaneous Ca2+ transients in interstitial cells of Cajal located within the deep muscular plexus of the murine small intestine

PubMed Central

Baker, Salah A.; Drumm, Bernard T.; Saur, Dieter; Hennig, Grant W.; Ward, Sean M.

2016-01-01

Key points Interstitial cells of Cajal at the level of the deep muscular plexus (ICC‐DMP) in the small intestine generate spontaneous Ca2+ transients that consist of localized Ca2+ events and limited propagating Ca2+ waves.Ca2+ transients in ICC‐DMP display variable characteristics: from discrete, highly localized Ca2+ transients to regionalized Ca2+ waves with variable rates of occurrence, amplitude, duration and spatial spread.Ca2+ transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells.Ca2+ transients in ICC‐DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s).Functional intracellular Ca2+ stores are essential for spontaneous Ca2+ transients, and the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump is necessary for maintenance of spontaneity.Ca2+ release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs). Release from these channels is interdependent.ICC express transcripts of multiple RyRs and InsP3Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression. Abstract Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC‐DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC‐DMP are mediated by activation of Ca2+‐activated Cl− channels; thus, Ca2+ signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca2+ transients in ICC‐DMP within intact jejunal muscles expressing a genetically encoded Ca2+ indicator (GCaMP3) selectively in ICC. ICC‐DMP displayed spontaneous Ca2+ transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca2+ transients was highly variable, and it was determined that firing was stochastic in nature. Ca2+ transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 μm) significantly increased the occurrence of Ca2+ transients, suggesting that ICC‐DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca2+ transients were minimally affected after 12 min in Ca2+ free solution, indicating these events do not depend immediately upon Ca2+ influx. However, inhibitors of sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels blocked ICC Ca2+ transients. These data suggest an interdependence between RyR and InsP3R in the generation of Ca2+ transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high‐resolution recording of the subcellular Ca2+ dynamics that control the behaviour of ICC‐DMP in situ. PMID:26824875

Memantine Can Reduce Ethanol-Induced Caspase-3 Activity and Apoptosis in H4 Cells by Decreasing Intracellular Calcium.

PubMed

Wang, Xiaolong; Chen, Jiajun; Wang, Hongbo; Yu, Hao; Wang, Changliang; You, Jiabin; Wang, Pengfei; Feng, Chunmei; Xu, Guohui; Wu, Xu; Zhao, Rui; Zhang, Guohua

2017-08-01

Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100 mM EtOH (in culture medium) for 2 days. For interaction studies, cells were treated with memantine (4 μM), EDTA (1 mM), or BAPTA-AM (10 μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.

The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses α Myosin Heavy-Chain Gene Expression

PubMed Central

Sucharov, Carmen C.; Helmke, Steve M.; Langer, Stephen J.; Perryman, M. Benjamin; Bristow, Michael; Leinwand, Leslie

2004-01-01

Human heart failure is accompanied by repression of genes such as α myosin heavy chain (αMyHC) and SERCA2A and the induction of fetal genes such as βMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human αMyHC promoter. We have now identified a region of the αMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human αMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases αMyHC mRNA expression and increases skeletal α-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the αMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human αMyHC promoter during heart failure. PMID:15367688

Motion of the Ca2+-pump captured.

PubMed

Yokokawa, Masatoshi; Takeyasu, Kunio

2011-09-01

Studies of ion pumps, such as ATP synthetase and Ca(2+)-ATPase, have a long history. The crystal structures of several kinds of ion pump have been resolved, and provide static pictures of mechanisms of ion transport. In this study, using fast-scanning atomic force microscopy, we have visualized conformational changes in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) in real time at the single-molecule level. The analyses of individual SERCA molecules in the presence of both ATP and free Ca(2+) revealed up-down structural changes corresponding to the Albers-Post scheme. This fluctuation was strongly affected by the ATP and Ca(2+) concentrations, and was prevented by an inhibitor, thapsigargin. Interestingly, at a physiological ATP concentrations, the up-down motion disappeared completely. These results indicate that SERCA does not transit through the shortest structure, and has a catalytic pathway different from the ordinary Albers-Post scheme under physiological conditions. © 2011 The Authors Journal compilation © 2011 FEBS.

Altered sarcoplasmic reticulum calcium cycling—targets for heart failure therapy

PubMed Central

Kho, Changwon; Lee, Ahyoung; Hajjar, Roger J.

2013-01-01

Cardiac myocyte function is dependent on the synchronized movements of Ca2+ into and out of the cell, as well as between the cytosol and sarcoplasmic reticulum. These movements determine cardiac rhythm and regulate excitation–contraction coupling. Ca2+ cycling is mediated by a number of critical Ca2+-handling proteins and transporters, such as L-type Ca2+ channels (LTCCs) and sodium/calcium exchangers in the sarcolemma, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a), ryanodine receptors, and cardiac phospholamban in the sarcoplasmic reticulum. The entry of Ca2+ into the cytosol through LTCCs activates the release of Ca2+ from the sarcoplasmic reticulum through ryanodine receptor channels and initiates myocyte contraction, whereas SERCA2a and cardiac phospholamban have a key role in sarcoplasmic reticulum Ca2+ sequesteration and myocyte relaxation. Excitation–contraction coupling is regulated by phosphorylation of Ca2+-handling proteins. Abnormalities in sarcoplasmic reticulum Ca2+ cycling are hallmarks of heart failure and contribute to the pathophysiology and progression of this disease. Correcting impaired intracellular Ca2+ cycling is a promising new approach for the treatment of heart failure. Novel therapeutic strategies that enhance myocyte Ca2+ homeostasis could prevent and reverse adverse cardiac remodeling and improve clinical outcomes in patients with heart failure. PMID:23090087

Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma.

PubMed

Chen, Jun; Miller, Marina; Unno, Hirotoshi; Rosenthal, Peter; Sanderson, Michael J; Broide, David H

2017-09-07

Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3 Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3 Zp3-Cre mice, which do not have a blood supply. Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3 Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Chronic Exposure to Low Doses of HgCl2 Avoids Calcium Handling Impairment in the Right Ventricle after Myocardial Infarction in Rats

PubMed Central

Faria, Thaís de Oliveira; Costa, Gustavo Pinto; Almenara, Camila Cruz Pereira; Angeli, Jhuli Keli; Vassallo, Dalton Valentim; Stefanon, Ivanita; Vassallo, Paula Frizera

2014-01-01

Right ventricle systolic dysfunction is a major risk factor for death and heart failure after myocardial infarction (MI). Heavy metal exposure has been associated with the development of several cardiovascular diseases, such as MI. The aim of this study was to investigate whether chronic exposure to low doses of mercury chloride (HgCl2) enhances the functional deterioration of right ventricle strips after MI. Male Wistar rats were divided into four groups: Control (vehicle); HgCl2 (exposure during 4 weeks- 1st dose 4.6 µg/kg, subsequent dose 0.07 µg/kg/day, i.m. to cover daily loss); MI surgery induced and HgCl2-MI groups. One week after MI, the morphological and hemodynamic measurements and isometric tension of right ventricle strips were investigated. The chronic HgCl2 exposure did not worsen the injury compared with MI alone in the morphological or hemodynamic parameters evaluated. At basal conditions, despite similar maximum isometric force at L-max, relaxation time was increased in the MI group but unaffected in the HgCl2-MI compared to the Control group. Impairment of the sarcoplasmic reticulum (SR) function and reduction in the sarcolemmal calcium influx were observed in MI group associated with SERCA2a reduction and increased PLB protein expression. Induction of MI in chronic HgCl2 exposed rats did not cause any alteration in the developed force at L-max, lusitropic function or −dF/dt except for a tendency of a reduction SR function. These findings could be partially explained by the normalization in the sarcolemmal calcium influx and the increase in NCX protein expression observed only in this group. These results suggest that chronic exposure to low doses of HgCl2 prevents the impaired SR function and the reduced sarcolemmal calcium influx observed in MI likely by acting on NCX, PLB and SERCA2a protein expression. PMID:24748367

Endoplasmic reticulum Chaperon Tauroursodeoxycholic Acid Alleviates Obesity-Induced Myocardial Contractile Dysfunction

PubMed Central

Ceylan-Isik, Asli F.; Sreejayan, Nair; Ren, Jun

2010-01-01

ER stress is involved in the pathophysiology of obesity although little is known about the role of ER stress on obesity-associated cardiac dysfunction. This study was designed to examine the effect of ER chaperone tauroursodeoxycholic acid (TUDCA) on obesity-induced myocardial dysfunction. Adult lean and ob/ob obese mice were treated TUDCA (50 mg/kg/d, p.o.) or vehicle for 5 wks. Oral glucose tolerance test (OGTT) was performed. Echocardiography, cardiomyocyte contractile and intracellular Ca2+ properties were assessed. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and protein expression of intracellular Ca2+ regulatory proteins were measured using 45Ca2+ uptake and Western blot analysis, respectively. Insulin signaling, ER stress markers and HSP90 were evaluated. Our results revealed that chronic TUDCA treatment lower systolic blood pressure and lessened glucose intolerance in obese mice. Obesity led to increased diastolic diameter, cardiac hypertrophy, compromised fractional shortening, cardiomyocyte contractile (peak shortening, maximal velocity of shortening/relengthening, and duration of contraction/relaxation) and intracellular Ca2+ properties, all of which were significantly attenuated by TUDCA. TUDCA reconciled obesity-associated decreased in SERCA activity and expression, and increase in serine phosphorylation of IRS, total and phosphorylated cJun, ER stress markers Bip, peIF2α and pPERK. Obesity-induced changes in phospholamban and HSP90 were unaffected by TUDCA. In vitro finding revealed that TUDCA ablated palmitic acid-induced cardiomyocyte contractile dysfunction. In summary, these data depicted a pivotal role of ER stress in obesity-associated cardiac contractile dysfunction, suggesting the therapeutic potential of ER stress as a target in the management of cardiac dysfunction in obesity. PMID:21035453

Deregulated Ca2+ cycling underlies the development of arrhythmia and heart disease due to mutant obscurin

PubMed Central

Hu, Li-Yen R.; Ackermann, Maegen A.; Hecker, Peter A.; Prosser, Benjamin L.; King, Brendan; O’Connell, Kelly A.; Grogan, Alyssa; Meyer, Logan C.; Berndsen, Christopher E.; Wright, Nathan T.; Jonathan Lederer, W.; Kontrogianni-Konstantopoulos, Aikaterini

2017-01-01

Obscurins are cytoskeletal proteins with structural and regulatory roles encoded by OBSCN. Mutations in OBSCN are associated with the development of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Specifically, the R4344Q mutation present in immunoglobulin domain 58 (Ig58) was the first to be linked with the development of HCM. To assess the effects of R4344Q in vivo, we generated the respective knock-in mouse model. Mutant obscurins are expressed and incorporated normally into sarcomeres. The expression patterns of sarcomeric and Ca2+-cycling proteins are unaltered in sedentary 1-year-old knock-in myocardia, with the exception of sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase 2 (SERCA2) and pentameric phospholamban whose levels are significantly increased and decreased, respectively. Isolated cardiomyocytes from 1-year-old knock-in hearts exhibit increased Ca2+-transients and Ca2+-load in the sarcoplasmic reticulum and faster contractility kinetics. Moreover, sedentary 1-year-old knock-in animals develop tachycardia accompanied by premature ventricular contractions, whereas 2-month-old knock-in animals subjected to pressure overload develop a DCM-like phenotype. Structural analysis revealed that the R4344Q mutation alters the distribution of electrostatic charges over the Ig58 surface, thus interfering with its binding capabilities. Consistent with this, wild-type Ig58 interacts with phospholamban modestly, and this interaction is markedly enhanced in the presence of R4344Q. Together, our studies demonstrate that under sedentary conditions, the R4344Q mutation results in Ca2+ deregulation and spontaneous arrhythmia, whereas in the presence of chronic, pathological stress, it leads to cardiac remodeling and dilation. We postulate that enhanced binding between mutant obscurins and phospholamban leads to SERCA2 disinhibition, which may underlie the observed pathological alterations. PMID:28630914

Transmural Heterogeneity and Remodeling of Ventricular Excitation-Contraction Coupling in Human Heart Failure

PubMed Central

Lou, Qing; Fedorov, Vadim V.; Glukhov, Alexey V.; Moazami, Nader; Fast, Vladimir G.; Efimov, Igor R.

2011-01-01

Background Excitation-contraction (EC) coupling is altered in the end-stage heart failure (HF). However, spatial heterogeneity of this remodeling has not been established at the tissue level in failing human heart. The objective is to study functional remodeling of EC coupling and calcium handling in failing and nonfailing human hearts. Methods and Results We simultaneously optically mapped action potentials (AP) and calcium transients (CaT) in coronary-perfused left ventricular wedge preparations from nonfailing (n = 6) and failing (n = 5) human hearts. Our major findings are: (1) CaT duration minus AP duration was longer at sub-endocardium in failing compared to nonfailing hearts during bradycardia (40 beats/min). (2) The transmural gradient of CaT duration was significantly smaller in failing hearts compared with nonfailing hearts at fast pacing rates (100 beats/min). (3) CaT in failing hearts had a flattened plateau at the midmyocardium; and exhibited a “two-component” slow rise at sub-endocardium in three failing hearts. (4) CaT relaxation was slower at sub-endocardium than that at sub-epicardium in both groups. Protein expression of sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) was lower at sub-endocardium than that at sub-epicardium in both nonfailing and failing hearts. SERCA2a protein expression at sub-endocardium was lower in hearts with ischemic cardiomyopathy compared with nonischemic cardiomyopathy. Conclusions For the first time, we present direct experimental evidence of transmural heterogeneity of EC coupling and calcium handling in human hearts. End-stage HF is associated with the heterogeneous remodeling of EC coupling and calcium handling. PMID:21502574

Differential calcium handling in two canine models of right ventricular pressure overload.

PubMed

Moon, Marc R; Aziz, Abdulhameed; Lee, Anson M; Moon, Cynthia J; Okada, Shoichi; Kanter, Evelyn M; Yamada, Kathryn A

2012-12-01

The purpose of this investigation was to characterize differential right atrial (RA) and ventricular (RV) molecular changes in Ca(2+)-handling proteins consequent to RV pressure overload and hypertrophy in two common, yet distinct models of pulmonary hypertension: dehydromonocrotaline (DMCT) toxicity and pulmonary artery (PA) banding. A total of 18 dogs underwent sternotomy in four groups: (1) DMCT toxicity (n = 5), (2) mild PA banding over 10 wk to match the RV pressure rise with DMCT (n = 5); (3) progressive PA banding to generate severe RV overload (n = 4); and (4) sternotomy only (n = 4). In the right ventricle, with DMCT, there was no change in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) or phospholamban (PLB), but we saw a trend toward down-regulation of phosphorylated PLB at serine-16 (p[Ser-16]PLB) (P = 0.07). Similarly, with mild PA banding, there was no change in SERCA or PLB, but p(Ser-16)PLB was down-regulated by 74% (P < 0.001). With severe PA banding, there was no change in PLB, but SERCA fell by 57% and p(Ser-16)PLB fell by 67% (P < 0.001). In the right atrium, with DMCT, there were no significant changes. With both mild and severe PA banding, p(Ser-16)PLB fell (P < 0.001), but SERCA and PLB did not change. Perturbations in Ca(2+)-handling proteins depend on the degree of RV pressure overload and the model used to mimic the RV effects of pulmonary hypertension. They are similar, but blunted, in the atrium compared with the ventricle. Copyright © 2012 Elsevier Inc. All rights reserved.

Target Deconvolution Efforts on Wnt Pathway Screen Reveal Dual Modulation of Oxidative Phosphorylation and SERCA2.

PubMed

Casás-Selves, Matias; Zhang, Andrew X; Dowling, James E; Hallén, Stefan; Kawatkar, Aarti; Pace, Nicholas J; Denz, Christopher R; Pontz, Timothy; Garahdaghi, Farzin; Cao, Qing; Sabirsh, Alan; Thakur, Kumar; O'Connell, Nichole; Hu, Jun; Cornella-Taracido, Iván; Weerapana, Eranthie; Zinda, Michael; Goodnow, Robert A; Castaldi, M Paola

2017-06-21

Wnt signaling is critical for development, cell proliferation and differentiation, and mutations in this pathway resulting in constitutive signaling have been implicated in various cancers. A pathway screen using a Wnt-dependent reporter identified a chemical series based on a 1,2,3-thiadiazole-5-carboxamide (TDZ) core with sub-micromolar potency. Herein we report a comprehensive mechanism-of-action deconvolution study toward identifying the efficacy target(s) and biological implication of this chemical series involving bottom-up quantitative chemoproteomics, cell biology, and biochemical methods. Through observing the effects of our probes on metabolism and performing confirmatory cellular and biochemical assays, we found that this chemical series inhibits ATP synthesis by uncoupling the mitochondrial potential. Affinity chemoproteomics experiments identified sarco(endo)plasmic reticulum Ca 2+ -dependent ATPase (SERCA2) as a binding partner of the TDZ series, and subsequent validation studies suggest that the TDZ series can act as ionophores through SERCA2 toward Wnt pathway inhibition. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Long-Term Oral Administration of Theaphenon-E Improves Cardiomyocyte Mechanics and Calcium Dynamics by Affecting Phospholamban Phosphorylation and ATP Production.

PubMed

Bocchi, Leonardo; Savi, Monia; Naponelli, Valeria; Vilella, Rocchina; Sgarbi, Gianluca; Baracca, Alessandra; Solaini, Giancarlo; Bettuzzi, Saverio; Rizzi, Federica; Stilli, Donatella

2018-06-15

Dietary polyphenols from green tea have been shown to possess cardio-protective activities in different experimental models of heart diseases and age-related ventricular dysfunction. The present study was aimed at evaluating whether long term in vivo administration of green tea extracts (GTE), can exert positive effects on the normal heart, with focus on the underlying mechanisms. The study population consisted of 20 male adult Wistar rats. Ten animals were given 40 mL/day tap water solution of GTE (concentration 0.3%) for 4 weeks (GTE group). The same volume of water was administered to the 10 remaining control rats (CTRL). Then, in vivo and ex vivo measurements of cardiac function were performed in the same animal, at the organ (hemodynamics) and cellular (cardiomyocyte mechanical properties and intracellular calcium dynamics) levels. On cardiomyocytes and myocardial tissue samples collected from the same in vivo studied animals, we evaluated: (1) the intracellular content of ATP, (2) the endogenous mitochondrial respiration, (3) the expression levels of the Sarcoplasmic Reticulum Ca2+-dependent ATPase 2a (SERCA2), the Phospholamban (PLB) and the phosphorylated form of PLB, the L-type Ca2+ channel, the Na+-Ca2+ exchanger, and the ryanodine receptor 2. GTE cardiomyocytes exhibited a hyperdynamic contractility compared with CTRL (the rate of shortening and re-lengthening, the fraction of shortening, the amplitude of calcium transient, and the rate of cytosolic calcium removal were significantly increased). A faster isovolumic relaxation was also observed at the organ level. Consistent with functional data, we measured a significant increase in the intracellular ATP content supported by enhanced endogenous mitochondrial respiration in GTE cardiomyocytes, as well as higher values of the ratios phosphorylated-PLB/PLB and SERCA2/PLB. Long-term in vivo administration of GTE improves cell mechanical properties and intracellular calcium dynamics in normal cardiomyocytes, by increasing energy availability and removing the inhibitory effect of PLB on SERCA2. © 2018 The Author(s). Published by S. Karger AG, Basel.

Facilitation of cytosolic calcium wave propagation by local calcium uptake into the sarcoplasmic reticulum in cardiac myocytes.

PubMed

Maxwell, Joshua T; Blatter, Lothar A

2012-12-01

The widely accepted paradigm for cytosolic Ca(2+) wave propagation postulates a 'fire-diffuse-fire' mechanism where local Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca(2+) release channels diffuses towards and activates neighbouring release sites, resulting in a propagating Ca(2+) wave. A recent challenge to this paradigm proposed the requirement for an intra-SR 'sensitization' Ca(2+) wave that precedes the cytosolic Ca(2+) wave and primes RyRs from the luminal side to CICR. Here, we tested this hypothesis experimentally with direct simultaneous measurements of cytosolic ([Ca(2+)](i); rhod-2) and intra-SR ([Ca(2+)](SR); fluo-5N) calcium signals during wave propagation in rabbit ventricular myocytes, using high resolution fluorescence confocal imaging. The increase in [Ca(2+)](i) at the wave front preceded depletion of the SR at each point along the calcium wave front, while during this latency period a transient increase of [Ca(2+)](SR) was observed. This transient elevation of [Ca(2+)](SR) could be identified at individual release junctions and depended on the activity of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA). Increased SERCA activity (β-adrenergic stimulation with 1 μM isoproterenol (isoprenaline)) decreased the latency period and increased the amplitude of the transient elevation of [Ca(2+)](SR), whereas inhibition of SERCA (3 μM cyclopiazonic acid) had the opposite effect. In conclusion, the data provide experimental evidence that local Ca(2+) uptake by SERCA into the SR facilitates the propagation of cytosolic Ca(2+) waves via luminal sensitization of the RyR, and supports a novel paradigm of a 'fire-diffuse-uptake-fire' mechanism for Ca(2+) wave propagation in cardiac myocytes.

Hyperthyroidism causes mechanical insufficiency of myocardium with possibly increased SR Ca2+-ATPase activity.

PubMed

Takeuchi, Koh; Minakawa, M; Otaki, M; Odagiri, S; Itoh, K; Murakami, A; Yaku, H; Kitamura, N

2003-12-01

Hyperthyroidism is known to affect multiple organ functions, and thyroid hormone has been known to improve myocardial function in a failing heart. The purpose of this study is to elucidate the functional and metabolic effects of thyroid hormone on myocardium in a rat model exposed to long-term excess thyroid hormone, particularly focusing on the SR Ca(2+)-ATPase (SERCA2) function. 3,5,3'-Triiodo-L-thyronine (T3), or the vehicle, was subcutaneously given for 4 weeks (T3 and control [C] group). Bolus I.V. Thapsigargin (TG) was used to test the SERCA2 function (C-TG and T3-TG) in Langendorff perfused heart. Myocardial functions such as LV-developed pressure (LVDP; mmHg), +/- dP/dt (mmHg/s), tau (ms), and oxygen consumption (MVO(2); ml/min/g wt) were measured. SERCA2 and GLUT4 protein level were also evaluated by Western immunoblotting. Left ventricle to body weight (LV/BW) ratio was significantly higher in the T3 group. Both negative dP/dt and tau were significantly decreased by TG. It is interesting that the decrement of negative dP/dt and tau attained by TG was significantly larger in the hyperthyroid group (T3-TG) than in a normal heart (C-TG). SERCA2 and GLUT4 protein levels were not significantly different between control and the T3 group. We conclude that prolonged exposure to thyroid hormone causes hypertrophy of the myocardium and an augmentation of the SR Ca(2+) ATPase activity. Care must be taken in hyperthyroid heart during the ischemia-reperfusion process where the SRECA2 function is inhibited.

Facilitation of cytosolic calcium wave propagation by local calcium uptake into the sarcoplasmic reticulum in cardiac myocytes

PubMed Central

Maxwell, Joshua T; Blatter, Lothar A

2012-01-01

The widely accepted paradigm for cytosolic Ca2+ wave propagation postulates a ‘fire-diffuse-fire’ mechanism where local Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca2+ release channels diffuses towards and activates neighbouring release sites, resulting in a propagating Ca2+ wave. A recent challenge to this paradigm proposed the requirement for an intra-SR ‘sensitization’ Ca2+ wave that precedes the cytosolic Ca2+ wave and primes RyRs from the luminal side to CICR. Here, we tested this hypothesis experimentally with direct simultaneous measurements of cytosolic ([Ca2+]i; rhod-2) and intra-SR ([Ca2+]SR; fluo-5N) calcium signals during wave propagation in rabbit ventricular myocytes, using high resolution fluorescence confocal imaging. The increase in [Ca2+]i at the wave front preceded depletion of the SR at each point along the calcium wave front, while during this latency period a transient increase of [Ca2+]SR was observed. This transient elevation of [Ca2+]SR could be identified at individual release junctions and depended on the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). Increased SERCA activity (β-adrenergic stimulation with 1 μm isoproterenol (isoprenaline)) decreased the latency period and increased the amplitude of the transient elevation of [Ca2+]SR, whereas inhibition of SERCA (3 μm cyclopiazonic acid) had the opposite effect. In conclusion, the data provide experimental evidence that local Ca2+ uptake by SERCA into the SR facilitates the propagation of cytosolic Ca2+ waves via luminal sensitization of the RyR, and supports a novel paradigm of a ‘fire-diffuse-uptake-fire’ mechanism for Ca2+ wave propagation in cardiac myocytes. PMID:22988145

Detection of Sequence-Specific Tyrosine Nitration of Manganese SOD and SERCA in Cardiovascular Disease and Aging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Shanqin; Ying, Jia; Jiang, Bingbing

2006-06-01

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies towards site-specific nY-modified proteins and to use histochemical and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies towards peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with angiotensin II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum withmore » known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite that chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site, but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates, but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.« less

Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak

PubMed Central

Sankaranarayanan, Rajiv; Li, Yatong; Greensmith, David J.; Eisner, David A.

2016-01-01

Key points Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay.In the presence of β‐adrenergic stimulation, RyR‐mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase.Two forms of Ca leak have been studied, Ca‐sensitising (induced by caffeine) and non‐sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient.Only Ca‐sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine.Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient. Abstract In heart failure, a reduction in Ca transient amplitude and contractile dysfunction can by caused by Ca leak through the sarcoplasmic reticulum (SR) Ca channel (ryanodine receptor, RyR) and/or decreased activity of the SR Ca ATPase (SERCA). We have characterised the effects of two forms of Ca leak (Ca‐sensitising and non‐sensitising) on calcium cycling and compared with those of SERCA inhibition. We measured [Ca2+]i with fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing SR leak with either caffeine (to sensitise the RyR to Ca activation) or ryanodine (non‐sensitising) had similar effects to SERCA inhibition: decreased systolic [Ca2+]i, increased diastolic [Ca2+]i and slowed decay. However, in the presence of isoproterenol, leak produced a biphasic decay of the Ca transient in the majority of cells while SERCA inhibition produced monophasic decay. Tetracaine reversed the effects of caffeine but not of ryanodine. When caffeine (1 mmol l−1) was added to a cell which displayed Ca waves, the wave frequency initially increased before waves disappeared and biphasic decay developed. Eventually (at higher caffeine concentrations), the biphasic decay was replaced by slow decay. We conclude that, in the presence of adrenergic stimulation, Ca leak can produce biphasic decay; the slow phase results from the leak opposing Ca uptake by SERCA. The degree of leak determines whether decay of Ca waves, biphasic or monophasic, occurs. PMID:26537441

Levosimendan reduces myocardial damage and improves cardiodynamics in streptozotocin induced diabetic cardiomyopathy via SERCA2a/NCX1 pathway.

PubMed

Akhtar, Md Sayeed; Pillai, Krishna Kolappa; Hassan, Md Quamrul; Dhyani, Neha; Ismail, Md Vasim; Najmi, Abul Kalam

2016-05-15

Diabetic cardiomyopathy (DCM) is one of the most common causes of mortality. Its pathophysiology is not fully understood and involve number of factors including, cardiovascular and metabolic disorders. The present study was designed to study the pathogenesis of DCM and to explore the effects of levosimendan along with either ramipril or insulin in the long term management of DCM. Streptozotocin (STZ) was used to develop DCM in Wistar rats at the dose of 25mg/kg body weight for three consecutive days. Rats were randomly divided into 9 groups and treatments were started after 2weeks of STZ administration. Persistent hyperglycemia was observed in STZ treated rats, leading to significant contractile dysfunction as evidenced by decreased left ventricular pressure (LVP), +LV (dp/dt), -LV (dp/dt) as well as elevated Tau and LVEDP. Marked myocardial damage such as fibrosis, increased wall tension, depletion of contractile proteins were observed as evidenced by increased levels of TGF-β, BNP, cTroponin-I, as well as decreased expression of SERCA2a and NCX1 proteins in diabetic rats. The levosimendan alone and also in combination with either ramipril or insulin significantly normalized the myocardial dysfunctions developed during the course of persistent hyperglycemia. The study suggests that levosimendan treatment improves cardiac dysfunction significantly. Its combined use with ramipril proves better than with insulin in correcting myocardial performance as well as reduction in myocardial damage. Copyright © 2016 Elsevier Inc. All rights reserved.

Intrinsic attenuation of post-irradiation calcium and ER stress imparts significant radioprotection to lepidopteran insect cells.

PubMed

Guleria, Ayushi; Thukral, Neha; Chandna, Sudhir

2018-04-15

Sf9 lepidopteran insect cells are 100-200 times more radioresistant than mammalian cells. This distinctive feature thus makes them suitable for studies exploring radioprotective molecular mechanisms. It has been established from previous studies of our group that downstream mitochondrial apoptotic signaling pathways in Sf9 cells are quite similar to mammalian cells, implicating the upstream signaling pathways in their extensive radioresistance. In the present study, intracellular and mitochondrial calcium levels remained unaltered in Sf9 cells in response to radiation, in sharp contrast to human (HEK293T) cells. The isolated mitochondria from Sf9 cells exhibited nearly 1.5 times greater calcium retention capacity than mammalian cells, highlighting their inherent stress resilience. Importantly, UPR/ER stress marker proteins (p-eIF2α, GRP4 and SERCA) remained unaltered by radiation and suggested highly attenuated ER and calcium stress. Lack of SERCA induction further corroborates the lack of radiation-induced calcium mobilization in these cells. The expression of CaMKII, an important effector molecule of calcium signaling, did not alter in response to radiation. Inhibiting CaMKII by KN-93 or suppressing CaM by siRNA failed to alter Sf9 cells response to radiation and suggests CaM-CaMKII independent radiation signaling. Therefore, this study suggests that attenuated calcium signaling/ER stress is an important determinant of lepidopteran cell radioresistance. Copyright © 2018 Elsevier Inc. All rights reserved.

SGK3 sustains ERα signaling and drives acquired aromatase inhibitor resistance through maintaining endoplasmic reticulum homeostasis.

PubMed

Wang, Yuanzhong; Zhou, Dujin; Phung, Sheryl; Warden, Charles; Rashid, Rumana; Chan, Nymph; Chen, Shiuan

2017-02-21

Many estrogen receptor alpha (ERα)-positive breast cancers initially respond to aromatase inhibitors (AIs), but eventually acquire resistance. Here, we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives acquired AI resistance. SGK3 is up-regulated and essential for endoplasmic reticulum (EnR) homeostasis through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function in AI-resistant cells. We have further found that EnR stress response down-regulates ERα expression through the protein kinase RNA-like EnR kinase (PERK) arm, and SGK3 retains ERα expression and signaling by preventing excessive EnR stress. Our study reveals regulation of ERα expression mediated by the EnR stress response and the feed-forward regulation between SGK3 and ERα in breast cancer. Given SGK3 inhibition reduces AI-resistant cell survival by eliciting excessive EnR stress and also depletes ERα expression/function, we propose SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer.

Systems approach to the study of stretch and arrhythmias in right ventricular failure induced in rats by monocrotaline

PubMed Central

Benoist, David; Stones, Rachel; Benson, Alan P.; Fowler, Ewan D.; Drinkhill, Mark J.; Hardy, Matthew E.L.; Saint, David A.; Cazorla, Olivier; Bernus, Olivier; White, Ed

2014-01-01

We demonstrate the synergistic benefits of using multiple technologies to investigate complex multi-scale biological responses. The combination of reductionist and integrative methodologies can reveal novel insights into mechanisms of action by tracking changes of in vivo phenomena to alterations in protein activity (or vice versa). We have applied this approach to electrical and mechanical remodelling in right ventricular failure caused by monocrotaline-induced pulmonary artery hypertension in rats. We show arrhythmogenic T-wave alternans in the ECG of conscious heart failure animals. Optical mapping of isolated hearts revealed discordant action potential duration (APD) alternans. Potential causes of the arrhythmic substrate; structural remodelling and/or steep APD restitution and dispersion were observed, with specific remodelling of the Right Ventricular Outflow Tract. At the myocyte level, [Ca2+]i transient alternans were observed together with decreased activity, gene and protein expression of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Computer simulations of the electrical and structural remodelling suggest both contribute to a less stable substrate. Echocardiography was used to estimate increased wall stress in failure, in vivo. Stretch of intact and skinned single myocytes revealed no effect on the Frank-Starling mechanism in failing myocytes. In isolated hearts acute stretch-induced arrhythmias occurred in all preparations. Significant shortening of the early APD was seen in control but not failing hearts. These observations may be linked to changes in the gene expression of candidate mechanosensitive ion channels (MSCs) TREK-1 and TRPC1/6. Computer simulations incorporating MSCs and changes in ion channels with failure, based on altered gene expression, largely reproduced experimental observations. PMID:25016242

Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaakinen, Mika; Papponen, Hinni; Metsikkoe, Kalervo

2008-01-15

The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca{sup 2+}-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped virusesmore » indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.« less

Characterization of ryanodine receptor and Ca2+-ATPase isoforms in the thermogenic heater organ of blue marlin (Makaira nigricans).

PubMed

Morrissette, Jeffery M; Franck, Jens P G; Block, Barbara A

2003-03-01

A thermogenic organ is found beneath the brain of billfishes (Istiophoridae), swordfish (Xiphiidae) and the butterfly mackerel (Scombridae). The heater organ has been shown to warm the brain and eyes up to 14 degrees C above ambient water temperature. Heater cells are derived from extraocular muscle fibers and express a modified muscle phenotype with an extensive transverse-tubule (T-tubule) network and sarcoplasmic reticulum (SR) enriched in Ca(2+)-ATPase (SERCA) pumps and ryanodine receptors (RyRs). Heater cells have a high mitochondria content but have lost most of the contractile myofilaments. Thermogenesis has been hypothesized to be associated with release and reuptake of Ca(2+). In this study, Ca(2+) fluxes in heater SR vesicles derived from blue marlin (Makaira nigricans) were measured using fura-2 fluorescence. Upon the addition of MgATP, heater SR vesicles rapidly sequestered Ca(2+). Uptake of Ca(2+) was thapsigargin sensitive, and maximum loading ranged between 0.8 micro mol Ca(2+) mg(-1) protein and 1.0 micro mol Ca(2+) mg(-1) protein. Upon the addition of 10 mmol l(-1) caffeine or 350 micro mol l(-1) ryanodine, heater SR vesicles released only a small fraction of the loaded Ca(2+). However, ryanodine could elicit a much larger Ca(2+) release event when the activity of the SERCA pumps was reduced. RNase protection assays revealed that heater tissue expresses an RyR isoform that is also expressed in fish slow-twitch skeletal muscle but is distinct from the RyR expressed in fish fast-twitch skeletal muscle. The heater and slow-twitch muscle RyR isoform has unique physiological properties. In the presence of adenine nucleotides, this RyR remains open even though cytoplasmic Ca(2+) is elevated, a condition that normally closes RyRs. The fast Ca(2+) sequestration by the heater SR, coupled with a physiologically unique RyR, is hypothesized to promote Ca(2+) cycling, ATP turnover and heat generation. A branch of the oculomotor nerve innervates heater organs, and, in this paper, we demonstrate that heater cells contain large 'endplate-like' clusters of acetylcholine receptors that appear to provide a mechanism for nervous control of thermogenesis.

Overexpression Myocardial Inducible Nitric Oxide Synthase Exacerbates Cardiac Dysfunction and Beta-Adrenergic Desensitization in Experimental Hypothyroidism☆,☆☆

PubMed Central

Shao, Qun; Cheng, Heng-Jie; Callahan, Michael F.; Kitzman, Dalane W; Li, Wei-Min; Cheng, Che Ping

2015-01-01

Background Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cadiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca2+]i transient ([Ca2+]iT), and β-adrenergic hyporesponsiveness. Methods and Results We simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca2+]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400W, 10−5 mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca2+]iT. In hypothyroidism, isoproterenol (10−8 M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca2+]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca2+]iT. Conclusions Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cadiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism. PMID:26681542

Overexpression myocardial inducible nitric oxide synthase exacerbates cardiac dysfunction and beta-adrenergic desensitization in experimental hypothyroidism.

PubMed

Shao, Qun; Cheng, Heng-Jie; Callahan, Michael F; Kitzman, Dalane W; Li, Wei-Min; Cheng, Che Ping

2016-02-01

Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cardiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca(2+)]i transient ([Ca(2+)]iT), and β-adrenergic hyporesponsiveness. We simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca(2+)]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400 W, 10(-5)mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca(2+)]iT. In hypothyroidism, isoproterenol (10(-8)M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca(2+)]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca(2+)]iT. Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cardiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

β-Arrestin2 Improves Post-Myocardial Infarction Heart Failure via Sarco(endo)plasmic Reticulum Ca2+-ATPase-Dependent Positive Inotropy in Cardiomyocytes.

PubMed

McCrink, Katie A; Maning, Jennifer; Vu, Angela; Jafferjee, Malika; Marrero, Christine; Brill, Ava; Bathgate-Siryk, Ashley; Dabul, Samalia; Koch, Walter J; Lymperopoulos, Anastasios

2017-11-01

Heart failure is the leading cause of death in the Western world, and new and innovative treatments are needed. The GPCR (G protein-coupled receptor) adapter proteins βarr (β-arrestin)-1 and βarr-2 are functionally distinct in the heart. βarr1 is cardiotoxic, decreasing contractility by opposing β 1 AR (adrenergic receptor) signaling and promoting apoptosis/inflammation post-myocardial infarction (MI). Conversely, βarr2 inhibits apoptosis/inflammation post-MI but its effects on cardiac function are not well understood. Herein, we sought to investigate whether βarr2 actually increases cardiac contractility. Via proteomic investigations in transgenic mouse hearts and in H9c2 rat cardiomyocytes, we have uncovered that βarr2 directly interacts with SERCA2a (sarco[endo]plasmic reticulum Ca 2+ -ATPase) in vivo and in vitro in a β 1 AR-dependent manner. This interaction causes acute SERCA2a SUMO (small ubiquitin-like modifier)-ylation, increasing SERCA2a activity and thus, cardiac contractility. βarr1 lacks this effect. Moreover, βarr2 does not desensitize β 1 AR cAMP-dependent procontractile signaling in cardiomyocytes, again contrary to βarr1. In vivo, post-MI heart failure mice overexpressing cardiac βarr2 have markedly improved cardiac function, apoptosis, inflammation, and adverse remodeling markers, as well as increased SERCA2a SUMOylation, levels, and activity, compared with control animals. Notably, βarr2 is capable of ameliorating cardiac function and remodeling post-MI despite not increasing cardiac βAR number or cAMP levels in vivo. In conclusion, enhancement of cardiac βarr2 levels/signaling via cardiac-specific gene transfer augments cardiac function safely, that is, while attenuating post-MI remodeling. Thus, cardiac βarr2 gene transfer might be a novel, safe positive inotropic therapy for both acute and chronic post-MI heart failure. © 2017 American Heart Association, Inc.

Randomized Clinical Trials of Gene Transfer for Heart Failure with Reduced Ejection Fraction.

PubMed

Penny, William F; Hammond, H Kirk

2017-05-01

Despite improvements in drug and device therapy for heart failure, hospitalization rates and mortality have changed little in the past decade. Randomized clinical trials using gene transfer to improve function of the failing heart are the focus of this review. Four randomized clinical trials of gene transfer in heart failure with reduced ejection fraction (HFrEF) have been published. Each enrolled patients with stable symptomatic HFrEF and used either intracoronary delivery of a virus vector or endocardial injection of a plasmid. The initial CUPID trial randomized 14 subjects to placebo and 25 subjects to escalating doses of adeno-associated virus type 1 encoding sarcoplasmic reticulum calcium ATPase (AAV1.SERCA2a). AAV1.SERCA2a was well tolerated, and the high-dose group met a 6 month composite endpoint. In the subsequent CUPID-2 study, 243 subjects received either placebo or the high dose of AAV1.SERCA2a. AAV1.SERCA2a administration, while safe, failed to meet the primary or any secondary endpoints. STOP-HF used plasmid endocardial injection of stromal cell-derived factor-1 to promote stem-cell recruitment. In a 93-subject trial of patients with ischemic etiology heart failure, the primary endpoint (symptoms and 6 min walk distance) failed, but subgroup analyses showed improvements in subjects with the lowest ejection fractions. A fourth trial randomized 14 subjects to placebo and 42 subjects to escalating doses of adenovirus-5 encoding adenylyl cyclase 6 (Ad5.hAC6). There were no safety concerns, and patients in the two highest dose groups (combined) showed improvements in left ventricular function (left ventricular ejection fraction and -dP/dt). The safety data from four randomized clinical trials of gene transfer in patients with symptomatic HFrEF suggest that this approach can be conducted with acceptable risk, despite invasive delivery techniques in a high-risk population. Additional trials are necessary before the approach can be endorsed for clinical practice.

Calsequestrin content and SERCA determine normal and maximal Ca2+ storage levels in sarcoplasmic reticulum of fast- and slow-twitch fibres of rat.

PubMed

Murphy, Robyn M; Larkins, Noni T; Mollica, Janelle P; Beard, Nicole A; Lamb, Graham D

2009-01-15

Whilst calsequestrin (CSQ) is widely recognized as the primary Ca2+ buffer in the sarcoplasmic reticulum (SR) in skeletal muscle fibres, its total buffering capacity and importance have come into question. This study quantified the absolute amount of CSQ isoform 1 (CSQ1, the primary isoform) present in rat extensor digitorum longus (EDL) and soleus fibres, and related this to their endogenous and maximal SR Ca2+ content. Using Western blotting, the entire constituents of minute samples of muscle homogenates or segments of individual muscle fibres were compared with known amounts of purified CSQ1. The fidelity of the analysis was proven by examining the relative signal intensity when mixing muscle samples and purified CSQ1. The CSQ1 contents of EDL fibres, almost exclusively type II fibres, and soleus type I fibres [SOL (I)] were, respectively, 36 +/- 2 and 10 +/- 1 micromol (l fibre volume)(-1), quantitatively accounting for the maximal SR Ca2+ content of each. Soleus type II [SOL (II)] fibres (approximately 20% of soleus fibres) had an intermediate amount of CSQ1. Every SOL (I) fibre examined also contained some CSQ isoform 2 (CSQ2), which was absent in every EDL and other type II fibre except for trace amounts in one case. Every EDL and other type II fibre had a high density of SERCA1, the fast-twitch muscle sarco(endo)plasmic reticulum Ca2+-ATPase isoform, whereas there was virtually no SERCA1 in any SOL (I) fibre. Maximal SR Ca2+ content measured in skinned fibres increased with CSQ1 content, and the ratio of endogenous to maximal Ca2+ content was inversely correlated with CSQ1 content. The relative SR Ca2+ content that could be maintained in resting cytoplasmic conditions was found to be much lower in EDL fibres than in SOL (I) fibres (approximately 20 versus >60%). Leakage of Ca2+ from the SR in EDL fibres could be substantially reduced with a SR Ca2+ pump blocker and increased by adding creatine to buffer cytoplasmic [ADP] at a higher level, both results indicating that at least part of the Ca2+ leakage occurred through SERCA. It is concluded that CSQ1 plays an important role in EDL muscle fibres by providing a large total pool of releasable Ca2+ in the SR whilst maintaining free [Ca2+] in the SR at sufficiently low levels that Ca2+ leakage through the high density of SERCA1 pumps does not metabolically compromise muscle function.

Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.

PubMed

Pirkmajer, Sergej; Kirchner, Henriette; Lundell, Leonidas S; Zelenin, Pavel V; Zierath, Juleen R; Makarova, Kira S; Wolf, Yuri I; Chibalin, Alexander V

2017-07-15

Small transmembrane proteins such as FXYDs, which interact with Na + ,K + -ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca 2+ -ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny-based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport. Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1-12), which regulate Na + ,K + -ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence-based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony vertebrates (Euteleostomi). Diversification of SERCA regulators was much less extensive, indicating they operate under different evolutionary constraints. Finally, we found that FXYDs in extant vertebrates can be classified into 13 gene subfamilies, which do not always correspond to the established FXYD classification. We therefore propose a revised classification that is based on evolutionary history of FXYDs and that is consistent across vertebrate species. Collectively, our findings provide an improved framework for investigating the function of ion transport in health and disease. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Finite Element Estimation of Protein-Ligand Association Rates with Post-Encounter Effects: Applications to Calcium binding in Troponin C and SERCA

PubMed Central

Kekenes-Huskey, P. M.; Gillette, A.; Hake, J.; McCammon, J. A.

2012-01-01

We introduce a computational pipeline and suite of software tools for the approximation of diffusion-limited binding based on a recently developed theoretical framework. Our approach handles molecular geometries generated from high-resolution structural data and can account for active sites buried within the protein or behind gating mechanisms. Using tools from the FEniCS library and the APBS solver, we implement a numerical code for our method and study two Ca2+-binding proteins: Troponin C and the Sarcoplasmic Reticulum Ca2+ ATPase (SERCA). We find that a combination of diffusional encounter and internal ‘buried channel’ descriptions provide superior descriptions of association rates, improving estimates by orders of magnitude. PMID:23293662

Finite Element Estimation of Protein-Ligand Association Rates with Post-Encounter Effects: Applications to Calcium binding in Troponin C and SERCA.

PubMed

Kekenes-Huskey, P M; Gillette, A; Hake, J; McCammon, J A

2012-10-31

We introduce a computational pipeline and suite of software tools for the approximation of diffusion-limited binding based on a recently developed theoretical framework. Our approach handles molecular geometries generated from high-resolution structural data and can account for active sites buried within the protein or behind gating mechanisms. Using tools from the FEniCS library and the APBS solver, we implement a numerical code for our method and study two Ca(2+)-binding proteins: Troponin C and the Sarcoplasmic Reticulum Ca(2+) ATPase (SERCA). We find that a combination of diffusional encounter and internal 'buried channel' descriptions provide superior descriptions of association rates, improving estimates by orders of magnitude.

Dronedarone administration prevents body weight gain and increases tolerance of the heart to ischemic stress: a possible involvement of thyroid hormone receptor alpha1.

PubMed

Pantos, Constantinos; Mourouzis, Iordanis; Malliopoulou, Vassiliki; Paizis, Ioannis; Tzeis, Stylianos; Moraitis, Panagiotis; Sfakianoudis, Konstantinos; Varonos, Dennis D; Cokkinos, Dennis V

2005-01-01

Hypothyroid heart displays a phenotype of cardioprotection against ischemia and this study investigated whether administration of dronedarone, an amiodarone-like compound that has been shown to preferentially antagonize thyroid hormone binding to thyroid hormone receptor alpha1 (TRalpha1), results in a similar effect. Dronedarone was given in Wistar rats (90 mg/kg, once daily (od) for 2 weeks) (DRON), while untreated animals served as controls (CONT). Hypothyroidism (HYPO) was induced by propylthiouracil administration. Isolated rat hearts were perfused in Langendorff mode and subjected to 20 minutes of zero-flow global ischemia (I) followed by 45 minutes of reperfusion (R). 3,5,3' Triiodothyronine remained unchanged while body weight and food intake were reduced. alpha-Myosin heavy chain (alpha-MHC) decreased in DRON while beta-myosin heavy chain (beta-MHC) and sarcoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase) expression (SERCA) was similar to CONT. In HYPO, alpha-MHC and SERCA were decreased while beta-MHC was increased. Myocardial glycogen content was increased in both DRON and HYPO. In DRON, resting heart rate and contractility were reduced and ischemic contracture was significantly suppressed while postischemic left ventricular end-diastolic pressure and lactate dehydrogenase release (IU/L min) after I/R were significantly decreased. In conclusion, dronedarone treatment results in cardioprotection by selectively mimicking hypothyroidism. This is accompanied by a reduction in body weight because of the suppression of food intake. TRs might prove novel pharmacologic targets for the treatment of cardiovascular illnesses.

Exercise training prevents the development of cardiac dysfunction in the low-dose streptozotocin diabetic rats fed a high-fat diet.

PubMed

Epp, Riley A; Susser, Shanel E; Morissette, Marc P; Kehler, D Scott; Jassal, Davinder S; Duhamel, Todd A

2013-01-01

This study tested the hypothesis that exercise training would prevent the development of diabetes-induced cardiac dysfunction and altered expression of sarcoplasmic reticulum Ca(2 +)-transport proteins in the low-dose streptozotocin-induced diabetic rats fed a high-fat diet (HFD+STZ). Male Sprague-Dawley rats (4 weeks old; 125-150 g) were made diabetic using a high-fat diet (40% fat, w/w) and a low-dose of streptozotocin (35 mg·(kg body mass)(-1)) by intravenous injection. Diabetic animals were divided among a sedentary group (Sed+HFD+STZ) or an exercise-trained group (Ex+HFD+STZ) that accumulated 3554 ± 338 m·day(-1) of voluntary wheel running (mean ± SE). Sedentary animals fed a low-fat diet served as the control (Sed+LFD). Oral glucose tolerance was impaired in the sedentary diabetic group (1179 ± 29; area under the curve (a.u.c.)) compared with that in the sedentary control animals (1447 ± 42 a.u.c.). Although left ventricular systolic function was unchanged by diabetes, impaired E/A ratios (i.e., diastolic function) and rates of pressure decay (-dP/dt) indicated the presence of diastolic dysfunction. Diabetes also reduced SERCA2a protein content and maximal SERCA2a activity (V(max)) by 21% and 32%, respectively. In contrast, the change in each parameter was attenuated by exercise training. Based on these data, it appears that exercise training prevented the development of diabetic cardiomyopathy and the dysregulation of sarcoplasmic reticulum protein content in an inducible animal model of type 2 diabetes.

Temporal and spatial dynamics underlying capacitative calcium entry in human colonic smooth muscle.

PubMed

Kovac, Jason R; Chrones, Tom; Sims, Stephen M

2008-01-01

Following smooth muscle excitation and contraction, depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) entry (CCE) to replenish stores and sustain cytoplasmic Ca(2+) (Ca(2+)(i)) elevations. The objectives of the present study were to characterize CCE and the Ca(2+)(i) dynamics underlying human colonic smooth muscle contraction by using tension recordings, fluorescent Ca(2+)-indicator dyes, and patch-clamp electrophysiology. The neurotransmitter acetylcholine (ACh) contracted tissue strips and, in freshly isolated colonic smooth muscle cells (SMCs), caused elevation of Ca(2+)(i) as well as activation of nonselective cation currents. To deplete Ca(2+)(i) stores, the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid were added to a Ca(2+)-free bathing solution. Under these conditions, addition of extracellular Ca(2+) (3 mM) elicited increased tension that was inhibited by the cation channel blockers SKF-96365 (10 microM) and lanthanum (100 microM), suggestive of CCE. In a separate series of experiments on isolated SMCs, SERCA inhibition generated a gradual and sustained inward current. When combined with high-speed Ca(2+)-imaging techniques, the CCE-evoked rise of Ca(2+)(i) was associated with inward currents carrying Ca(2+) that were inhibited by SKF-96365. Regional specializations in Ca(2+) influx and handling during CCE were observed. Distinct "hotspot" regions of Ca(2+) rise and plateau were evident in 70% of cells, a feature not previously recognized in smooth muscle. We propose that store-operated Ca(2+) entry occurs in hotspots contributing to localized Ca(2+) elevations in human colonic smooth muscle.

Transport mechanism of the sarcoplasmic reticulum Ca2+ -ATPase pump.

PubMed

Møller, Jesper V; Nissen, Poul; Sørensen, Thomas L-M; le Maire, Marc

2005-08-01

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) belongs to the group of P-type ATPases, which actively transport inorganic cations across membranes at the expense of ATP hydrolysis. Three-dimensional structures of several transport intermediates of SERCA1a, stabilized by structural analogues of ATP and phosphoryl groups, are now available at atomic resolution. This has enabled the transport cycle of the protein to be described, including the coupling of Ca(2+) occlusion and phosphorylation by ATP, and of proton counter-transport and dephosphorylation. From these structures, Ca(2+)-ATPase gradually emerges as a molecular mechanical device in which some of the transmembrane segments perform Ca(2+) transport by piston-like movements and by the transmission of reciprocating movements that affect the chemical reactivity of the cytosolic globular domains.

Woodpecker drumming behavior is linked to the elevated expression of genes that encode calcium handling proteins in the neck musculature.

PubMed

Schuppe, Eric R; Petersen, John O; Fuxjager, Matthew J

2018-05-31

Many animals perform elaborate physical displays for social communication. Identifying molecular mechanisms that co-evolve with these complex behavioral signals can therefore help reveal how forces of selection shape animal design. To study this issue, we examine gene expression profiles in select skeletal muscles that actuate woodpecker drum displays. This remarkable whole-body signal is produced when individuals rapidly hammer their bill against trees. We find that, compared to muscles that play no part in producing this behavior, the main muscle used to drum abundantly expresses two genes that encode proteins that support myocytic calcium (Ca 2+ ) handling dynamics-namely parvalbumin (PV) and sarcoplasmic reticulum Ca 2+ ATPase (SERCA1). Meanwhile, we find no such difference in the expression of another gene similarly vital to Ca 2+ handling, the ryanodine receptor (RYR1). These differences are not present in a non-woodpecker species, which readily produce much slower drum-like movements for foraging (but not social signaling). Our data therefore point to an association between the fast drum displays of woodpeckers and muscle-specific expression of genes whose protein products enhance select aspects of myocytic Ca 2+ handling. © 2018. Published by The Company of Biologists Ltd.

Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

NASA Astrophysics Data System (ADS)

Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

1999-07-01

Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

Isoflurane-Induced Caspase-3 Activation Is Dependent on Cytosolic Calcium and Can Be Attenuated by Memantine

PubMed Central

Zhang, Guohua; Dong, Yuanlin; Zhang, Bin; Ichinose, Fumito; Wu, Xu; Culley, Deborah J.; Crosby, Gregory

2008-01-01

Increasing evidence indicates that caspase activation and apoptosis are associated with a variety of neurodegenerative disorders, including Alzheimer's disease. We reported that anesthetic isoflurane can induce apoptosis, alter processing of the amyloid precursor protein (APP), and increase amyloid-β protein (Aβ) generation. However, the mechanism by which isoflurane induces apoptosis is primarily unknown. We therefore set out to assess effects of extracellular calcium concentration on isoflurane-induced caspase-3 activation in H4 human neuroglioma cells stably transfected to express human full-length APP (H4-APP cells). In addition, we tested effects of RNA interference (RNAi) silencing of IP3 receptor, NMDA receptor, and endoplasmic reticulum (ER) calcium pump, sacro-/ER calcium ATPase (SERCA1). Finally, we examined the effects of the NMDA receptor partial antagonist, memantine, in H4-APP cells and brain tissue of naive mice. EDTA (10 mm), BAPTA (10 μm), and RNAi silencing of IP3 receptor, NMDA receptor, or SERCA1 attenuated capase-3 activation. Memantine (4 μm) inhibited isoflurane-induced elevations in cytosolic calcium levels and attenuated isoflurane-induced caspase-3 activation, apoptosis, and cell viability. Memantine (20 mg/kg, i.p.) reduced isoflurane-induced caspase-3 activation in brain tissue of naive mice. These results suggest that disruption of calcium homeostasis underlies isoflurane-induced caspase activation and apoptosis. We also show for the first time that the NMDA receptor partial antagonist, memantine, can prevent isoflurane-induced caspase-3 activation and apoptosis in vivo and in vitro. These findings, indicating that isoflurane-induced caspase activation and apoptosis are dependent on cytosolic calcium levels, should facilitate the provision of safer anesthesia care, especially for Alzheimer's disease and elderly patients. PMID:18434534

Store-operated Ca2+ entry supports contractile function in hearts of hibernators

PubMed Central

Nakipova, Olga V.; Averin, Alexey S.; Evdokimovskii, Edward V.; Pimenov, Oleg Yu.; Kosarski, Leonid; Ignat’ev, Dmitriy; Anufriev, Andrey; Kokoz, Yuri M.; Reyes, Santiago; Terzic, Andre; Alekseev, Alexey E.

2017-01-01

Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA). We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C–7°C–30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states. PMID:28531217

Increased LDL electronegativity in chronic kidney disease disrupts calcium homeostasis resulting in cardiac dysfunction.

PubMed

Chang, Kuan-Cheng; Lee, An-Sheng; Chen, Wei-Yu; Lin, Yen-Nien; Hsu, Jing-Fang; Chan, Hua-Chen; Chang, Chia-Ming; Chang, Shih-Sheng; Pan, Chia-Chi; Sawamura, Tatsuya; Chang, Chi-Tzong; Su, Ming-Jai; Chen, Chu-Huang

2015-07-01

Chronic kidney disease (CKD), an independent risk factor for cardiovascular disease, is associated with abnormal lipoprotein metabolism. We examined whether electronegative low-density lipoprotein (LDL) is mechanistically linked to cardiac dysfunction in patients with early CKD. We compared echocardiographic parameters between patients with stage 2 CKD (n = 88) and normal controls (n = 89) and found that impaired relaxation was more common in CKD patients. Reduction in estimated glomerular filtration rate was an independent predictor of left ventricular relaxation dysfunction. We then examined cardiac function in a rat model of early CKD induced by unilateral nephrectomy (UNx) by analyzing pressure-volume loop data. The time constant of isovolumic pressure decay was longer and the maximal velocity of pressure fall was slower in UNx rats than in controls. When we investigated the mechanisms underlying relaxation dysfunction, we found that LDL from CKD patients and UNx rats was more electronegative than LDL from their respective controls and that LDL from UNx rats induced intracellular calcium overload in H9c2 cardiomyocytes in vitro. Furthermore, chronic administration of electronegative LDL, which signals through lectin-like oxidized LDL receptor-1 (LOX-1), induced relaxation dysfunction in wild-type but not LOX-1(-/-) mice. In in vitro and in vivo experiments, impaired cardiac relaxation was associated with increased calcium transient resulting from nitric oxide (NO)-dependent nitrosylation of SERCA2a due to increases in inducible NO synthase expression and endothelial NO synthase uncoupling. In conclusion, LDL becomes more electronegative in early CKD. This change disrupts SERCA2a-regulated calcium homeostasis, which may be the mechanism underlying cardiorenal syndrome. Copyright © 2015 Elsevier Ltd. All rights reserved.

Overexpression of human mutated G93A SOD1 changes dynamics of the ER mitochondria calcium cycle specifically in mouse embryonic motor neurons.

PubMed

Lautenschläger, Janin; Prell, Tino; Ruhmer, Julia; Weidemann, Lisa; Witte, Otto W; Grosskreutz, Julian

2013-09-01

Motor neurons vulnerable to the rapidly progressive deadly neurodegenerative disease amyotrophic lateral sclerosis (ALS) inherently express low amounts of calcium binding proteins (CaBP), likely to allow physiological motor neuron firing frequency modulation. At the same time motor neurons are susceptible to AMPA receptor mediated excitotoxicity and internal calcium deregulation which is not fully understood. We analysed ER mitochondria calcium cycle (ERMCC) dynamics with subsecond resolution in G93A hSOD1 overexpressing motor neurons as a model of ALS using fluorescent calcium imaging. When comparing vulnerable motor neurons and non-motor neurons from G93A hSOD1 mice and their non-transgenic littermates, we found a decelerated cytosolic calcium clearance in the presence of G93A hSOD1. While both non-transgenic as well as G93A hSOD1 motor neurons displayed large mitochondrial calcium uptake by the mitochondrial uniporter (mUP), the mitochondrial calcium extrusion system was altered in the presence of G93A hSOD1. In addition, ER calcium uptake by the sarco-/endoplasmic reticulum ATPase (SERCA) was increased in G93A hSOD1 motor neurons. In survival assays, blocking the mitochondrial sodium calcium exchanger (mNCE) by CGP37157 as well as inhibiting SERCA by cyclopiazonic acid showed protective effects against kainate induced excitotoxicity. Thus, our study shows for the first time that the functional consequence of G93A hSOD1 overexpression in intact motor neurons is indeed a disturbance of the ER mitochondria calcium cycle, and identified two promising targets for therapeutic intervention in the pathology of ALS. Copyright © 2013 Elsevier Inc. All rights reserved.

Genetic deletion of muscle RANK or selective inhibition of RANKL is not as effective as full-length OPG-fc in mitigating muscular dystrophy.

PubMed

Dufresne, Sébastien S; Boulanger-Piette, Antoine; Bossé, Sabrina; Argaw, Anteneh; Hamoudi, Dounia; Marcadet, Laetitia; Gamu, Daniel; Fajardo, Val A; Yagita, Hideo; Penninger, Josef M; Russell Tupling, A; Frenette, Jérôme

2018-04-24

Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that regulates synchronously bone and skeletal muscle physiopathology is still elusive. Receptor-activator of nuclear factor κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are the key regulators of osteoclast differentiation and bone remodelling. We thus hypothesized that RANK/RANKL/OPG, which is a key pathway for bone regulation, is involved in Duchenne muscular dystrophy (DMD) physiopathology. Our results show that muscle-specific RANK deletion (mdx-RANK mko ) in dystrophin deficient mdx mice improves significantly specific force [54% gain in force] of EDL muscles with no protective effect against eccentric contraction-induced muscle dysfunction. In contrast, full-length OPG-Fc injections restore the force of dystrophic EDL muscles [162% gain in force], protect against eccentric contraction-induced muscle dysfunction ex vivo and significantly improve functional performance on downhill treadmill and post-exercise physical activity. Since OPG serves a soluble receptor for RANKL and as a decoy receptor for TRAIL, mdx mice were injected with anti-RANKL and anti-TRAIL antibodies to decipher the dual function of OPG. Injections of anti-RANKL and/or anti-TRAIL increase significantly the force of dystrophic EDL muscle [45% and 17% gains in force, respectively]. In agreement, truncated OPG-Fc that contains only RANKL domains produces similar gains, in terms of force production, than anti-RANKL treatments. To corroborate that full-length OPG-Fc also acts independently of RANK/RANKL pathway, dystrophin/RANK double-deficient mice were treated with full-length OPG-Fc for 10 days. Dystrophic EDL muscles exhibited a significant gain in force relative to untreated dystrophin/RANK double-deficient mice, indicating that the effect of full-length OPG-Fc is in part independent of the RANKL/RANK interaction. The sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) activity is significantly depressed in dysfunctional and dystrophic muscles and full-length OPG-Fc treatment increased SERCA activity and SERCA-2a expression. These findings demonstrate the superiority of full-length OPG-Fc treatment relative to truncated OPG-Fc, anti-RANKL, anti-TRAIL or muscle RANK deletion in improving dystrophic muscle function, integrity and protection against eccentric contractions. In conclusion, full-length OPG-Fc represents an efficient alternative in the development of new treatments for muscular dystrophy in which a single therapeutic approach may be foreseeable to maintain both bone and skeletal muscle functions.

Rhodiola Inhibits Atrial Arrhythmogenesis in a Heart Failure Model.

PubMed

Liu, Shuen-Hsin; Hsiao, Ya-Wen; Chong, Eric; Singhal, Rahul; Fong, Man-Cai; Tsai, Yung-Nan; Hsu, Chiao-Po; Chen, Yao-Chang; Chen, Yi-Jen; Chiou, Chuen-Wang; Chiang, Shuo-Ju; Chang, Shih-Lin; Chen, Shih-Ann

2016-09-01

Rhodiola, a popular plant in Tibet, has been proven to decrease arrhythmia. The aim of this study was to elucidate the molecular mechanism and electrophysiological properties of rhodiola in the suppression of atrial fibrillation. This study consisted of 3 groups as follows: Group 1: normal control rabbits (n = 5); Group 2: rabbits with heart failure (HF) created by coronary ligation and who received 2 weeks of water orally as a placebo (n = 5); and Group 3: rabbits with HF who received 2 weeks of a rhodiola 270 mg/kg/day treatment orally (n = 5). The monophasic action potential, histology, and real-time polymerase chain reaction (RT-PCR) analysis of ionic channels and PI3K/AKT/eNOS were examined. Compared with the HF group, attenuated atrial fibrosis (35.4 ± 17.4% vs. 16.9 ± 8.4%, P = 0.05) and improved left ventricular (LV) ejection fraction (51.6 ± 3.4% vs. 68.0 ± 0.5%, P = 0.001) were observed in the rhodiola group. The rhodiola group had a shorter ERP (85.3 ± 6.8 vs. 94.3 ± 1.2, P = 0.002), APD90 (89.3 ± 1.5 vs. 112.7 ± 0.7, P < 0.001) in the left atrium (LA), and decreased AF inducibility (0.90 ± 0.04 vs. 0.42 ± 0.04, P < 0.001) compared with the HF group. The mRNA expressions of Kv1.4, Kv1.5, Kv4.3, KvLQT1, Cav1.2, and SERCA2a in the HF LA were up-regulated after rhodiola treatment. The rhodiola-treated HF LA demonstrated higher mRNA expression of PI3K-AKT compared with the HF group. Rhodiola reversed LA electrical remodeling, attenuated atrial fibrosis and suppressed AF in rabbits with HF. The beneficial electrophysiological effect of rhodiola may be related to upregulation of Kv1.4, Kv1.5, Kv4.3, KvLQT1, Cav1.2, SERCA2a, and activation of PI3K/AKT signaling. © 2016 Wiley Periodicals, Inc.

Phosphorylation of serine96 of histidine-rich calcium-binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia

PubMed Central

Pollak, Adam J.; Haghighi, Kobra; Kunduri, Swati; Arvanitis, Demetrios A.; Liu, Guan-Sheng; Singh, Vivek P.; Gonzalez, David J.; Sanoudou, Despina; Wiley, Sandra E.; Dixon, Jack E.; Kranias, Evangelia G.

2017-01-01

Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores. Histidine-rich Ca-binding protein (HRC) resides in the lumen of the SR, where it contributes to the regulation of Ca cycling by protecting stressed or failing hearts. The common Ser96Ala human genetic variant of HRC strongly correlates with life-threatening ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. However, the underlying molecular pathways of this disease remain undefined. Here, we demonstrate that family with sequence similarity 20C (Fam20C), a recently characterized protein kinase in the secretory pathway, phosphorylates HRC on Ser96. HRC Ser96 phosphorylation was confirmed in cells and human hearts. Furthermore, a Ser96Asp HRC variant, which mimics constitutive phosphorylation of Ser96, diminished delayed aftercontractions in HRC null cardiac myocytes. This HRC phosphomimetic variant was also able to rescue the aftercontractions elicited by the Ser96Ala variant, demonstrating that phosphorylation of Ser96 is critical for the cardioprotective function of HRC. Phosphorylation of HRC on Ser96 regulated the interactions of HRC with both triadin and SERCA2a, suggesting a unique mechanism for regulation of SR Ca homeostasis. This demonstration of the role of Fam20C-dependent phosphorylation in heart disease will open new avenues for potential therapeutic approaches against arrhythmias. PMID:28784772

Obesity Alters Molecular and Functional Cardiac Responses to Ischemia-Reperfusion and Glucagon-Like Peptide-1 Receptor Agonism

PubMed Central

Sassoon, Daniel J; Goodwill, Adam G; Noblet, Jillian N; Conteh, Abass M; Herring, B. Paul; McClintick, Jeanette N; Tune, Johnathan D; Mather, Kieren J

2016-01-01

This study tested the hypothesis that obesity alters the cardiac response to ischemia/reperfusion and/or glucagon like peptide-1 (GLP-1) receptor activation, and that these differences are associated with alterations in the obese cardiac proteome and microRNA (miR) transcriptome. Ossabaw swine were fed normal chow or obesogenic diet for 6 months. Cardiac function was assessed at baseline, during a 30-min coronary occlusion, and during 2 hours of reperfusion in anesthetized swine treated with saline or exendin-4 for 24 hours. Cardiac biopsies were obtained from normal and ischemia/reperfusion territories. Fat-fed animals were heavier, and exhibited hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. Plasma troponin-I concentration (index of myocardial injury) was increased following ischemia/reperfusion and decreased by exendin-4 treatment in both groups. Ischemia/reperfusion produced reductions in systolic pressure and stroke volume in lean swine. These indices were higher in obese hearts at baseline and relatively maintained throughout ischemia/reperfusion. Exendin-4 administration increased systolic pressure in lean swine but did not affect blood pressure in obese swine. End-diastolic volume was reduced by exendin-4 following ischemia/reperfusion in obese swine. These divergent physiologic responses were associated with obesity-related differences in proteins related to myocardial structure/function (e.g. titin) and calcium handling (e.g. SERCA2a, histidine-rich Ca2+ binding protein). Alterations in expression of cardiac miRs in obese hearts included miR-15, miR-27, miR-130, miR-181, and let-7. Taken together, these observations validate this discovery approach and reveal novel associations that suggest previously undiscovered mechanisms contributing to the effects of obesity on the heart and contributing to the actions of GLP-1 following ischemia/reperfusion. PMID:27234258

Impaired Ca2+ cycling of nonischemic myocytes contributes to sarcomere dysfunction early after myocardial infarction.

PubMed

Kronenbitter, Annette; Funk, Florian; Hackert, Katarzyna; Gorreßen, Simone; Glaser, Dennis; Boknik, Peter; Poschmann, Gereon; Stühler, Kai; Isić, Malgorzata; Krüger, Martina; Schmitt, Joachim P

2018-06-01

Changes in the nonischemic remote myocardium of the heart contribute to left ventricular dysfunction after ischemia and reperfusion (I/R). Understanding the underlying mechanisms early after I/R is crucial to improve the adaptation of the viable myocardium to increased mechanical demands. Here, we investigated the role of myocyte Ca 2+ handling in the remote myocardium 24 h after 60 min LAD occlusion. Cardiomyocytes isolated from the basal noninfarct-related parts of wild type mouse hearts demonstrated depressed beat-to-beat Ca 2+ handling. The amplitude of the Ca 2+ transients as well as the kinetics of Ca 2+ transport were reduced by up to 25%. These changes were associated with impaired sarcomere contraction. While expression levels of Ca 2+ regulatory proteins were unchanged in remote myocardium compared to the corresponding regions of sham-operated hearts, mobility shift analyses of phosphorylated protein showed 2.9 ± 0.4-fold more unphosphorylated phospholamban (PLN) monomers, the PLN species that inhibits the Ca 2+ ATPase SERCA2a (P ≤ 0.001). Phospho-specific antibodies revealed normal phosphorylation of PLN at T17 in remote myocardium, but markedly reduced phosphorylation at its PKA-dependent phosphorylation site, S16 (P ≤ 0.01). The underlying cause involved enhanced activity of protein phosphatases, particularly PP2A (P ≤ 0.01). In contrast, overall PKA activity was normal. The PLN interactome, as determined by co-immunoprecipitation and mass spectrometry, and the phosphorylation state of PKA targets other than PLN were also unchanged. Isoproterenol enhanced cellular Ca 2+ cycling much stronger in remote myocytes than in healthy controls and improved sarcomere function. We conclude that the reduced phosphorylation state of PLN at S16 impairs myocyte Ca 2+ cycling in the remote myocardium 24 h after I/R and contributes to contractile dysfunction. Copyright © 2018 Elsevier Ltd. All rights reserved.

Geographic Structuring of the Plasmodium falciparum Sarco(endo)plasmic Reticulum Ca2+ ATPase (PfSERCA) Gene Diversity

PubMed Central

Pinto, João; Gribaldo, Simonetta; Legrand, Eric; Niang, Makhtar; Kim, Nimol; Pharath, Lim; Volnay, Béatrice; Ekala, Marie Therese; Bouchier, Christiane; Fandeur, Thierry; Berzosa, Pedro; Benito, Agustin; Ferreira, Isabel Dinis; Ferreira, Cynthia; Vieira, Pedro Paulo; Alecrim, Maria das Graças; Mercereau-Puijalon, Odile; Cravo, Pedro

2010-01-01

Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp), of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates). This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte. PMID:20195531

Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity.

PubMed

Mortimer, Nathan T; Goecks, Jeremy; Kacsoh, Balint Z; Mobley, James A; Bowersock, Gregory J; Taylor, James; Schlenke, Todd A

2013-06-04

Because parasite virulence factors target host immune responses, identification and functional characterization of these factors can provide insight into poorly understood host immune mechanisms. The fruit fly Drosophila melanogaster is a model system for understanding humoral innate immunity, but Drosophila cellular innate immune responses remain incompletely characterized. Fruit flies are regularly infected by parasitoid wasps in nature and, following infection, flies mount a cellular immune response culminating in the cellular encapsulation of the wasp egg. The mechanistic basis of this response is largely unknown, but wasps use a mixture of virulence proteins derived from the venom gland to suppress cellular encapsulation. To gain insight into the mechanisms underlying wasp virulence and fly cellular immunity, we used a joint transcriptomic/proteomic approach to identify venom genes from Ganaspis sp.1 (G1), a previously uncharacterized Drosophila parasitoid species, and found that G1 venom contains a highly abundant sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. Accordingly, we found that fly immune cells termed plasmatocytes normally undergo a cytoplasmic calcium burst following infection, and that this calcium burst is required for activation of the cellular immune response. We further found that the plasmatocyte calcium burst is suppressed by G1 venom in a SERCA-dependent manner, leading to the failure of plasmatocytes to become activated and migrate toward G1 eggs. Finally, by genetically manipulating plasmatocyte calcium levels, we were able to alter fly immune success against G1 and other parasitoid species. Our characterization of parasitoid wasp venom proteins led us to identify plasmatocyte cytoplasmic calcium bursts as an important aspect of fly cellular immunity.

Disorder of endoplasmic reticulum calcium channel components is associated with the increased apoptotic potential in pale, soft, exudative pork.

PubMed

Guo, Bing; Zhang, Wangang; Tume, Ron K; Hudson, Nicholas J; Huang, Feng; Yin, Yan; Zhou, Guanghong

2016-05-01

Eight pale, soft and exudative (PSE) and eight reddish-pink, firm and non-exudative (RFN) porcine longissimus muscle samples were selected based on pH and L* at 1h postmortem (PM), and drip loss at 24h PM, and used to evaluate the cellular calcium and apoptosis status. We found that SERCA1 was decreased, while IP3R was decreased in PSE meat (P<0.05), indicative of the overloaded sarcoplasmic calcium status. In PSE meat, the pro-apoptotic factor BAX was increased while the anti-apoptotic factor Bcl-2 was decreased (P<0.05). The significantly increased activity of caspase 3 and the expression of its cleavage fragment suggested higher apoptotic potential in PSE meat compared with RFN meat (P<0.05). Moreover, the significantly higher expression level of cytochrome C (P<0.05) suggests the important role of mitochondria during apoptosis appearance in PSE meat. Taken together, our data inferred that the calcium channel disorder present in PSE meat was associated with the increased apoptotic potential. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanisms of intracellular calcium homeostasis in developing and mature bovine corpora lutea.

PubMed

Wright, Marietta F; Bowdridge, Elizabeth; McDermott, Erica L; Richardson, Samuel; Scheidler, James; Syed, Qaisar; Bush, Taylor; Inskeep, E Keith; Flores, Jorge A

2014-03-01

Although calcium (Ca(2+)) is accepted as an intracellular mediator of prostaglandin F2 alpha (PGF2alpha) actions on luteal cells, studies defining mechanisms of Ca(2+) homeostasis in bovine corpora lutea (CL) are lacking. The increase in intracellular Ca(2+) concentration ([Ca(2+)]i) induced by PGF2alpha in steroidogenic cells from mature CL is greater than in those isolated from developing CL. Our hypothesis is that differences in signal transduction associated with developing and mature CL contribute to the increased efficacy of PGF2alpha to induce a Ca(2+) signal capable of inducing regression in mature CL. To test this hypothesis, major genes participating in Ca(2+) homeostasis in the bovine CL were identified, and expression of mRNA, protein, or activity, in the case of phospholipase Cbeta (PLCbeta), in developing and mature bovine CL was compared. In addition, we examined the contribution of external and internal Ca(2+) to the PGF2alpha stimulated rise in [Ca(2+)]i in LLCs isolated from developing and mature bovine CL. Three differences were identified in mechanisms of calcium homeostasis between developing and mature CL, which could account for the lesser increase in [Ca(2+)]i in response to PGF2alpha in developing than in mature CL. First, there were lower concentrations of inositol 1,4,5-trisphosphate (IP3) after similar PGF2alpha challenge, indicating reduced phospholipase C beta (PLCbeta) activity, in developing than mature CL. Second, there was an increased expression of sorcin (SRI) in developing than in mature CL. This cytoplasmic Ca(2+) binding protein modulates the endoplasmic reticulum (ER) Ca(2+) release channel, ryanodine receptor (RyR), to be in the closed configuration. Third, there was greater expression of ATP2A2 or SERCA, which causes calcium reuptake into the ER, in developing than in mature CL. Developmental differences in expression detected in whole CL were confirmed by Western blots using protein samples from steroidogenic cells isolated from developing and mature CL. Localization of these genes in steroidogenic luteal cells was confirmed by immunohistochemistry. Therefore, it is concluded that the cellular mechanisms that allow PGF2alpha to induce a calcium signal of greater magnitude in mature than in developing CL involve 1) greater PLCbeta activity with enhanced generation of IP3, 2) an enhanced Ca(2+) release from the ER via unrestrained RYR2 due to a decrease in SRI expression, and 3) a reduction in calcium reuptake to the ER due to lower expression of ATP2A2. Accordingly, the increase in [Ca(2+)]i induced by PGF2alpha in mature large steroidogenic cells had less dependency from extracellular calcium than in those isolated from immature CL.

Longitudinal monitoring of Gaussia and Nano luciferase activities to concurrently assess ER calcium homeostasis and ER stress in vivo.

PubMed

Wires, Emily S; Henderson, Mark J; Yan, Xiaokang; Bäck, Susanne; Trychta, Kathleen A; Lutrey, Molly H; Harvey, Brandon K

2017-01-01

The endoplasmic reticulum (ER) is essential to many cellular processes including protein processing, lipid metabolism and calcium storage. The ability to longitudinally monitor ER homeostasis in the same organism would offer insight into progressive molecular and cellular adaptations to physiologic or pathologic states, but has been challenging. We recently described the creation of a Gaussia luciferase (GLuc)-based secreted ER calcium-modulated protein (SERCaMP or GLuc-SERCaMP) to longitudinally monitor ER calcium homeostasis. Here we describe a complementary tool to measure the unfolded protein response (UPR), utilizing a UPRE-driven secreted Nano luciferase (UPRE-secNLuc) to examine the activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1) pathways of the UPR. We observed an upregulation of endogenous ATF6- and XBP1-regulated genes following pharmacologically-induced ER stress that was consistent with responsiveness of the UPRE sensor. Both GLuc and NLuc-based reporters have favorable properties for in vivo studies, however, they are not easily used in combination due to overlapping substrate activities. We describe a method to measure the enzymatic activities of both reporters from a single sample and validated the approach using culture medium and rat blood samples to measure GLuc-SERCaMP and UPRE-secNLuc. Measuring GLuc and NLuc activities from the same sample allows for the robust and quantitative measurement of two cellular events or cell populations from a single biological sample. This study is the first to describe the in vivo measurement of UPRE activation by sampling blood, using an approach that allows concurrent interrogation of two components of ER homeostasis.

Ca2+-induced delayed afterdepolarizations are triggered by dyadic subspace Ca2+ affirming that increasing SERCA reduces aftercontractions

PubMed Central

Noble, Penelope J.; Noble, Denis

2011-01-01

Ca2+-induced delayed afterdepolarizations (DADs) are depolarizations that occur after full repolarization. They have been observed across multiple species and cell types. Experimental results have indicated that the main cause of DADs is Ca2+ overload. The main hypothesis as to their initiation has been Ca2+ overflow from the overloaded sarcoplasmic reticulum (SR). Our results using 37 previously published mathematical models provide evidence that Ca2+-induced DADs are initiated by the same mechanism as Ca2+-induced Ca2+ release, i.e., the modulation of the opening of ryanodine receptors (RyR) by Ca2+ in the dyadic subspace; an SR overflow mechanism was not necessary for the induction of DADs in any of the models. The SR Ca2+ level is better viewed as a modulator of the appearance of DADs and the magnitude of Ca2+ release. The threshold for the total Ca2+ level within the cell (not only the SR) at which Ca2+ oscillations arise in the models is close to their baseline level (∼1- to 3-fold). It is most sensitive to changes in the maximum sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump rate (directly proportional), the opening probability of RyRs, and the Ca2+ diffusion rate from the dyadic subspace into the cytosol (both indirectly proportional), indicating that the appearance of DADs is multifactorial. This shift in emphasis away from SR overload as the trigger for DADs toward a multifactorial analysis could explain why SERCA overexpression has been shown to suppress DADs (while increasing contractility) and why DADs appear during heart failure (at low SR Ca2+ levels). PMID:21666112

The vascular barrier-protecting hawthorn extract WS® 1442 raises endothelial calcium levels by inhibition of SERCA and activation of the IP3 pathway.

PubMed

Willer, Elisabeth A; Malli, Roland; Bondarenko, Alexander I; Zahler, Stefan; Vollmar, Angelika M; Graier, Wolfgang F; Fürst, Robert

2012-10-01

WS® 1442 has been proven as an effective and safe therapeutical to treat mild forms of congestive heart failure. Beyond this action, we have recently shown that WS® 1442 protects against thrombin-induced vascular barrier dysfunction and the subsequent edema formation by affecting endothelial calcium signaling. The aim of the study was to analyze the influence of WS® 1442 on intracellular calcium concentrations [Ca(2+)](i) in the human endothelium and to investigate the underlying mechanisms. Using ratiometric calcium measurements and a FRET sensor, we found that WS® 1442 concentration-dependently increased basal [Ca(2+)](i) by depletion of the endoplasmic reticulum (ER) and inhibited a subsequent histamine-triggered rise of [Ca(2+)](i). Interestingly, the augmented [Ca(2+)](i) did neither trigger an activation of the contractile machinery nor led to a barrier breakdown (macromolecular permeability). It also did not impair endothelial cell viability. As assessed by patch clamp recordings, WS® 1442 did only slightly affect endothelial Na(+)/K(+)-ATPase, but increased [Ca(2+)](i) by inhibiting the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) and by activating the inositol 1,4,5-trisphosphate (IP(3)) pathway. Most importantly, WS® 1442 did not induce store-operated calcium entry (SOCE), but even irreversibly prevented histamine-induced SOCE. Taken together, WS® 1442 prevented the deleterious hyperpermeability-associated rise of [Ca(2+)](i) by a preceding, non-toxic release of Ca(2+) from the ER. WS® 1442 interfered with SERCA and the IP(3) pathway without inducing SOCE. The elucidation of this intriguing mechanism helps to understand the complex pharmacology of the cardiovascular drug WS® 1442. Copyright © 2012 Elsevier Ltd. All rights reserved.

Infrasound-induced hemodynamics, ultrastructure, and molecular changes in the rat myocardium.

PubMed

Pei, Zhaohui; Sang, Hanfei; Li, Ruiman; Xiao, Pingxi; He, Jiangui; Zhuang, Zhiqiang; Zhu, Miaozhang; Chen, Jingzao; Ma, Hong

2007-04-01

Recent interest in adverse effects of infrasound on organisms arises from health concerns. We assessed the association between infrasound exposure of 5 Hz at 130 dB and changes of cardiac ultrastructure and function in rats. Thirty-two Sprague-Dawley rats were randomized into control, 1, 7, and 14 days groups for 2 h of infrasound once daily according to planned schedules. Changes of cardiac ultrastructure, hemodynamics indices, intracellular Ca(2+) concentrations ([Ca(2+)](i)), and sarcoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) were detected. Heart rates in 1 day group were significantly increased compared with control group and no significant changes in other groups. Left ventricular systolic pressures were significantly increased with time. Left ventricular diastolic end pressure and maximum rising rates of left ventricular pressure (+dl/dt) were significantly increased in 7 and 14 days groups and not changed in 1 day group, compared with control group. Maximum dropping rates of left ventricular pressure (-dl/dt) were significantly decreased in 7 and 14 days groups and not changed in 1 day group, compared with control group. In heart cells, there were several swelled mitochondria in 1 day group, more swelled mitochondria in 7 days group, platelet aggregation in the intercellular substance in 14 days group. [Ca(2+)](i) were significantly increased with time. There was a significant increase in SERCA2 in 1 day group, while a significant decrease in 7 and 14 days groups, compared with control group. Infrasound of 5 Hz at 130 dB can damage cardiac ultrastructure and function. Changes of [Ca(2+)](i) and SERCA2 play an important role in the secondary cardiac damage. (c) 2007 Wiley Periodicals, Inc.

Effects of Electrical Stimulation on Skeletal Muscle of Old Sedentary People

PubMed Central

Mosole, Simone; Zampieri, Sandra; Furlan, Sandra; Carraro, Ugo; Löefler, Stefan; Kern, Helmut; Volpe, Pompeo

2018-01-01

Physical activity plays an important role in preventing muscle atrophy and chronic diseases in adults and in the elderly. Calcium (Ca2+) cycling and activation of specific molecular pathways are essential in contraction-induced muscle adaptation. This study attains human muscle sections and total homogenates prepared from biopsies obtained before (control) and after 9 weeks of training by electrical stimulation (ES) on a group of volunteers. The aim of the study was to investigate about the molecular mechanisms that support functional muscle improvement by ES. Evidences of kinase/phosphatase pathways activation after ES were obtained. Moreover, expression of Sarcalumenin, Calsequestrin and sarco/endoplasmic reticulum Ca2+-ATPase (Serca) isoforms was regulated by training. In conclusion, this work shows that neuromuscular ES applied to vastus lateralis muscle of sedentary seniors combines fiber remodeling with activation of Ca2+-Calmodulin molecular pathways and modulation of key Ca2+-handling proteins. PMID:29662923

Altered Left Ventricular Ion Channel Transcriptome in a High-Fat-Fed Rat Model of Obesity: Insight into Obesity-Induced Arrhythmogenesis

PubMed Central

Yon, Marianne; Pickavance, Lucy; Yanni Gerges, Joseph; Davis, Gershan; Wilding, John; Jian, Kun; Hart, George; Boyett, Mark

2016-01-01

Introduction. Obesity is increasingly common and is associated with an increased prevalence of cardiac arrhythmias. The aim of this study was to see whether in obesity there is proarrhythmic gene expression of ventricular ion channels and related molecules. Methods and Results. Rats were fed on a high-fat diet and compared to control rats on a normal diet (n = 8). After 8 weeks, rats on the high-fat diet showed significantly greater weight gain and higher adiposity. Left ventricle samples were removed at 8 weeks and mRNA expression of ion channels and other molecules was measured using qPCR. Obese rats had significant upregulation of Cav1.2, HCN4, Kir2.1, RYR2, NCX1, SERCA2a, and RYR2 mRNA and downregulation of ERG mRNA. In the case of HCN4, it was confirmed that there was a significant increase in protein expression. The potential effects of the mRNA changes on the ventricular action potential and intracellular Ca2+ transient were predicted using computer modelling. Modelling predicted prolongation of the ventricular action potential and an increase in the intracellular Ca2+ transient, both of which would be expected to be arrhythmogenic. Conclusion. High-fat diet causing obesity results in arrhythmogenic cardiac gene expression of ion channels and related molecules. PMID:27747100

Rate-dependent Ca2+ signalling underlying the force-frequency response in rat ventricular myocytes: a coupled electromechanical modeling study

PubMed Central

2013-01-01

Background Rate-dependent effects on the Ca2+ sub-system in a rat ventricular myocyte are investigated. Here, we employ a deterministic mathematical model describing various Ca2+ signalling pathways under voltage clamp (VC) conditions, to better understand the important role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP) as they affect various intracellular targets. In particular, we study the frequency dependence of the peak force generated by the myofilaments, the force-frequency response (FFR). Methods Our cell model incorporates frequency-dependent CaM-mediated spatially heterogenous interaction of CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors and the SERCA pump). It also accounts for the rate-dependent effects of phospholamban (PLB) on the SERCA pump; the rate-dependent role of cAMP in up-regulation of the L-type Ca2+ channel (ICa,L); and the enhancement in SERCA pump activity via phosphorylation of PLB. Results Our model reproduces positive peak FFR observed in rat ventricular myocytes during voltage-clamp studies both in the presence/absence of cAMP mediated β-adrenergic stimulation. This study provides quantitative insight into the rate-dependence of Ca2+-induced Ca2+-release (CICR) by investigating the frequency-dependence of the trigger current (ICa,L) and RyR-release. It also highlights the relative role of the sodium-calcium exchanger (NCX) and the SERCA pump at higher frequencies, as well as the rate-dependence of sarcoplasmic reticulum (SR) Ca2+ content. A rigorous Ca2+ balance imposed on our investigation of these Ca2+ signalling pathways clarifies their individual roles. Here, we present a coupled electromechanical study emphasizing the rate-dependence of isometric force developed and also investigate the temperature-dependence of FFR. Conclusions Our model provides mechanistic biophysically based explanations for the rate-dependence of CICR, generating useful and testable hypotheses. Although rat ventricular myocytes exhibit a positive peak FFR in the presence/absence of beta-adrenergic stimulation, they show a characteristic increase in the positive slope in FFR due to the presence of Norepinephrine or Isoproterenol. Our study identifies cAMP-mediated stimulation, and rate-dependent CaMKII-mediated up-regulation of ICa,L as the key mechanisms underlying the aforementioned positive FFR. PMID:24020888

Insulin-Like Growth Factor I (IGF-1) Deficiency Ameliorates Sex Difference in Cardiac Contractile Function and Intracellular Ca2+ Homeostasis

PubMed Central

Ceylan-Isik, Asli F.; Li, Qun; Ren, Jun

2011-01-01

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (± dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), fura-fluorescence intensity (FFI) and intracellular Ca2+ clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ± dL/dt, longer TPS, TR90 and intracellular Ca2+ clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na+-Ca2+ exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca2+ regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca2+ regulation. PMID:21763763

Insulin-like growth factor I (IGF-1) deficiency ameliorates sex difference in cardiac contractile function and intracellular Ca(2+) homeostasis.

PubMed

Ceylan-Isik, Asli F; Li, Qun; Ren, Jun

2011-10-10

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (±dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR(90)), fura-fluorescence intensity (FFI) and intracellular Ca(2+) clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ±dL/dt, longer TPS, TR(90) and intracellular Ca(2+) clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na(+)-Ca(2+) exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca(2+) regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca(2+) regulation. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Burst discharges in neurons of the thalamic reticular nucleus are shaped by calcium-induced calcium release.

PubMed

Coulon, Philippe; Herr, David; Kanyshkova, Tatyana; Meuth, Patrick; Budde, Thomas; Pape, Hans-Christian

2009-01-01

The nucleus reticularis thalami (NRT) is a layer of inhibitory neurons that surrounds the dorsal thalamus. It appears to be the 'pacemaker' of certain forms of slow oscillations in the thalamus and was proposed to be a key determinant of the internal attentional searchlight as well as the origin of hypersynchronous activity during absence seizures. Neurons of the NRT exhibit a transient depolarization termed low threshold spike (LTS) following sustained hyperpolarization. This is caused by the activation of low-voltage-activated Ca2+ channels (LVACC). Although the role of these channels in thalamocortical oscillations was studied in great detail, little is known about the downstream intracellular Ca2+ signalling pathways and their feedback onto the oscillations. A signalling triad consisting of the sarco(endo)plasmic reticulum calcium ATPase (SERCA), Ca2+ activated K+ channels (SK2), and LVACC is active in dendrites of NRT neurons and shapes rhythmic oscillations. The aim of our study was to find out (i) if and how Ca2+-induced Ca2+ release (CICR) via ryanodine receptors (RyR) can be evoked in NRT neurons and (ii) how the released Ca2+ affects burst activity. Combining electrophysiological, immunohistochemical, and two-photon Ca2+ imaging techniques, we show that CICR in NRT neurons takes place by a cell-type specific coupling of LVACC and RyR. CICR could be evoked by the application of caffeine, by activation of LVACC, or by repetitive LTS generation. During the latter, CICR contributed 30% to the resulting build-up of [Ca2+]i. CICR was abolished by cyclopiazonic acid, a specific blocker for SERCA, or by high concentrations of ryanodine (50 microM). Unlike other thalamic nuclei, in the NRT the activation of high-voltage-activated Ca2+ channels failed to evoke CICR. While action potentials contributed little to the build-up of [Ca2+]i upon repetitive LTS generation, the Ca2+ released via RyR significantly reduced the number of action potentials during an LTS and reduced the neurons' low threshold activity, thus potentially reducing hypersynchronicity. This effect persisted in the presence of the SK2 channel blocker apamin. We conclude that the activation of LVACC specifically causes CICR via RyR in neurons of the NRT, thereby adding a Ca2+-dependent intracellular route to the mechanisms determining rhythmic oscillatory bursting in this nucleus.

The effect of taurine and β-alanine supplementation on taurine transporter protein and fatigue resistance in skeletal muscle from mdx mice.

PubMed

Horvath, Deanna M; Murphy, Robyn M; Mollica, Janelle P; Hayes, Alan; Goodman, Craig A

2016-11-01

This study investigated the effect of taurine and β-alanine supplementation on muscle function and muscle taurine transporter (TauT) protein expression in mdx mice. Wild-type (WT) and mdx mice (5 months) were supplemented with taurine or β-alanine for 4 weeks, after which in vitro contractile properties, fatigue resistance and force recovery, and the expression of the TauT protein and proteins involved in excitation-contraction (E-C) coupling were examined in fast-twitch muscle. There was no difference in basal TauT protein expression or basal taurine content between mdx than WT muscle. Supplementation with taurine and β-alanine increased and reduced taurine content, respectively, in muscle from WT and mdx mice but had no effect of TauT protein. Taurine supplementation reduced body and muscle mass, and enhanced fatigue resistance and force recovery in mdx muscle. β-Alanine supplementation enhanced fatigue resistance in WT and mdx muscle. There was no difference in the basal expression of key E-C coupling proteins [ryanodine receptor 1 (RyR1), dihydropyridine receptor (DHPR), sarco(endo)plasmic reticulum Ca 2+ -ATPase 1 (SERCA1) or calsequestrin 1 (CSQ1)] between WT and mdx mice, and the expression of these proteins was not altered by taurine or β-alanine supplementation. These findings suggest that TauT protein expression is relatively insensitive to changes in muscle taurine content in WT and mdx mice, and that taurine and β-alanine supplementation may be viable therapeutic strategies to improve fatigue resistance of dystrophic skeletal muscle.

CYP2E1 overexpression inhibits microsomal Ca2+-ATPase activity in HepG2 cells.

PubMed

Caro, Andres A; Evans, Kerry L; Cederbaum, Arthur I

2009-01-31

Cytochrome P450 2E1 (CYP2E1) is a microsomal enzyme that generates reactive oxygen species during its catalytic cycle. We previously found an important role for calcium in CYP2E1-potentiated injury in HepG2 cells. The possibility that CYP2E1 may oxidatively damage and inactivate the microsomal Ca2+-ATPase in intact liver cells was evaluated, in order to explain why calcium is elevated during CYP2E1 toxicity. Microsomes were isolated by differential centrifugation from two liver cell line: E47 cells (HepG2 cells transfected with the pCI neo expression vector containing the human CYP2E1 cDNA, which overexpress active microsomal CYP2E1), and control C34 cells (HepG2 cells transfected with the pCI neo expression vector alone, which do not express significantly any cytochrome P450). The Ca2+-dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca2+-dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes versus 4.7 nmol Pi/min/mg protein in microsomes). Saturation curves with Ca2+ or ATP showed that CYP2E1 overexpression produced a decrease in Vmax but did not affect the Km for either Ca2+ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATP-dependent microsomal calcium uptake was evaluated by fluorimetry using fluo-3 as the fluorogenic probe. Calcium uptake rate in E47 microsomes was 28% lower than in C34 microsomes. Treatment of E47 cells with 2mM N-acetylcysteine prevented the decrease in microsomal Ca2+-ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca2+-ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.

Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production

PubMed Central

Poksay, Karen S.; Sheffler, Douglas J.; Spilman, Patricia; Campagna, Jesus; Jagodzinska, Barbara; Descamps, Olivier; Gorostiza, Olivia; Matalis, Alex; Mullenix, Michael; Bredesen, Dale E.; Cosford, Nicholas D. P.; John, Varghese

2017-01-01

Alzheimer’s disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds – identified here using cells and tissues expressing wt human APP – in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects. PMID:28261092

Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients.

PubMed

Pelzl, Lisann; Elsir, Bhaeldin; Sahu, Itishri; Bissinger, Rosi; Singh, Yogesh; Sukkar, Basma; Honisch, Sabina; Schoels, Ludger; Jemaà, Mohamed; Lang, Elisabeth; Storch, Alexander; Hermann, Andreas; Stournaras, Christos; Lang, Florian

2017-01-01

The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment. © 2017 The Author(s). Published by S. Karger AG, Basel.

Targeted GLUT-4 deficiency in the heart induces cardiomyocyte hypertrophy and impaired contractility linked with Ca(2+) and proton flux dysregulation.

PubMed

Domenighetti, Andrea A; Danes, Vennetia R; Curl, Claire L; Favaloro, Jennifer M; Proietto, Joseph; Delbridge, Lea M D

2010-04-01

There is clinical evidence to suggest that impaired myocardial glucose uptake contributes to the pathogenesis of hypertrophic, insulin-resistant cardiomyopathy. The goal of this study was to determine whether cardiac deficiency of the insulin-sensitive glucose transporter, GLUT4, has deleterious effect on cardiomyocyte excitation-contraction coupling. Cre-Lox mouse models of cardiac GLUT4 knockdown (KD, 85% reduction) and knockout (KO, >95% reduction), which exhibit similar systemic hyperinsulinemic and hyperglycemic states, were investigated. The Ca(2+) current (I(Ca)) and Na(+)-Ca(2+) exchanger (NCX) fluxes, Na(+)-H(+) exchanger (NHE) activity, and contractile performance of GLUT4-deficient myocytes was examined using whole-cell patch-clamp, epifluorescence, and imaging techniques. GLUT4-KO exhibited significant cardiac enlargement characterized by cardiomyocyte hypertrophy (40% increase in cell area) and fibrosis. GLUT4-KO myocyte contractility was significantly diminished, with reduced mean maximum shortening (5.0+/-0.4% vs. 6.2+/-0.6%, 5 Hz). Maximal rates of shortening and relaxation were also reduced (20-25%), and latency was delayed. In GLUT4-KO myocytes, the I(Ca) density was decreased (-2.80+/-0.29 vs. -5.30+/-0.70 pA/pF), and mean I(NCX) was significantly increased in both outward (by 60%) and inward (by 100%) directions. GLUT4-KO expression levels of SERCA2 and RyR2 were reduced by approximately 50%. NHE-mediated H(+) flux in response to NH(4)Cl acid loading was markedly elevated GLUT4-KO myocytes, associated with doubled expression of NHE1. These findings demonstrate that, independent of systemic endocrinological disturbance, cardiac GLUT4 deficiency per se provides a lesion sufficient to induce profound alterations in cardiomyocyte Ca(2+) and pH homeostasis. Our investigation identifies the cardiac GLUT4 as a potential primary molecular therapeutic target in ameliorating the functional deficits associated with insulin-resistant cardiomyopathy. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Cardiac gene transfer of short hairpin RNA directed against phospholamban effectively knocks down gene expression but causes cellular toxicity in canines.

PubMed

Bish, Lawrence T; Sleeper, Meg M; Reynolds, Caryn; Gazzara, Jeffrey; Withnall, Elanor; Singletary, Gretchen E; Buchlis, George; Hui, Daniel; High, Katherine A; Gao, Guangping; Wilson, James M; Sweeney, H Lee

2011-08-01

Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways.

Cardiac Gene Transfer of Short Hairpin RNA Directed Against Phospholamban Effectively Knocks Down Gene Expression but Causes Cellular Toxicity in Canines

PubMed Central

Sleeper, Meg M.; Reynolds, Caryn; Gazzara, Jeffrey; Withnall, Elanor; Singletary, Gretchen E.; Buchlis, George; Hui, Daniel; High, Katherine A.; Gao, Guangping; Wilson, James M.; Sweeney, H. Lee

2011-01-01

Abstract Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways. PMID:21542669

Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity

PubMed Central

Bublitz, Maike; Kjellerup, Lasse; Cohrt, Karen O’Hanlon; Gordon, Sandra; Mortensen, Anne Louise; Clausen, Johannes D.; Pallin, Thomas David; Hansen, John Bondo; Fuglsang, Anja Thoe; Dalby-Brown, William

2018-01-01

We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H+-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca2+-ATPase (SERCA) and Na+,K+-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 Å resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model of the Candida albicans H+-ATPase based on this crystal structure, indicates that the compounds could bind to the same pocket and identifies pocket extensions that could be exploited for selectivity enhancement. The results of this study will aid further optimization towards selective H+-ATPase inhibitors as a new class of antifungal agents. PMID:29293507

Store-operated Ca²⁺ entry and depolarization explain the anomalous behaviour of myometrial SR: effects of SERCA inhibition on electrical activity, Ca²⁺ and force.

PubMed

Noble, Debbie; Borysova, Lyudmyla; Wray, Susan; Burdyga, Theodor

2014-09-01

In the myometrium SR Ca(2+) depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca(2+) sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca(2+)-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca(2+) transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca(2+)]i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca(2+) spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca(2+) and depolarization continued a point was reached when electrical and Ca(2+) spikes and phasic contractions ceased, and a maintained, tonic force and Ca(2+) was produced. Lanthanum, a non-selective blocker of store-operated Ca(2+) entry, but not the L-type Ca(2+) channel blocker nifedipine (1-10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca(2+) depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca(2+) release is coupled to marked Ca(2+) entry, via store operated Ca(2+) channels, leading to depolarization and enhanced electrical and mechanical activity. Copyright © 2014. Published by Elsevier Ltd.

SERCA plays a crucial role in the toxicity of a betulinic acid derivative with potential antimalarial activity.

PubMed

Diedrich, Denise; Wildner, Andreia C; Silveira, Thayse F; Silva, Gloria N S; Santos, Francine Dos; da Silva, Elenilson F; do Canto, Vanessa P; Visioli, Fernanda; Gosmann, Grace; Bergold, Ana M; Zimmer, Aline R; Netz, Paulo A; Gnoatto, Simone C B

2018-05-01

Malaria is one of the most significant infectious diseases that affect poor populations in tropical areas throughout the world. Plants have been shown to be a good source for the development of new antimalarial chemotherapeutic agents, as shown for the discovery of quinine and artemisinin derivatives. Our research group has been working with semisynthetic triterpene derivatives that show potential antimalarial activity toward different strains of Plasmodium falciparum by specifically modulating calcium pathways in the parasite. Promising results were obtained for nanomolar concentrations of the semisynthetic betulinic acid derivative LAFIS13 against the P. falciparum 3D7 strain in vitro, with a selectivity index of 18 compared to a mammalian cell line. Continuing these studies, we present here in vitro and in vivo toxicological evaluations of this compound, followed by docking studies with PfATP6, a sarco/endoplasmic reticulum Ca +2 -ATPase (SERCA) protein. LAFIS13 showed an LD 50 between 300 and 50 mg/kg, and the acute administration of 50 mg/kg (i.p.) had no negative effects on hematological, biochemical and histopathological parameters. Based on the results of the in vitro assays, LAFIS13 not exerted significant effects on coagulation parameters of human peripheral blood, but a hemolytic activity was verified at higher concentrations. According to the molecular docking study, the PfATP6 protein may be a target for LAFIS13, which corroborates its previously reported modulatory effects on calcium homeostasis in the parasite. Notably, LAFIS13 showed a higher selectivity for the mammalian SERCA protein than for PfATP6, thus impairing the selectivity between parasite and host. In summary, the direct interaction with calcium pumps and the hemolytic potential of the compound proved to be plausible mechanism of LAFIS13 toxicity. Copyright © 2018 Elsevier B.V. All rights reserved.

Neuronal calcium wave propagation varies with changes in endoplasmic reticulum parameters: a computer model

PubMed Central

Neymotin, Samuel A.; McDougal, Robert A.; Sherif, Mohamed A.; Fall, Christopher P.; Hines, Michael L.; Lytton, William W.

2015-01-01

Calcium (Ca2+) waves provide a complement to neuronal electrical signaling, forming a key part of a neuron’s second messenger system. We developed a reaction-diffusion model of an apical dendrite with diffusible inositol triphosphate (IP3), diffusible Ca2+, IP3 receptors (IP3Rs), endoplasmic reticulum (ER) Ca2+ leak, and ER pump (SERCA) on ER. Ca2+ is released from ER stores via IP3Rs upon binding of IP3 and Ca2+. This results in Ca2+-induced-Ca2+-release (CICR) and increases Ca2+ spread. At least two modes of Ca2+ wave spread have been suggested: a continuous mode based on presumed relative homogeneity of ER within the cell; and a pseudo-saltatory model where Ca2+ regeneration occurs at discrete points with diffusion between them. We compared the effects of three patterns of hypothesized IP3R distribution: 1. continuous homogeneous ER, 2. hotspots with increased IP3R density (IP3R hotspots), 3. areas of increased ER density (ER stacks). All three modes produced Ca2+ waves with velocities similar to those measured in vitro (~50–90µm /sec). Continuous ER showed high sensitivity to IP3R density increases, with time to onset reduced and speed increased. Increases in SERCA density resulted in opposite effects. The measures were sensitive to changes in density and spacing of IP3R hotspots and stacks. Increasing the apparent diffusion coefficient of Ca2+ substantially increased wave speed. An extended electrochemical model, including voltage gated calcium channels and AMPA synapses, demonstrated that membrane priming via AMPA stimulation enhances subsequent Ca2+ wave amplitude and duration. Our modeling suggests that pharmacological targeting of IP3Rs and SERCA could allow modulation of Ca2+ wave propagation in diseases where Ca2+ dysregulation has been implicated. PMID:25734493

Human cardiomyocyte calcium handling and transverse tubules in mid-stage of post-myocardial-infarction heart failure.

PubMed

Høydal, Morten Andre; Kirkeby-Garstad, Idar; Karevold, Asbjørn; Wiseth, Rune; Haaverstad, Rune; Wahba, Alexander; Stølen, Tomas L; Contu, Riccardo; Condorelli, Gianluigi; Ellingsen, Øyvind; Smith, Godfrey L; Kemi, Ole J; Wisløff, Ulrik

2018-06-01

Cellular processes in the heart rely mainly on studies from experimental animal models or explanted hearts from patients with terminal end-stage heart failure (HF). To address this limitation, we provide data on excitation contraction coupling, cardiomyocyte contraction and relaxation, and Ca 2+ handling in post-myocardial-infarction (MI) patients at mid-stage of HF. Nine MI patients and eight control patients without MI (non-MI) were included. Biopsies were taken from the left ventricular myocardium and processed for further measurements with epifluorescence and confocal microscopy. Cardiomyocyte function was progressively impaired in MI cardiomyocytes compared with non-MI cardiomyocytes when increasing electrical stimulation towards frequencies that simulate heart rates during physical activity (2 Hz); at 3 Hz, we observed almost total breakdown of function in MI. Concurrently, we observed impaired Ca 2+ handling with more spontaneous Ca 2+ release events, increased diastolic Ca 2+ , lower Ca 2+ amplitude, and prolonged time to diastolic Ca 2+ removal in MI (P < 0.01). Significantly reduced transverse-tubule density (-35%, P < 0.01) and sarcoplasmic reticulum Ca 2+ adenosine triphosphatase 2a (SERCA2a) function (-26%, P < 0.01) in MI cardiomyocytes may explain the findings. Reduced protein phosphorylation of phospholamban (PLB) serine-16 and threonine-17 in MI provides further mechanisms to the reduced function. Depressed cardiomyocyte contraction and relaxation were associated with impaired intracellular Ca 2+ handling due to impaired SERCA2a activity caused by a combination of alteration in the PLB/SERCA2a ratio and chronic dephosphorylation of PLB as well as loss of transverse tubules, which disrupts normal intracellular Ca 2+ homeostasis and handling. This is the first study that presents these mechanisms from viable and intact cardiomyocytes isolated from the left ventricle of human hearts at mid-stage of post-MI HF. © 2018 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.

Protein Aggregates and Novel Presenilin Gene Variants in Idiopathic Dilated Cardiomyopathy

PubMed Central

Gianni, Davide; Li, Airong; Tesco, Giuseppina; McKay, Kenneth M.; Moore, John; Raygor, Kunal; Rota, Marcello; Gwathmey, Judith K; Dec, G William; Aretz, Thomas; Leri, Annarosa; Semigran, Marc J; Anversa, Piero; Macgillivray, Thomas E; Tanzi, Rudolph E.; Monte, Federica del

2010-01-01

Background Heart failure (HF) is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as Idiopathic Dilated Cardiomyopathy (iDCM), the origin of HF is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of β-amyloid impair cell function and lead to cell death. Methods and Results We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca2+ homeostasis. Additionally, we have identified two new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 co-immunoprecipitates with SERCA2a. Conclusions Based on these findings we propose that two mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca2+ handling and a direct effect of PSEN1 sequence variants on EC-coupling protein function. PMID:20194882

Quantification and characterization of grouped type I myofibers in human aging.

PubMed

Kelly, Neil A; Hammond, Kelley G; Stec, Michael J; Bickel, C Scott; Windham, Samuel T; Tuggle, S Craig; Bamman, Marcas M

2018-01-01

Myofiber type grouping is a histological hallmark of age-related motor unit remodeling. Despite the accepted concept that denervation-reinnervation events lead to myofiber type grouping, the completeness of those conversions remains unknown. Type I myofiber grouping was assessed in vastus lateralis biopsies from Young (26 ± 4 years; n = 27) and Older (66 ± 4 years; n = 91) adults. Grouped and ungrouped type I myofibers were evaluated for phenotypic differences. Higher type I grouping in Older versus Young was driven by more myofibers per group (i.e., larger group size) (P < 0.05). In Older only, grouped type I myofibers displayed larger cross-sectional area, more myonuclei, lower capillary supply, and more sarco(endo)plasmic reticulum calcium ATPase I (SERCA I) expression (P < 0.05) than ungrouped type I myofibers. Grouped type I myofibers retain type II characteristics suggesting that conversion during denervation-reinnervation events is either progressive or incomplete. Muscle Nerve 57: E52-E59, 2018. © 2017 Wiley Periodicals, Inc.

Estrogen reduces endoplasmic reticulum stress to protect against glucotoxicity induced-pancreatic β-cell death.

PubMed

Kooptiwut, Suwattanee; Mahawong, Pitchnischa; Hanchang, Wanthanee; Semprasert, Namoiy; Kaewin, Suchada; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-Thai

2014-01-01

Estrogen can improve glucose homeostasis not only in diabetic rodents but also in humans. However, the molecular mechanism by which estrogen prevents pancreatic β-cell death remains unclear. To investigate this issue, INS-1 cells, a rat insulinoma cell line, were cultured in medium with either 11.1mM or 40mM glucose in the presence or the absence of estrogen. Estrogen significantly reduced apoptotic β-cell death by decreasing nitrogen-induced oxidative stress and the expression of the ER stress markers GRP 78, ATF6, P-PERK, PERK, uXBP1, sXBP1, and CHOP in INS-1 cells after prolonged culture in medium with 40mM glucose. In contrast, estrogen increased the expression of survival proteins, including sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA-2), Bcl-2, and P-p38, in INS-1 cells after prolonged culture in medium with 40mM glucose. The cytoprotective effect of estrogen was attenuated by addition of the estrogen receptor (ERα and ERβ) antagonist ICI 182,780 and the estrogen membrane receptor inhibitor G15. We showed that estrogen decreases not only oxidative stress but also ER stress to protect against 40mM glucose-induced pancreatic β-cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

Elucidating a molecular mechanism that the deterioration of porcine meat quality responds to increased cortisol based on transcriptome sequencing.

PubMed

Wan, Xuebin; Wang, Dan; Xiong, Qi; Xiang, Hong; Li, Huanan; Wang, Hongshuai; Liu, Zezhang; Niu, Hongdan; Peng, Jian; Jiang, Siwen; Chai, Jin

2016-11-11

Stress response is tightly linked to meat quality. The current understanding of the intrinsic mechanism of meat deterioration under stress is limited. Here, male piglets were randomly assigned to cortisol and control groups. Our results showed that when serum cortisol level was significantly increased, the meat color at 1 h postmortem, muscle bundle ratio, apoptosis rate, and gene expression levels of calcium channel and cell apoptosis including SERCA1, IP3R1, BAX, Bcl-2, and Caspase-3, were notably increased. However, the value of drip loss at 24 h postmortem and serum CK were significantly decreased. Additionally, a large number of differentially expressed genes (DEGs) in GC regulation mechanism were screened out using transcriptome sequencing technology. A total of 223 DEGs were found, including 80 up-regulated genes and 143 down-regulated genes. A total of 204 genes were enriched in GO terms, and 140 genes annotated into in KEGG database. Numerous genes were primarily involved in defense, inflammatory and wound responses. This study not only identifies important genes and signalling pathways that may affect the meat quality but also offers a reference for breeding and feeding management to provide consumers with better quality pork products.

UTP – Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells

PubMed Central

Liñán-Rico, Andromeda; Ochoa-Cortes, Fernando; Zuleta-Alarcon, Alix; Alhaj, Mazin; Tili, Esmerina; Enneking, Josh; Harzman, Alan; Grants, Iveta; Bergese, Sergio; Christofi, Fievos L.

2017-01-01

Background: Enterochromaffin cells (EC) synthesize and release 5-HT and ATP to trigger or modulate gut neural reflexes and transmit information about visceral/pain sensation. Alterations in 5-HT signaling mechanisms may contribute to the pathogenesis of IBD or IBS, but the pharmacologic or molecular mechanisms modulating Ca2+-dependent 5-HT release are not understood. Previous studies indicated that purinergic signaling via ATP and ADP is an important mechanism in modulation of 5-HT release. However, EC cells also respond to UTP and UDP suggesting uridine triphosphate receptor and signaling pathways are involved as well. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells. Methods: UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca2+imaging, patch-clamp, pharmacological analysis, immunohistochemistry, western blots and qPCR. 5-HT release was monitored in BON or EC isolated from human gut surgical specimens (hEC). Results: UTP, UTPγS, UDP or ATP induced Ca2+oscillations in BON. UTP evoked a biphasic concentration-dependent Ca2+response. Cells responded in the order of UTP, ATP > UTPγS > UDP >> MRS2768, BzATP, α,β-MeATP > MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y4, 50% cells), UDP (P2Y6, 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Ca2+responses were blocked with inhibitors of PLC, IP3R, SERCA Ca2+pump, La3+sensitive Ca2+channels or chelation of intracellular free Ca2+ by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca2+pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca2+currents (ICa), Vm-depolarization and inhibited IK (not IA) currents. An IKv7.2/7.3 K+ channel blocker XE-991 mimicked UTP-induced Vm-depolarization and blocked UTP-responses. XE-991 blocked IK and UTP caused further reduction. La3+ or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca2+buffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y4/P2Y6R. Zero-Ca2+buffer augmented Ca2+responses and 5-HT release. Conclusion: UTP activates a predominant P2Y4R pathway to trigger Ca2+oscillations via internal Ca2+mobilization through a PLC/IP3/IP3R/SERCA Ca2+signaling pathway to stimulate 5-HT release; Ca2+influx is inhibitory. UTP-induced Vm-depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca2+oscillations or Ica/VOCC). UTP-gated signaling pathways triggered by activation of P2Y4R stimulate 5-HT release. PMID:28751862

UTP - Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells.

PubMed

Liñán-Rico, Andromeda; Ochoa-Cortes, Fernando; Zuleta-Alarcon, Alix; Alhaj, Mazin; Tili, Esmerina; Enneking, Josh; Harzman, Alan; Grants, Iveta; Bergese, Sergio; Christofi, Fievos L

2017-01-01

Background: Enterochromaffin cells (EC) synthesize and release 5-HT and ATP to trigger or modulate gut neural reflexes and transmit information about visceral/pain sensation. Alterations in 5-HT signaling mechanisms may contribute to the pathogenesis of IBD or IBS, but the pharmacologic or molecular mechanisms modulating Ca 2+ -dependent 5-HT release are not understood. Previous studies indicated that purinergic signaling via ATP and ADP is an important mechanism in modulation of 5-HT release. However, EC cells also respond to UTP and UDP suggesting uridine triphosphate receptor and signaling pathways are involved as well. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells. Methods: UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca 2+ imaging, patch-clamp, pharmacological analysis, immunohistochemistry, western blots and qPCR. 5-HT release was monitored in BON or EC isolated from human gut surgical specimens (hEC). Results: UTP, UTPγS, UDP or ATP induced Ca 2+ oscillations in BON. UTP evoked a biphasic concentration-dependent Ca 2+ response. Cells responded in the order of UTP, ATP > UTPγS > UDP > MRS2768, BzATP, α,β-MeATP > MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y 4 , 50% cells), UDP (P2Y 6 , 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Ca 2+ responses were blocked with inhibitors of PLC, IP3R, SERCA Ca 2+ pump, La 3+ sensitive Ca 2+ channels or chelation of intracellular free Ca 2+ by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca 2+ pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca 2+ currents (I Ca ), V m -depolarization and inhibited I K (not I A ) currents. An I Kv 7.2/7.3 K + channel blocker XE-991 mimicked UTP-induced V m -depolarization and blocked UTP-responses. XE-991 blocked I K and UTP caused further reduction. La 3+ or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca 2+ buffer did not. UTP stimulated 5-HT release in hEC expressing TPH1, 5-HT, P2Y 4 /P2Y 6 R. Zero-Ca 2+ buffer augmented Ca 2+ responses and 5-HT release. Conclusion: UTP activates a predominant P2Y 4 R pathway to trigger Ca 2+ oscillations via internal Ca 2+ mobilization through a PLC/IP 3 /IP3R/SERCA Ca 2+ signaling pathway to stimulate 5-HT release; Ca 2+ influx is inhibitory. UTP-induced V m -depolarization depends on PLC signaling and an unidentified K channel (which appears independent of Ca 2+ oscillations or I ca /VOCC). UTP-gated signaling pathways triggered by activation of P2Y 4 R stimulate 5-HT release.

17β-Estradiol and/or estrogen receptor alpha signaling blocks protein phosphatase 1 mediated ISO induced cardiac hypertrophy.

PubMed

Fang, Hsin-Yuan; Hung, Meng-Yu; Lin, Yueh-Min; Pandey, Sudhir; Chang, Chia-Chien; Lin, Kuan-Ho; Shen, Chia-Yao; Viswanadha, Vijaya Padma; Kuo, Wei-Wen; Huang, Chih-Yang

2018-01-01

Earlier studies have shown that estrogen possess protective function against the development of pathological cardiac hypertrophy. However, the molecular mechanisms of estrogens (E2) protective effect are poorly understood. Additionally, abnormal activation of β-adrenergic signaling have been implicated in the development of pathological cardiac remodeling. However, the role of serine/threonine protein phosphatase 1 (PP1) in pathological cardiac remodeling under the influence of β-adrenergic signaling have been sparsely investigated. In this study, we assessed the downstream effects of abnormal activation of PP1 upon isoproterenol (ISO) induced pathological cardiac changes. We found that pre-treatment of 17β-estradiol (E2), tet-on estrogen receptor-α, or both significantly inhibited ISO-induced increase in cell size, hypertrophy marker gene expression and cytosolic calcium accumulation in H9c2 cells. Additionally, treatment with estrogen receptor inhibitor (ICI) reversed those effects, implicating role of E2 in inhibiting pathological cardiac remodeling. However, specific inhibition of ERα using melatonin, reduced ISO-induced PP1c expression and enhanced the level of ser-16 phosphorylated phospholamban (PLB), responsible for regulation of sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity. Furthermore, hypertrophic effect caused by overexpression of PP1cα was reduced by treatment with specific inhibitor of ERα. Collectively, we found that estrogen and estrogen receptor-α have protective effect against pathological cardiac changes by suppressing PP1 expression and its downstream signaling pathway, which further needs to be elucidated.

Tetanic Ca2+ transient differences between slow- and fast-twitch mouse skeletal muscle fibres: a comprehensive experimental approach.

PubMed

Calderón, Juan C; Bolaños, Pura; Caputo, Carlo

2014-12-01

One hundred and eighty six enzymatically dissociated murine muscle fibres were loaded with Mag-Fluo-4 AM, and adhered to laminin, to evaluate the effect of modulating cytosolic Ca(2+) buffers and sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA), mitochondria, and Na(+)/Ca(2+) exchanger (NCX) on the differential tetanic Ca(2+) transient kinetics found in different fibre types. Tetanic Ca(2+) transients were classified as morphology type I (MT-I) or type II (MT-II) according to their shape. The first peak of the MT-I (n = 25) and MT-II (n = 23) tetanic Ca(2+) transients had an amplitude (∆F/F) of 0.41 ± 0.03 and 0.83 ± 0.05 and a rise time (ms) of 1.35 and 0.98, respectively. MT-I signals had a time constant of decay (τ1, ms) of 75.9 ± 4.2 while MT-II transients showed a double exponential decay with time constants of decay (τ1 and τ2, ms) of 18.3 ± 1.4 and 742.2 ± 130.3. Sarcoendoplasmic reticulum Ca(2+) ATPase inhibition demonstrated that the decay phase of the tetanic transients mostly rely on SERCA function. Adding Ca(2+) chelators in the AM form to MT-I fibres changed the morphology of the initial five peaks to a MT-II one, modifying the decay phase of the signal in a dose-dependent manner. Mitochondria and NCX function have a minor role in explaining differences in tetanic Ca(2+) transients among fibre types but still help in removing Ca(2+) from the cytosol in both MT-I and MT-II fibres. Cytoplasmic Ca(2+) buffering capacity and SERCA function explain most of the different kinetics found in tetanic Ca(2+) transients from different fibre types, but mitochondria and NCX have a measurable role in shaping tetanic Ca(2+) responses in both slow and fast-twitch muscle fibre types. We provided experimental evidence on the mechanisms that help understand the kinetics of tetanic Ca(2+) transients themselves and explain kinetic differences found among fibre types.

Nitric Oxide Synthase 1 Modulates Basal and β-Adrenergic-Stimulated Contractility by Rapid and Reversible Redox-Dependent S-Nitrosylation of the Heart

PubMed Central

Vielma, Alejandra Z.; León, Luisa; Fernández, Ignacio C.; González, Daniel R.

2016-01-01

S-nitrosylation of several Ca2+ regulating proteins in response to β-adrenergic stimulation was recently described in the heart; however the specific nitric oxide synthase (NOS) isoform and signaling pathways responsible for this modification have not been elucidated. NOS-1 activity increases inotropism, therefore, we tested whether β-adrenergic stimulation induces NOS-1-dependent S-nitrosylation of total proteins, the ryanodine receptor (RyR2), SERCA2 and the L-Type Ca2+ channel (LTCC). In the isolated rat heart, isoproterenol (10 nM, 3-min) increased S-nitrosylation of total cardiac proteins (+46±14%) and RyR2 (+146±77%), without affecting S-nitrosylation of SERCA2 and LTCC. Selective NOS-1 blockade with S-methyl-L-thiocitrulline (SMTC) and Nω-propyl-l-arginine decreased basal contractility and relaxation (−25–30%) and basal S-nitrosylation of total proteins (−25–60%), RyR2, SERCA2 and LTCC (−60–75%). NOS-1 inhibition reduced (−25–40%) the inotropic response and protein S-nitrosylation induced by isoproterenol, particularly that of RyR2 (−85±7%). Tempol, a superoxide scavenger, mimicked the effects of NOS-1 inhibition on inotropism and protein S-nitrosylation; whereas selective NOS-3 inhibitor L-N5-(1-Iminoethyl)ornithine had no effect. Inhibition of NOS-1 did not affect phospholamban phosphorylation, but reduced its oligomerization. Attenuation of contractility was abolished by PKA blockade and unaffected by guanylate cyclase inhibition. Additionally, in isolated mouse cardiomyocytes, NOS-1 inhibition or removal reduced the Ca2+-transient amplitude and sarcomere shortening induced by isoproterenol or by direct PKA activation. We conclude that 1) normal cardiac performance requires basal NOS-1 activity and S-nitrosylation of the calcium-cycling machinery; 2) β-adrenergic stimulation induces rapid and reversible NOS-1 dependent, PKA and ROS-dependent, S-nitrosylation of RyR2 and other proteins, accounting for about one third of its inotropic effect. PMID:27529477

p53 and Ca(2+) signaling from the endoplasmic reticulum: partners in anti-cancer therapies.

PubMed

Bittremieux, Mart; Bultynck, Geert

2015-01-01

Ca(2+) transfer from the endoplasmic reticulum (ER) to the mitochondria critically controls cell survival and cell death decisions. Different oncogenes and deregulation of tumor suppressors exploit this mechanism to favor the survival of altered, malignant cells. Two recent studies of the Pinton team revealed a novel, non-transcriptional function of cytosolic p53 in cell death. During cell stress, p53 is recruited to the ER and the ER-mitochondrial contact sites. This results in augmented ER Ca(2+) levels by enhancing sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity, ultimately promoting mitochondrial Ca(2+) overload. The boosting of "toxic" Ca(2+) signaling by p53 appears to be a critical component of the cell death-inducing properties of chemotherapeutic agents and anti-cancer treatments, like photodynamic stress. Strikingly, the resistance of p53-deficient cancer cells to these treatments could be overcome by facilitating Ca(2+) transfer between the ER and the mitochondria via overexpression of SERCA or of the mitochondrial Ca(2+) uniporter (MCU). Importantly, these concepts have also been supported by in vivo Ca(2+) measurements in tumor masses in mice. Collectively, these studies link for the first time the major tumor suppressor, p53, to Ca(2+) signaling in dictating cell-death outcomes and by the success of anti-cancer treatments.

Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak.

PubMed

Leon-Aparicio, Daniel; Pacheco, Jonathan; Chavez-Reyes, Jesus; Galindo, Jose M; Valdes, Jesus; Vaca, Luis; Guerrero-Hernandez, Agustin

2017-07-01

We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca 2+ leak using synthetic Ca 2+ indicators that report changes in both the cytoplasmic ([Ca 2+ ] i ) and the luminal ER ([Ca 2+ ] ER ) Ca 2+ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca 2+ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca 2+ ] ER but did not decrease the ER Ca 2+ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP 3 R and only Orai3 channel supported the 2-APB-induced ER Ca 2+ leak, while Orai1 and Orai2 inhibited this type of ER Ca 2+ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca 2+ leak but did not modify the ER Ca 2+ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca 2+ ] i response after TG but only when the ER store had been overloaded with Ca 2+ by eliminating the acidic internal Ca 2+ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca 2+ leak but forms an ER Ca 2+ leak channel that is limiting the overloading with Ca 2+ of the ER store. Copyright © 2017 Elsevier Ltd. All rights reserved.

Disease-associated changes in the expression of ion channels, ion receptors, ion exchangers and Ca{sup 2+}-handling proteins in heart hypertrophy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zwadlo, Carolin; Borlak, Juergen

2005-09-15

The molecular pathology of cardiac hypertrophy is multifactorial with transcript regulation of ion channels, ion exchangers and Ca{sup 2+}-handling proteins being speculative. We therefore investigated disease-associated changes in gene expression of various ion channels and their receptors as well as ion exchangers, cytoskeletal proteins and Ca{sup 2+}-handling proteins in normotensive and spontaneously hypertensive (SHR) rats. We also compared experimental findings with results from hypertrophic human hearts, previously published (Borlak, J., and Thum, T., 2003. Hallmarks of ion channel gene expression in end-stage heart failure. FASEB J. 17, 1592-1608). We observed significant (P < 0.05) induction in transcript level of ATP-drivenmore » ion exchangers (Atp1A1, NCX-1, SERCA2a), ion channels (L-type Ca{sup 2+}-channel, K{sub ir}3.4, Na{sub v}1.5) and RyR-2 in hypertrophic hearts, while gene expression was repressed in diseased human hearts. Further, the genes coding for calreticulin and calmodulin, PMCA 1 and 4 as well as {alpha}-skeletal actin were significantly (P < 0.05) changed in hypertrophic human heart, but were unchanged in hypertrophic left ventricles of the rat heart. Notably, transcript level of {alpha}- and {beta}-MHC, calsequestrin, K{sub ir}6.1 (in the right ventricle only), phospholamban as well as troponin T were repressed in both diseased human and rat hearts. Our study enabled an identification of disease-associated candidate genes. Their regulation is likely to be the result of an imbalance between pressure load/stretch force and vascular tonus and the observed changes may provide a rational for the rhythm disturbances observed in patients with cardiac hypertrophy.« less

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.more » - Highlights: • Dexamethasone accelerates Ca{sup 2+} transient decay in hESC-CMs. • Dexamethasone enhances SERCA and NCX function in hESC-CMs. • Dexamethasone increases force of contraction and sarcomere length in hESC-CMs. • Dexamethasone does not alter I{sub Ca,L} and action potential characteristics in hESC-CMs.« less

Amalaki rasayana, a traditional Indian drug enhances cardiac mitochondrial and contractile functions and improves cardiac function in rats with hypertrophy.

PubMed

Kumar, Vikas; Aneesh, Kumar A; Kshemada, K; Ajith, Kumar G S; Binil, Raj S S; Deora, Neha; Sanjay, G; Jaleel, A; Muraleedharan, T S; Anandan, E M; Mony, R S; Valiathan, M S; Santhosh, Kumar T R; Kartha, C C

2017-08-17

We evaluated the cardioprotective effect of Amalaki Rasayana (AR), a rejuvenating Ayurvedic drug prepared from Phyllanthus emblica fruits in the reversal of remodeling changes in pressure overload left ventricular cardiac hypertrophy (LVH) and age-associated cardiac dysfunction in male Wistar rats. Six groups (aging groups) of 3 months old animals were given either AR or ghee and honey (GH) orally; seventh group was untreated. Ascending aorta was constricted using titanium clips in 3 months old rats (N = 24; AC groups) and after 6 months, AR or GH was given for further 12 months to two groups; one group was untreated. Histology, gene and protein expression analysis were done in heart tissues. Chemical composition of AR was analyzed by HPLC, HPTLC and LC-MS. AR intake improved (P < 0.05) cardiac function in aging rats and decreased LVH (P < 0.05) in AC rats as well as increased (P < 0.05) fatigue time in treadmill exercise in both groups. In heart tissues of AR administered rats of both the groups, SERCA2, CaM, Myh11, antioxidant, autophagy, oxidative phosphorylation and TCA cycle proteins were up regulated. ADRB1/2 and pCREB expression were increased; pAMPK, NF-kB were decreased. AR has thus a beneficial effect on myocardial energetics, muscle contractile function and exercise tolerance capacity.

Functional Effects of Hyperthyroidism on Cardiac Papillary Muscle in Rats.

PubMed

Vieira, Fabricio Furtado; Olivoto, Robson Ruiz; Silva, Priscyla Oliveira da; Francisco, Julio Cesar; Fogaça, Rosalvo Tadeu Hochmuller

2016-12-01

Hyperthyroidism is currently recognized to affect the cardiovascular system, leading to a series of molecular and functional changes. However, little is known about the functional influence of hyperthyroidism in the regulation of cytoplasmic calcium and on the sodium/calcium exchanger (NCX) in the cardiac muscle. To evaluate the functional changes in papillary muscles isolated from animals with induced hyperthyroidism. We divided 36 Wistar rats into a group of controls and another of animals with hyperthyroidism induced by intraperitoneal T3 injection. We measured in the animals' papillary muscles the maximum contraction force, speed of contraction (+df/dt) and relaxation (-df/dt), contraction and relaxation time, contraction force at different concentrations of extracellular sodium, post-rest potentiation (PRP), and contraction force induced by caffeine. In hyperthyroid animals, we observed decreased PRP at all rest times (p < 0.05), increased +df/dt and -df/dt (p < 0.001), low positive inotropic response to decreased concentration of extracellular sodium (p < 0.001), reduction of the maximum force in caffeine-induced contraction (p < 0.003), and decreased total contraction time (p < 0.001). The maximal contraction force did not differ significantly between groups (p = 0.973). We hypothesize that the changes observed are likely due to a decrease in calcium content in the sarcoplasmic reticulum, caused by calcium leakage, decreased expression of NCX, and increased expression of a-MHC and SERCA2.

Effects of atorvastatin and losartan on monocrotaline-induced pulmonary artery remodeling in rats.

PubMed

Xie, Liangdi; Lin, Peisen; Xie, Hong; Xu, Changsheng

2010-01-01

Structural remodeling of pulmonary artery plays an important role in maintaining sustained pulmonary arterial hypertension (PAH). The anti-remodeling effects of statins have been reported in systemic hypertension. In this study, we studied the effects of atovastatin (Ato) or losartan (Los) in monocrotaline (MCL)-induced pulmonary artery remodeling using a rat model. Forty Sprague-Dawley (SD) rats were randomly assigned into four groups (n = 10): normal control (Ctr), PAH, PAH treated with Los, and PAH treated with Ato. We found that in the Los- or Ato-treated group, the mean pulmonary arterial pressure, right heart hypertrophy index, ratio of wall/lumen thickness (WT%), as well as the wall/lumen area (WA%) were significantly reduced compared to the PAH group. Also in pulmonary arteries dissected from rats in the Ato- or Los-treated group, in both mRNA and protein levels, the expression of α1C subunit of voltage-gated calcium channel (Ca(v)α1c) was downregulated, while sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA-2a) and inositol 1,4,5 triphosphate receptor 1 (IP3R-1) upregulated. However, the mRNA level of RyR-3 subunit of calcium regulating channel was increased, whereas its protein level was reduced in the treated groups. Our results suggest that atorvastatin or losartan may regress the remodeling of the pulmonary artery in pulmonary hypertensive rats, with differential expression of calcium regulating channels.

Comparative molecular analyses of select pH- and osmoregulatory genes in three freshwater crayfish Cherax quadricarinatus, C. destructor and C. cainii.

PubMed

Ali, Muhammad Y; Pavasovic, Ana; Dammannagoda, Lalith K; Mather, Peter B; Prentis, Peter J

2017-01-01

Systemic acid-base balance and osmotic/ionic regulation in decapod crustaceans are in part maintained by a set of transport-related enzymes such as carbonic anhydrase (CA), Na + /K + -ATPase (NKA), H + -ATPase (HAT), Na + /K + /2Cl - cotransporter (NKCC), Na + /Cl - /HCO[Formula: see text] cotransporter (NBC), Na + /H + exchanger (NHE), Arginine kinase (AK), Sarcoplasmic Ca +2 -ATPase (SERCA) and Calreticulin (CRT). We carried out a comparative molecular analysis of these genes in three commercially important yet eco-physiologically distinct freshwater crayfish , Cherax quadricarinatus, C. destructor and C. cainii , with the aim to identify mutations in these genes and determine if observed patterns of mutations were consistent with the action of natural selection. We also conducted a tissue-specific expression analysis of these genes across seven different organs, including gills, hepatopancreas, heart, kidney, liver, nerve and testes using NGS transcriptome data. The molecular analysis of the candidate genes revealed a high level of sequence conservation across the three Cherax sp. Hyphy analysis revealed that all candidate genes showed patterns of molecular variation consistent with neutral evolution. The tissue-specific expression analysis showed that 46% of candidate genes were expressed in all tissue types examined, while approximately 10% of candidate genes were only expressed in a single tissue type. The largest number of genes was observed in nerve (84%) and gills (78%) and the lowest in testes (66%). The tissue-specific expression analysis also revealed that most of the master genes regulating pH and osmoregulation (CA, NKA, HAT, NKCC, NBC, NHE) were expressed in all tissue types indicating an important physiological role for these genes outside of osmoregulation in other tissue types. The high level of sequence conservation observed in the candidate genes may be explained by the important role of these genes as well as potentially having a number of other basic physiological functions in different tissue types.

Frequency-dependent acceleration of relaxation in mammalian heart: a property not relying on phospholamban and SERCA2a phosphorylation

PubMed Central

Valverde, Carlos A; Mundiña-Weilenmann, Cecilia; Said, Matilde; Ferrero, Paola; Vittone, Leticia; Salas, Margarita; Palomeque, Julieta; Petroff, Martín Vila; Mattiazzi, Alicia

2005-01-01

An increase in stimulation frequency causes an acceleration of myocardial relaxation (FDAR). Several mechanisms have been postulated to explain this effect, among which is the Ca2+–calmodulin-dependent protein kinase (CaMKII)-dependent phosphorylation of the Thr17 site of phospholamban (PLN). To gain further insights into the mechanisms of FDAR, we studied the FDAR and the phosphorylation of PLN residues in perfused rat hearts, cat papillary muscles and isolated cat myocytes. This allowed us to sweep over a wide range of frequencies, in species with either positive or negative force–frequency relationships, as well as to explore the FDAR under isometric (or isovolumic) and isotonic conditions. Results were compared with those produced by isoprenaline, an intervention known to accelerate relaxation (IDAR) via PLN phosphorylation. While IDAR occurs tightly associated with a significant increase in the phosphorylation of Ser16 and Thr17 of PLN, FDAR occurs without significant changes in the phosphorylation of PLN residues in the intact heart and cat papillary muscles. Moreover, in intact hearts, FDAR was not associated with any significant change in the CaMKII-dependent phosphorylation of sarcoplasmic/endoplasmic Ca2+ ATPase (SERCA2a), and was not affected by the presence of the CaMKII inhibitor, KN-93. In isolated myocytes, FDAR occurred associated with an increase in Thr17 phosphorylation. However, for a similar relaxant effect produced by isoprenaline, the phosphorylation of PLN (Ser16 and Thr17) was significantly higher in the presence of the β-agonist. Moreover, the time course of Thr17 phosphorylation was significantly delayed with respect to the onset of FDAR. In contrast, the time course of Ser16 phosphorylation, the first residue that becomes phosphorylated with isoprenaline, was temporally associated with IDAR. Furthermore, KN-93 significantly decreased the phosphorylation of Thr17 that was evoked by increasing the stimulation frequency, but failed to affect FDAR. Taken together, the results provide direct evidence indicating that CaMKII phosphorylation pathways are not involved in FDAR and that FDAR and IDAR do not share a common underlying mechanism. More likely, a CaMKII-independent mechanism could be involved, whereby increasing stimulation frequency would disrupt the SERCA2a–PLN interaction, leading to an increase in SR Ca2+ uptake and myocardial relaxation. PMID:15528241

Characterization of Beta-leptinotarsin-h and the Effects of Calcium Flux Antagonists on its Activity

DTIC Science & Technology

2005-04-07

A alone. a IP3R, IP3 receptor ; LO, ligand -operated; RyR, ryanodine receptor ; SERCA, sarcoplasmic reticulum endoplasmic reticulum Ca 2C ATPase; SO...observation eliminated non-selective cation channels such as nicotinic, glutamatergic, purinergic P2X , and serotoni- nergic 5-HT3 ligand -operated Ca 2C...nicardipine, nifedipine, SNX-482) was inhibitory. Selective inhibitors of ligand -operated, store-operated, and transduction-operated channels were also not

Iron-overload injury and cardiomyopathy in acquired and genetic models is attenuated by resveratrol therapy.

PubMed

Das, Subhash K; Wang, Wang; Zhabyeyev, Pavel; Basu, Ratnadeep; McLean, Brent; Fan, Dong; Parajuli, Nirmal; DesAulniers, Jessica; Patel, Vaibhav B; Hajjar, Roger J; Dyck, Jason R B; Kassiri, Zamaneh; Oudit, Gavin Y

2015-12-07

Iron-overload cardiomyopathy is a prevalent cause of heart failure on a world-wide basis and is a major cause of mortality and morbidity in patients with secondary iron-overload and genetic hemochromatosis. We investigated the therapeutic effects of resveratrol in acquired and genetic models of iron-overload cardiomyopathy. Murine iron-overload models showed cardiac iron-overload, increased oxidative stress, altered Ca(2+) homeostasis and myocardial fibrosis resulting in heart disease. Iron-overload increased nuclear and acetylated levels of FOXO1 with corresponding inverse changes in SIRT1 levels in the heart corrected by resveratrol therapy. Resveratrol, reduced the pathological remodeling and improved cardiac function in murine models of acquired and genetic iron-overload at varying stages of iron-overload. Echocardiography and hemodynamic analysis revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. Myocardial SERCA2a levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCA2a levels and corrected altered Ca(2+) homeostasis. Iron-mediated pro-oxidant and pro-fibrotic effects in human and murine cardiomyocytes and cardiofibroblasts were suppressed by resveratrol which correlated with reduction in iron-induced myocardial oxidative stress and myocardial fibrosis. Resveratrol represents a clinically and economically feasible therapeutic intervention to reduce the global burden from iron-overload cardiomyopathy at early and chronic stages of iron-overload.

Divergent Mechanisms Leading to Signaling Dysfunction in Embryonic Muscle by Bisphenol A and Tetrabromobisphenol A

PubMed Central

Pessah, Isaac N.

2017-01-01

Bisphenol A (BPA) and its brominated derivative tetrabromobisphenol A (TBBPA) are high production volume chemicals used in the manufacture of various consumer products. Although regarded as endocrine disruptors, these chemicals are suspected to exert nongenomic actions on muscle function that are not well understood. Using skeletal muscle microsomes, we examined the effects of BPA and TBBPA on ryanodine receptor type 1 (RyR1), dihydropyridine receptor (DHPR), and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). We assessed the impact of these chemicals on Ca2+ dynamics and signaling in embryonic skeletal myotubes through fluorescent Ca2+ imaging and measurement of resting membrane potential (Vm). TBBPA activated RyR1 and inhibited DHPR and SERCA, inducing a net efflux of Ca2+ from loaded microsomes, whereas BPA exhibited little or no activity at these targets. Regardless, both compounds disrupted the function of intact myotubes. TBBPA diminished and eventually abrogated Ca2+ transients, altered intracellular Ca2+ equilibrium, and caused Vm depolarization. For some cells, BPA caused rapid Ca2+ transient loss without marked changes in cytosolic and sarcoplasmic reticulum Ca2+ levels, likely owing to altered cellular excitability as a result of BPA-induced Vm hyperpolarization. BPA and TBBPA both interfere with skeletal muscle function through divergent mechanisms that impair excitation-contraction coupling and may be exemplary of their adverse outcomes in other muscle types. PMID:28143888

Two Dimensional Finite Element Model to Study Calcium Distribution in Oocytes

NASA Astrophysics Data System (ADS)

Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2015-06-01

Cytosolic free calcium concentration is a key regulatory factor and perhaps the most widely used means of controlling cellular function. Calcium can enter cells through different pathways which are activated by specific stimuli including membrane depolarization, chemical signals and calcium depletion of intracellular stores. One of the important components of oocyte maturation is differentiation of the Ca2+ signaling machinery which is essential for egg activation after fertilization. Eggs acquire the ability to produce the fertilization-specific calcium signal during oocyte maturation. The calcium concentration patterns required during different stages of oocyte maturation are still not completely known. Also the mechanisms involved in calcium dynamics in oocyte cell are still not well understood. In view of above a two dimensional FEM model has been proposed to study calcium distribution in an oocyte cell. The parameters such as buffers, ryanodine receptor, SERCA pump and voltage gated calcium channel are incorporated in the model. Based on the biophysical conditions the initial and boundary conditions have been framed. The model is transformed into variational form and Ritz finite element method has been employed to obtain the solution. A program has been developed in MATLAB 7.10 for the entire problem and executed to obtain numerical results. The numerical results have been used to study the effect of buffers, RyR, SERCA pump and VGCC on calcium distribution in an oocyte cell.

Iron-overload injury and cardiomyopathy in acquired and genetic models is attenuated by resveratrol therapy

PubMed Central

Das, Subhash K.; Wang, Wang; Zhabyeyev, Pavel; Basu, Ratnadeep; McLean, Brent; Fan, Dong; Parajuli, Nirmal; DesAulniers, Jessica; Patel, Vaibhav B.; Hajjar, Roger J.; Dyck, Jason R. B.; Kassiri, Zamaneh; Oudit, Gavin Y.

2015-01-01

Iron-overload cardiomyopathy is a prevalent cause of heart failure on a world-wide basis and is a major cause of mortality and morbidity in patients with secondary iron-overload and genetic hemochromatosis. We investigated the therapeutic effects of resveratrol in acquired and genetic models of iron-overload cardiomyopathy. Murine iron-overload models showed cardiac iron-overload, increased oxidative stress, altered Ca2+ homeostasis and myocardial fibrosis resulting in heart disease. Iron-overload increased nuclear and acetylated levels of FOXO1 with corresponding inverse changes in SIRT1 levels in the heart corrected by resveratrol therapy. Resveratrol, reduced the pathological remodeling and improved cardiac function in murine models of acquired and genetic iron-overload at varying stages of iron-overload. Echocardiography and hemodynamic analysis revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. Myocardial SERCA2a levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCA2a levels and corrected altered Ca2+ homeostasis. Iron-mediated pro-oxidant and pro-fibrotic effects in human and murine cardiomyocytes and cardiofibroblasts were suppressed by resveratrol which correlated with reduction in iron-induced myocardial oxidative stress and myocardial fibrosis. Resveratrol represents a clinically and economically feasible therapeutic intervention to reduce the global burden from iron-overload cardiomyopathy at early and chronic stages of iron-overload. PMID:26638758

Gene expression in mdx mouse muscle in relation to age and exercise: aberrant mechanical-metabolic coupling and implications for pre-clinical studies in Duchenne muscular dystrophy.

PubMed

Camerino, Giulia Maria; Cannone, Maria; Giustino, Arcangela; Massari, Ada Maria; Capogrosso, Roberta Francesca; Cozzoli, Anna; De Luca, Annamaria

2014-11-01

Weakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. Mechanical activity modulates gene expression and muscle plasticity. Here, we investigated the outcome of 4 (T4, 8 weeks of age) and 12 (T12, 16 weeks of age) weeks of either exercise or cage-based activity on a large set of genes in the gastrocnemius muscle of mdx and wild-type (WT) mice using quantitative real-time PCR. Basal expression of the exercise-sensitive genes peroxisome-proliferator receptor γ coactivator 1α (Pgc-1α) and Sirtuin1 (Sirt1) was higher in mdx versus WT mice at both ages. Exercise increased Pgc-1α expression in WT mice; Pgc-1α was downregulated by T12 exercise in mdx muscles, along with Sirt1, Pparγ and the autophagy marker Bnip3. Sixteen weeks old mdx mice showed a basal overexpression of the slow Mhc1 isoform and Serca2; T12 exercise fully contrasted this basal adaptation as well as the high expression of follistatin and myogenin. Conversely, T12 exercise was ineffective in WT mice. Damage-related genes such as gp91-phox (NADPH-oxidase2), Tgfβ, Tnfα and c-Src tyrosine kinase were overexpressed in mdx muscles and not affected by exercise. Likewise, the anti-inflammatory adiponectin was lower in T12-exercised mdx muscles. Chronic exercise with minor adaptive effects in WT muscles leads to maladaptation in mdx muscles with a disequilibrium between protective and damaging signals. Increased understanding of the pathways involved in the altered mechanical-metabolic coupling may help guide appropriate physical therapies while better addressing pharmacological interventions in translational research. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Thyroid Hormone Signaling in Male Mouse Skeletal Muscle Is Largely Independent of D2 in Myocytes

PubMed Central

Werneck-de-Castro, Joao P.; Fonseca, Tatiana L.; Ignacio, Daniele L.; Fernandes, Gustavo W.; Andrade-Feraud, Cristina M.; Lartey, Lattoya J.; Ribeiro, Marcelo B.; Ribeiro, Miriam O.; Gereben, Balazs

2015-01-01

The type 2 deiodinase (D2) activates the prohormone T4 to T3. D2 is expressed in skeletal muscle (SKM), and its global inactivation (GLOB-D2KO mice) reportedly leads to skeletal muscle hypothyroidism and impaired differentiation. Here floxed Dio2 mice were crossed with mice expressing Cre-recombinase under the myosin light chain 1f (cre-MLC) to disrupt D2 expression in the late developmental stages of skeletal myocytes (SKM-D2KO). This led to a loss of approximately 50% in D2 activity in neonatal and adult SKM-D2KO skeletal muscle and about 75% in isolated SKM-D2KO myocytes. To test the impact of Dio2 disruption, we measured soleus T3 content and found it to be normal. We also looked at the expression of T3-responsive genes in skeletal muscle, ie, myosin heavy chain I, α-actin, myosin light chain, tropomyosin, and serca 1 and 2, which was preserved in neonatal SKM-D2KO hindlimb muscles, at a time that coincides with a peak of D2 activity in control animals. In adult soleus the baseline level of D2 activity was about 6-fold lower, and in the SKM-D2KO soleus, the expression of only one of five T3-responsive genes was reduced. Despite this, adult SKM-D2KO animals performed indistinguishably from controls on a treadmill test, running for approximately 16 minutes and reached a speed of about 23 m/min; muscle strength was about 0.3 mN/m·g body weight in SKM-D2KO and control ankle muscles. In conclusion, there are multiple sources of D2 in the mouse SKM, and its role is limited in postnatal skeletal muscle fibers. PMID:26214036

Three-dimensional contractile muscle tissue consisting of human skeletal myocyte cell line.

PubMed

Shima, Ai; Morimoto, Yuya; Sweeney, H Lee; Takeuchi, Shoji

2018-06-18

This paper describes a method to construct three-dimensional (3D) contractile human skeletal muscle tissues from a cell line. The 3D tissue was fabricated as a fiber-based structure and cultured for two weeks under tension by anchoring its both ends. While myotubes from the immortalized human skeletal myocytes used in this study never contracted in the conventional two-dimensional (2D) monolayer culture, myotubes in the 3D tissue showed spontaneous contraction at a high frequency and also reacted to the electrical stimulation. Immunofluorescence revealed that the myotubes in the 3D tissues had sarcomeres and expressed ryanodine receptor (RyR) and sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). In addition, intracellular calcium oscillations in the myotubes in the 3D tissue were observed. These results indicated that the 3D culture enabled the myocyte cell line to reach a more highly matured state compared to 2D culture. Since contraction is the most significant feature of skeletal muscle, we believe that our 3D human muscle tissue with the contractile ability would be a useful tool for both basic biology research and drug discovery as one of the muscle-on-chips. Copyright © 2018. Published by Elsevier Inc.

Cardioprotective effects of early and late aerobic exercise training in experimental pulmonary arterial hypertension.

PubMed

Moreira-Gonçalves, Daniel; Ferreira, Rita; Fonseca, Hélder; Padrão, Ana Isabel; Moreno, Nuno; Silva, Ana Filipa; Vasques-Nóvoa, Francisco; Gonçalves, Nádia; Vieira, Sara; Santos, Mário; Amado, Francisco; Duarte, José Alberto; Leite-Moreira, Adelino F; Henriques-Coelho, Tiago

2015-11-01

Clinical studies suggest that aerobic exercise can exert beneficial effects in pulmonary arterial hypertension (PAH), but the underlying mechanisms are largely unknown. We compared the impact of early or late aerobic exercise training on right ventricular function, remodeling and survival in experimental PAH. Male Wistar rats were submitted to normal cage activity (SED), exercise training in early (EarlyEX) and in late stage (LateEX) of PAH induced by monocrotaline (MCT, 60 mg/kg). Both exercise interventions resulted in improved cardiac function despite persistent right pressure-overload, increased exercise tolerance and survival, with greater benefits in EarlyEX+MCT. This was accompanied by improvements in the markers of cardiac remodeling (SERCA2a), neurohumoral activation (lower endothelin-1, brain natriuretic peptide and preserved vascular endothelial growth factor mRNA), metabolism and mitochondrial oxidative stress in both exercise interventions. EarlyEX+MCT provided additional improvements in fibrosis, tumor necrosis factor-alpha/interleukin-10 and brain natriuretic peptide mRNA, and beta/alpha myosin heavy chain protein expression. The present study demonstrates important cardioprotective effects of aerobic exercise in experimental PAH, with greater benefits obtained when exercise training is initiated at an early stage of the disease.

A Thapsigargin-Resistant Intracellular Calcium Sequestering Compartment in Rat Brain

DTIC Science & Technology

2000-03-31

touted as inhibitors of the PMCAs, but their effects have yet to be thoroughly examined on all P-type ATPases [23, 125]. SERCAs: a family ofP·type ATPases... effective in reducing the sensitivity to TG by 1aOO-fold [151]. The potent, seledive blockade of Ca2+ accumulation in the ER by TG has made it possible...accumulation while the calcium ionophore A23187 is 100%) effective [140]. Thus, 10-150/0 of net Ca2+ uptake into brain microsomal compartments is

Glucose-6-phosphate Reduces Calcium Accumulation in Rat Brain Endoplasmic Reticulum

DTIC Science & Technology

2012-04-01

Ekman and Jager, 1993) adapted from the ammonium molybdate/ malachite green method quan- tified Pi production by SERCA activity. Colorimetric reagent was...prepared by mixing one volume of 10% (w/v) (NH4)6Mo7O24– 4 H2O in 4M HCl with three volumes of 0.2% (w/v) malachite green in 4M HCl, followed by...seryl and threonyl residues in phosphoproteins using alkaline hydrolysis and malachite green. Anal. Biochem. 214, 138. Fulceri, R., Romani, A

Protein Changes Contributing to Right Ventricular Cardiomyocyte Diastolic Dysfunction in Pulmonary Arterial Hypertension

PubMed Central

Rain, Silvia; Bos, Denielli da Silva Goncalves; Handoko, M. Louis; Westerhof, Nico; Stienen, Ger; Ottenheijm, Coen; Goebel, Max; Dorfmüller, Peter; Guignabert, Christophe; Humbert, Marc; Bogaard, Harm‐Jan; dos Remedios, Cris; Saripalli, Chandra; Hidalgo, Carlos G.; Granzier, Henk L.; Vonk‐Noordegraaf, Anton; van der Velden, Jolanda; de Man, Frances S.

2014-01-01

Background Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca2+ sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. Methods and Results RV samples from PAH patients undergoing heart‐lung transplantation were compared to non‐failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western‐blot analyses using antibodies to protein‐kinase‐A (PKA), Cα (PKCα) and Ca2+/calmoduling‐dependent‐kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P<0.0001). To test the functional relevance of PKA‐, PKCα‐, and CamKIIδ‐mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca2+ sensitivity is mediated by sarcomeric troponin I (cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P<0.05) and reduced PKA‐mediated cTnI phosphorylation (Ser22/23) (P<0.001). Finally, alterations in Ca2+‐handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca2+ clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P<0.05). Conclusions Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca2+ handling proteins contribute to RV diastolic dysfunction in PAH. PMID:24895160

Peripheral Serotonin Regulates Maternal Calcium Trafficking in Mammary Epithelial Cells during Lactation in Mice

PubMed Central

Laporta, Jimena; Keil, Kimberly P.; Vezina, Chad M.; Hernandez, Laura L.

2014-01-01

Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of the mammary alveolar epithelium (blood) into the ductal lumen (milk). Regulation of calcium transport during lactation is critical for maternal and neonatal health. The monoamine serotonin (5-HT) is synthesized by the mammary gland and functions as a homeostatic regulation of lactation. Genetic ablation of tryptophan hydroxylase 1 (Tph1), which encodes the rate-limiting enzyme in non-neuronal serotonin synthesis, causes a deficiency in circulating serotonin. As a consequence maternal calcium concentrations decrease, mammary epithelial cell morphology is altered, and cell proliferation is decreased during lactation. Here we demonstrate that serotonin deficiency decreases the expression and disrupts the normal localization of calcium transporters located in the apical (PMCA2) and basolateral (CaSR, ORAI-1) membranes of the lactating mammary gland. In addition, serotonin deficiency decreases the mRNA expression of calcium transporters located in intracellular compartments (SERCA2, SPCA1 and 2). Mammary expression of serotonin receptor isoform 2b and its downstream pathways (PLCβ3, PKC and MAP-ERK1/2) are also decreased by serotonin deficiency, which might explain the numerous phenotypic alterations described above. In most cases, addition of exogenous 5-hydroxy-L-tryptophan to the Tph1 deficient mice rescued the phenotype. Our data supports the hypothesis that serotonin is necessary for proper mammary gland structure and function, to regulate blood and mammary epithelial cell transport of calcium during lactation. These findings can be applicable to the treatment of lactation-induced hypocalcemia in dairy cows and can have profound implications in humans, given the wide-spread use of selective serotonin reuptake inhibitors as antidepressants during pregnancy and lactation. PMID:25299122

Cardiac microRNA-133 is down-regulated in thyroid hormone-mediated cardiac hypertrophy partially via Type 1 Angiotensin II receptor.

PubMed

Diniz, Gabriela Placoná; Lino, Caroline Antunes; Guedes, Elaine Castilho; Moreira, Luana do Nascimento; Barreto-Chaves, Maria Luiza Morais

2015-09-01

Elevated thyroid hormone (TH) levels induce cardiac hypertrophy partially via type 1 Angiotensin II receptor (AT1R). MicroRNAs (miRNAs) are key regulators of cardiac homeostasis, and miR-133 has been shown to be involved in cardiac hypertrophy. However, the potential role of miR-133 in cardiac growth induced by TH is unknown. Thus, we aimed to investigate the miR-133 expression, as well as its potential role in cardiac hypertrophy in response to TH. Wistar rats were subjected to hyperthyroidism combined or not with the AT1R blocker. T3 serum levels were assessed to confirm the hyperthyroid status. TH induced cardiac hypertrophy, as evidenced by higher cardiac weight/tibia length ratio and α-actin mRNA levels, which was prevented by AT1R blocker. miR-133 expression was decreased in TH-induced cardiac hypertrophy in part through the AT1R. Additionally, the cardiac mRNA levels of miR-133 targets, SERCA2a and calcineurin were increased in hyperthyroidism partially via AT1R, as evaluated by real-time RT-PCR. Interestingly, miR-133 levels were unchanged in T3-induced cardiomyocyte hypertrophy in vitro. However, a gain-of-function study revealed that miR-133 mimic blunted the T3-induced cardiomyocyte hypertrophy in vitro. Together, our data indicate that miR-133 expression is reduced in TH-induced cardiac hypertrophy partially by the AT1R and that miR-133 mimic prevents the cardiomyocyte hypertrophy in response to T3 in vitro. These findings provide new insights regarding the mechanisms involved in the cardiac growth mediated by TH, suggesting that miR-133 plays a key role in TH-induced cardiomyocyte hypertrophy.

Microscopic heat pulse-induced calcium dynamics in single WI-38 fibroblasts.

PubMed

Itoh, Hideki; Oyama, Kotaro; Suzuki, Madoka; Ishiwata, Shin'ichi

2014-01-01

Temperature-sensitive Ca(2+) dynamics occur primarily through transient receptor potential channels, but also by means of Ca(2+) channels and pumps on the endoplasmic reticulum membrane. As such, cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt) is re-equilibrated by changes in ambient temperature. The present study investigated the effects of heat pulses (heating duration: 2 s or 150 s) on [Ca(2+)]cyt in single WI-38 fibroblasts, which are considered as normal cells. We found that Ca(2+) burst occurred immediately after short (2 s) heat pulse, which is similar to our previous report on HeLa cells, but with less thermosensitivity. The heat pulses originated from a focused 1455-nm infrared laser light were applied in the vicinity of cells under the optical microscope. Ca(2+) bursts induced by the heat pulse were suppressed by treating cells with inhibitors for sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) or inositol trisphosphate receptor (IP3R). Long (150 s) heat pulses also induced Ca(2+) bursts after the onset of heating and immediately after re-cooling. Cells were more thermosensitive at physiological (37°C) than at room (25°C) temperature; however, at 37°C, cells were responsive at a higher temperature (ambient temperature+heat pulse). These results strongly suggest that the heat pulse-induced Ca(2+) burst is caused by a transient imbalance in Ca(2+) flow between SERCA and IP3R, and offer a potential new method for thermally controlling Ca(2+)-regulated cellular functions.

Inhibition of spontaneous activity of rabbit atrioventricular node cells by KB-R7943 and inhibitors of sarcoplasmic reticulum Ca2+ ATPase

PubMed Central

Cheng, Hongwei; Smith, Godfrey L.; Hancox, Jules C.; Orchard, Clive H.

2011-01-01

The atrioventricular node (AVN) can act as a subsidiary cardiac pacemaker if the sinoatrial node fails. In this study, we investigated the effects of the Na–Ca exchange (NCX) inhibitor KB-R7943, and inhibition of the sarcoplasmic reticulum calcium ATPase (SERCA), using thapsigargin or cyclopiazonic acid (CPA), on spontaneous action potentials (APs) and [Ca2+]i transients from cells isolated from the rabbit AVN. Spontaneous [Ca2+]i transients were monitored from undialysed AVN cells at 37 °C using Fluo-4. In separate experiments, spontaneous APs and ionic currents were recorded using the whole-cell patch clamp technique. Rapid application of 5 μM KB-R7943 slowed or stopped spontaneous APs and [Ca2+]i transients. However, in voltage clamp experiments in addition to blocking NCX current (INCX) KB-R7943 partially inhibited L-type calcium current (ICa,L). Rapid reduction of external [Na+] also abolished spontaneous activity. Inhibition of SERCA (using 2.5 μM thapsigargin or 30 μM CPA) also slowed or stopped spontaneous APs and [Ca2+]i transients. Our findings are consistent with the hypothesis that sarcoplasmic reticulum (SR) Ca2+ release influences spontaneous activity in AVN cells, and that this occurs via [Ca2+]i-activated INCX; however, the inhibitory action of KB-R7943 on ICa,L means that care is required in the interpretation of data obtained using this compound. PMID:21163524

Skeletal muscle proteins: a new approach to delimitate the time since death.

PubMed

Foditsch, Elena Esra; Saenger, Alexandra Maria; Monticelli, Fabio Carlo

2016-03-01

Skeletal muscle tissue is proposed as a forensic model tissue with strong potential, as it is easily accessible and its true-to-life state structure and function is well known. Despite this strong potential, skeletal muscle degradation studies are rare. The aim of this study was to test if a skeletal muscle-based protein analysis is applicable to delimitate the time since death. Under standard conditions, two pigs were stored either at 22 °C for 5 days or 4 °C for 21 days. Their Mm. biceps femori were sampled periodically for analyses of ten skeletal muscle proteins postmortem. All analyzed proteins can serve as markers for a delimitation of the time since death. Desmin, nebulin, titin, and SERCA 1 displayed distinct protein patterns at certain points of time. The other five proteins, α-actinin, calsequestrin-1, laminin, troponin T-C, and SERCA 2, showed no degradation patterns within the analyzed postmortem time frame. Referring to specific skeletal muscle proteins, results showed short-term stabilities for just a minority of analyzed proteins, while the majority of investigated proteins displayed characteristics as long-term markers. Due to specific patterns and the possibility to determine definite constraints of the presence, absence, or pattern alterations of single proteins, the feasibility of porcine skeletal muscle as forensic model tissue is outlined and the potential of skeletal muscle as forensic model tissue is underlined, especially with respect to later postmortem phases, which so far lack feasible methods to delimitate the time since death.

CD147 reinforces [Ca2+]i oscillations and promotes oncogenic progression in hepatocellular carcinoma.

PubMed

Tang, Juan; Guo, Yun-Shan; Yu, Xiao-Ling; Huang, Wan; Zheng, Ming; Zhou, Ying-Hui; Nan, Gang; Wang, Jian-Chao; Yang, Hai-Jiao; Yu, Jing-Min; Jiang, Jian-Li; Chen, Zhi-Nan

2015-10-27

Oscillations in intracellular Ca2+ concentrations ([Ca2+]i) mediate various cellular function. Although it is known that [Ca2+]i oscillations are susceptible to dysregulation in tumors, the tumor-specific regulators of [Ca2+]i oscillations are poorly characterized. We discovered that CD147 promotes hepatocellular carcinoma (HCC) metastasis and proliferation by enhancing the amplitude and frequency of [Ca2+]i oscillations in HCC cells. CD147 activates two distinct signaling pathways to regulate [Ca2+]i oscillations. By activating FAK-Src-IP3R1 signaling pathway, CD147 promotes Ca2+ release from endoplasmic reticulum (ER) and enhances the amplitude of [Ca2+]i oscillations. Furthermore, CD147 accelerates ER Ca2+refilling and enhances the frequency of [Ca2+]i oscillations through activating CaMKP-PAK1-PP2A-PLB-SERCA signaling pathway. Besides, CD147-promoted ER Ca2+ release and refilling are tightly regulated by changing [Ca2+]i. CD147 may activate IP3R1 channel under low [Ca2+]i conditions and CD147 may activate SERCA pump under high [Ca2+]i conditions. CD147 deletion suppresses HCC tumorigenesis and increases the survival rate of liver-specific CD147 knockout mice by regulating [Ca2+]i oscillations in vivo. Together, these results reveal that CD147 functions as a critical regulator of ER-dependent [Ca2+]i oscillations to promote oncogenic progression in HCC.

CD147 reinforces [Ca2+]i oscillations and promotes oncogenic progression in hepatocellular carcinoma

PubMed Central

Zheng, Ming; Zhou, Ying-Hui; Nan, Gang; Wang, Jian-Chao; Yang, Hai-Jiao; Yu, Jing-Min; Jiang, Jian-Li; Chen, Zhi-Nan

2015-01-01

Oscillations in intracellular Ca2+ concentrations ([Ca2+]i) mediate various cellular function. Although it is known that [Ca2+]i oscillations are susceptible to dysregulation in tumors, the tumor-specific regulators of [Ca2+]i oscillations are poorly characterized. We discovered that CD147 promotes hepatocellular carcinoma (HCC) metastasis and proliferation by enhancing the amplitude and frequency of [Ca2+]i oscillations in HCC cells. CD147 activates two distinct signaling pathways to regulate [Ca2+]i oscillations. By activating FAK-Src-IP3R1 signaling pathway, CD147 promotes Ca2+ release from endoplasmic reticulum (ER) and enhances the amplitude of [Ca2+]i oscillations. Furthermore, CD147 accelerates ER Ca2+ refilling and enhances the frequency of [Ca2+]i oscillations through activating CaMKP-PAK1-PP2A-PLB-SERCA signaling pathway. Besides, CD147-promoted ER Ca2+ release and refilling are tightly regulated by changing [Ca2+]i. CD147 may activate IP3R1 channel under low [Ca2+]i conditions and CD147 may activate SERCA pump under high [Ca2+]i conditions. CD147 deletion suppresses HCC tumorigenesis and increases the survival rate of liver-specific CD147 knockout mice by regulating [Ca2+]i oscillations in vivo. Together, these results reveal that CD147 functions as a critical regulator of ER-dependent [Ca2+]i oscillations to promote oncogenic progression in HCC. PMID:26498680

Caffeine-induced [Ca2+] oscillations in neurones of frog sympathetic ganglia

PubMed Central

Cseresnyés, Zoltán; Bustamante, Alexander I; Schneider, Martin F

1999-01-01

Single cell fluorimetry was used to monitor caffeine-induced oscillations of cytosolic [Ca2+] in frog sympathetic ganglion neurones in 2.0 mm K+ Ringer solution.[Ca2+] oscillations decreased in frequency and exhibited three different amplitude patterns after the first large peak of [Ca2+]: (a) a series of big oscillations (BOs) of constant large amplitude (300–;400 nm), (b) a series of much smaller oscillations (SOs) (40–60 nm), or (c) a series of decaying oscillations (DOs) of rapidly decreasing amplitude.A model in which the oscillation amplitude was determined by the Ca2+ content of the endoplasmic reticulum (ER) whereas the oscillation frequency was controlled by how rapidly the cytosolic [Ca2+] reached the threshold for Ca2+-induced Ca2+ release (CICR) was able to simulate each observed pattern by varying the level of activity of the ER Ca2+ pump (SERCA), CICR and release-activated Ca2+ transport (RACT). A cumulative, cytosolic Ca2+-dependent inactivation of the plasma membrane (PM) Ca2+ influx or of the Ca2+-sensitive leak coefficient of the ryanodine receptors caused the oscillation frequency to decrease in the model.Transitions between BOs and SOs and changes in [Ca2+] oscillations caused by ryanodine, thapsigargin, lanthanum and FCCP could also be simulated.We conclude that RACT, SERCA, CICR and Ca2+-dependent PM Ca2+ influx are major mechanisms underlying [Ca2+] oscillations in these neurones. PMID:9831718

Restoring the impaired cardiac calcium homeostasis and cardiac function in iron overload rats by the combined deferiprone and N-acetyl cysteine

PubMed Central

Wongjaikam, Suwakon; Kumfu, Sirinart; Khamseekaew, Juthamas; Chattipakorn, Siriporn C.; Chattipakorn, Nipon

2017-01-01

Intracellular calcium [Ca2+]i dysregulation plays an important role in the pathophysiology of iron overload cardiomyopathy. Although either iron chelators or antioxidants provide cardioprotection, a comparison of the efficacy of deferoxamine (DFO), deferiprone (DFP), deferasirox (DFX), N-acetyl cysteine (NAC) or a combination of DFP plus NAC on cardiac [Ca2+]i homeostasis in chronic iron overload has never been investigated. Male Wistar rats were fed with either a normal diet or a high iron (HFe) diet for 4 months. At 2 months, HFe rats were divided into 6 groups and treated with either a vehicle, DFO (25 mg/kg/day), DFP (75 mg/kg/day), DFX (20 mg/kg/day), NAC (100 mg/kg/day), or combined DFP plus NAC. At 4 months, the number of cardiac T-type calcium channels was increased, whereas cardiac sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA) was decreased, leading to cardiac iron overload and impaired cardiac [Ca2+]i homeostasis. All pharmacological interventions restored SERCA levels. Although DFO, DFP, DFX or NAC alone shared similar efficacy in improving cardiac [Ca2+]i homeostasis, only DFP + NAC restored cardiac [Ca2+]i homeostasis, leading to restoring left ventricular function in the HFe-fed rats. Thus, the combined DFP + NAC was more effective than any monotherapy in restoring cardiac [Ca2+]i homeostasis, leading to restored myocardial contractility in iron-overloaded rats. PMID:28287621

Comparative molecular analyses of select pH- and osmoregulatory genes in three freshwater crayfish Cherax quadricarinatus, C. destructor and C. cainii

PubMed Central

Pavasovic, Ana; Dammannagoda, Lalith K.; Mather, Peter B.; Prentis, Peter J.

2017-01-01

Systemic acid-base balance and osmotic/ionic regulation in decapod crustaceans are in part maintained by a set of transport-related enzymes such as carbonic anhydrase (CA), Na+/K+-ATPase (NKA), H+-ATPase (HAT), Na+/K+/2Cl− cotransporter (NKCC), Na+/Cl−/HCO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${}_{3}^{-}$\\end{document}3− cotransporter (NBC), Na+/H+ exchanger (NHE), Arginine kinase (AK), Sarcoplasmic Ca+2-ATPase (SERCA) and Calreticulin (CRT). We carried out a comparative molecular analysis of these genes in three commercially important yet eco-physiologically distinct freshwater crayfish, Cherax quadricarinatus, C. destructor and C. cainii, with the aim to identify mutations in these genes and determine if observed patterns of mutations were consistent with the action of natural selection. We also conducted a tissue-specific expression analysis of these genes across seven different organs, including gills, hepatopancreas, heart, kidney, liver, nerve and testes using NGS transcriptome data. The molecular analysis of the candidate genes revealed a high level of sequence conservation across the three Cherax sp. Hyphy analysis revealed that all candidate genes showed patterns of molecular variation consistent with neutral evolution. The tissue-specific expression analysis showed that 46% of candidate genes were expressed in all tissue types examined, while approximately 10% of candidate genes were only expressed in a single tissue type. The largest number of genes was observed in nerve (84%) and gills (78%) and the lowest in testes (66%). The tissue-specific expression analysis also revealed that most of the master genes regulating pH and osmoregulation (CA, NKA, HAT, NKCC, NBC, NHE) were expressed in all tissue types indicating an important physiological role for these genes outside of osmoregulation in other tissue types. The high level of sequence conservation observed in the candidate genes may be explained by the important role of these genes as well as potentially having a number of other basic physiological functions in different tissue types. PMID:28852583

Cardioprotection Via Modulation of Calcium Homeostasis by Thiopental in Hypoxia-Reoxygenated Neonatal Rat Cardiomyocytes

PubMed Central

Kim, Hyun-Soo; Hwang, Ki-Chul

2010-01-01

Purpose Ca2+ homeostasis plays an important role in myocardial cell injury induced by hypoxia-reoxygenation, and prevention of intracellular Ca2+ overload is key to cardioprotection. Even though thiopental is a frequently used anesthetic agent, little is known about its cardioprotective effects, particulary in association with Ca2+ homeostasis. We investigated whether thiopental protects cardiomyocytes against hypoxia-reoxygenation injury by regulating Ca2+ homeostasis. Materials and Methods Neonatal rat cardiomyocytes were isolated. Cardiomyocytes were exposed to different concentrations of thiopental and immediately replaced in the hypoxic chamber to maintain hypoxia. After 1 hour of exposure, a culture dish was transferred to the CO2 incubator and cells were incubated at 37℃ for 5 hours. At the end of the experiments, the authors assessed cell protection using immunoblot analysis and caspase activity. The mRNA of genes involved in Ca2+ homeostasis, mitochondrial membrane potential, and cellular Ca2+ levels were examined. Results In thiopental-treated cardiomyocytes, there was a decrease in expression of the proapoptotic protein Bax, caspase-3 activation, and intracellular Ca2+ content. In addition, both enhancement of anti-apoptotic protein Bcl-2 and activation of Erk concerned with survival were shown. Furthermore, thiopental attenuated alterations of genes involving Ca2+ regulation and significantly modulated abnormal changes of NCX and SERCA2a genes in hypoxia-reoxygenated neonatal cardiomyocytes. Thiopental suppressed disruption of mitochondrial membrane potential (ΔΨm) induced by hypoxia-reoxygenation. Conclusion Thiopental is likely to modulate expression of genes that regulate Ca2+ homeostasis, which reduces apoptotic cell death and results in cardioprotection. PMID:20191008

A novel interaction between calcium-modulating cyclophilin ligand and Basigin regulates calcium signaling and matrix metalloproteinase activities in human melanoma cells.

PubMed

Long, Tingting; Su, Juan; Tang, Wen; Luo, Zhongling; Liu, Shuang; Liu, Zhaoqian; Zhou, Honghao; Qi, Min; Zeng, Weiqi; Zhang, Jianglin; Chen, Xiang

2013-10-01

Intracellular free calcium is a ubiquitous second messenger regulating a multitude of normal and pathogenic cellular responses, including the development of melanoma. Upstream signaling pathways regulating the intracellular free calcium concentration ([Ca2+]i) may therefore have a significant impact on melanoma growth and metastasis. In this study, we demonstrate that the endoplasmic reticulum (ER)-associated protein calcium-modulating cyclophilin ligand (CAML) is bound to Basigin, a widely expressed integral plasma membrane glycoprotein and extracellular matrix metalloproteinase inducer (EMMPRIN, or CD147) implicated in melanoma proliferation, invasiveness, and metastasis. This interaction between CAML and Basigin was first identified using yeast two-hybrid screening and further confirmed by co-immunoprecipitation. In human A375 melanoma cells, CAML and Basigin were co-localized to the ER. Knockdown of Basigin in melanoma cells by siRNA significantly decreased resting [Ca2+]i and the [Ca2+]i increase induced by the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), indicating that the interaction between CAML and Basigin regulates ER-dependent [Ca2+]i signaling. Meanwhile upregulating the [Ca2+]i either by TG or phorbol myristate acetate (PMA) could stimulate the production of MMP-9 in A375 cells with the expression of Basigin. Our study has revealed a previously uncharacterized [Ca2+]i signaling pathway that may control melanoma invasion, and metastasis. Disruption of this pathway may be a novel therapeutic strategy for melanoma treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Cardiac and peripheral adjustments induced by early exercise training intervention were associated with autonomic improvement in infarcted rats: role in functional capacity and mortality.

PubMed

Jorge, Luciana; Rodrigues, Bruno; Rosa, Kaleizu Teodoro; Malfitano, Christiane; Loureiro, Tatiana Carolina Alba; Medeiros, Alessandra; Curi, Rui; Brum, Patricia Chakur; Lacchini, Silvia; Montano, Nicola; De Angelis, Kátia; Irigoyen, Maria-Cláudia

2011-04-01

To test the effects of early exercise training (ET) on left ventricular (LV) and autonomic functions, haemodynamics, tissues blood flows (BFs), maximal oxygen consumption (VO(2) max), and mortality after myocardial infarction (MI) in rats. Male Wistar rats were divided into: control (C), sedentary-infarcted (SI), and trained-infarcted (TI). One week after MI, TI group underwent an ET protocol (90 days, 50-70% VO(2) max). Left ventricular function was evaluated non-invasively and invasively. Baroreflex sensitivity, heart rate variability, and pulse interval were measured. Cardiac output (CO) and regional BFs were determined using coloured microspheres. Infarcted area was reduced in TI (19 ± 6%) compared with SI (34 ± 5%) after ET. Exercise training improved the LV and autonomic functions, the CO and regional BF changes induced by MI, as well as increased SERCA2 expression and mRNA vascular endothelial growth factor levels. These changes brought about by ET resulted in mortality rate reduction in the TI (13%) group compared with the SI (54%) group. Early aerobic ET reduced cardiac and peripheral dysfunctions and preserved cardiovascular autonomic control after MI in trained rats. Consequently, these ET-induced changes resulted in improved functional capacity and survival after MI.

Heterogeneous expression of Ca(2+) handling proteins in rabbit sinoatrial node.

PubMed

Musa, Hanny; Lei, Ming; Honjo, Hauro; Jones, Sandra A; Dobrzynski, Halina; Lancaster, Mathew K; Takagishi, Yoshiko; Henderson, Zaineb; Kodama, Itsuo; Boyett, Mark R

2002-03-01

We investigated the densities of the L-type Ca(2+) current, i(Ca,L), and various Ca(2+) handling proteins in rabbit sinoatrial (SA) node. The density of i(Ca,L), recorded with the whole-cell patch-clamp technique, varied widely in sinoatrial node cells. The density of i(Ca,L) was significantly (p<0.001) correlated with cell capacitance (measure of cell size) and the density was greater in larger cells (likely to be from the periphery of the SA node) than in smaller cells (likely to be from the center of the SA node). Immunocytochemical labeling of the L-type Ca(2+) channel, Na(+)-Ca(2+) exchanger, sarcoplasmic reticulum Ca(2+) release channel (RYR2), and sarcoplasmic reticulum Ca(2+) pump (SERCA2) also varied widely in SA node cells. In all cases there was significantly (p<0.05) denser labeling of cells from the periphery of the SA node than of cells from the center. In contrast, immunocytochemical labeling of the Na(+)-K(+) pump was similar in peripheral and central cells. We conclude that Ca(2+) handling proteins are sparse and poorly organized in the center of the SA node (normally the leading pacemaker site), whereas they are more abundant in the periphery (at the border of the SA node with the surrounding atrial muscle).

Patient-Specific Induced Pluripotent Stem Cell as a Model for Familial Dilated Cardiomyopathy

PubMed Central

Sun, Ning; Yazawa, Masayuki; Liu, Jianwei; Han, Leng; Sanchez-Freire, Veronica; Abilez, Oscar J.; Navarrete, Enrique G.; Hu, Shijun; Wang, Li; Lee, Andrew; Pavlovic, Aleksandra; Lin, Shin; Chen, Rui; Hajjar, Roger J.; Snyder, Michael P.; Dolmetsch, Ricardo E.; Butte, Manish J.; Ashley, Euan A.; Longaker, Michael T.; Robbins, Robert C.; Wu, Joseph C.

2013-01-01

Dilated cardiomyopathy (DCM) is the most common cardiomyopathy, characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific models, investigating underlying mechanisms, and optimizing therapy. Here we generated cardiomyocytes (CMs) from iPSCs derived from patients of a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to the control healthy individuals in the same family cohort, DCM iPSC-CMs exhibited altered Ca2+ handling, decreased contractility, and abnormal sarcomeric α-actinin distribution. When stimulated with β-adrenergic agonist, DCM iPSC-CMs showed characteristics of failure such as reduced beating rates, compromised contraction, and significantly more cells with abnormal sarcomeric α-actinin distribution. β-adrenergic blocker treatment and over-expression of sarcoplasmic reticulum Ca2+ ATPase (Serca2a) improved DCM iPSC-CMs function. Our study demonstrated that human DCM iPSC-CMs recapitulated to some extent the disease phenotypes morphologically and functionally, and thus can serve as a useful platform for exploring molecular and cellular mechanisms and optimizing treatment of this particular disease. PMID:22517884

Microscopic heat pulse-induced calcium dynamics in single WI-38 fibroblasts

PubMed Central

Itoh, Hideki; Oyama, Kotaro; Suzuki, Madoka; Ishiwata, Shin’ichi

2014-01-01

Temperature-sensitive Ca2+ dynamics occur primarily through transient receptor potential channels, but also by means of Ca2+ channels and pumps on the endoplasmic reticulum membrane. As such, cytoplasmic Ca2+ concentration ([Ca2+]cyt) is re-equilibrated by changes in ambient temperature. The present study investigated the effects of heat pulses (heating duration: 2 s or 150 s) on [Ca2+]cyt in single WI-38 fibroblasts, which are considered as normal cells. We found that Ca2+ burst occurred immediately after short (2 s) heat pulse, which is similar to our previous report on HeLa cells, but with less thermosensitivity. The heat pulses originated from a focused 1455-nm infrared laser light were applied in the vicinity of cells under the optical microscope. Ca2+ bursts induced by the heat pulse were suppressed by treating cells with inhibitors for sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) or inositol trisphosphate receptor (IP3R). Long (150 s) heat pulses also induced Ca2+ bursts after the onset of heating and immediately after re-cooling. Cells were more thermosensitive at physiological (37°C) than at room (25°C) temperature; however, at 37°C, cells were responsive at a higher temperature (ambient temperature+heat pulse). These results strongly suggest that the heat pulse-induced Ca2+ burst is caused by a transient imbalance in Ca2+ flow between SERCA and IP3R, and offer a potential new method for thermally controlling Ca2+-regulated cellular functions. PMID:27493505

Activation of endoplasmic reticulum calcium leak by 2-APB depends on the luminal calcium concentration.

PubMed

Leon-Aparicio, Daniel; Chavez-Reyes, Jesus; Guerrero-Hernandez, Agustin

2017-07-01

It has been shown that 2-APB is a nonspecific modulator of ion channel activity, while most of the channels are inhibited by this compound, there are few examples of channels that are activated by 2-APB. Additionally, it has been shown that, 2-APB leads to a reduction in the luminal endoplasmic reticulum Ca 2+ level ([Ca 2+ ] ER ) and we have carried out simultaneous recordings of both [Ca 2+ ] i and the [Ca 2+ ] ER in HeLa cell suspensions to assess the mechanism involved in this effect. This approach allowed us to determine that 2-APB induces a reduction in the [Ca 2+ ] ER by activating an ER-resident Ca 2+ permeable channel more than by inhibiting the activity of SERCA pumps. Interestingly, this effect of 2-APB of reducing the [Ca 2+ ] ER is auto-limited because depends on a replete ER Ca 2+ store; a condition that thapsigargin does not require to decrease the [Ca 2+ ] ER . Additionally, our data indicate that the ER Ca 2+ permeable channel activated by 2-APB does not seem to participate in the ER Ca 2+ leak revealed by inhibiting SERCA pump with thapsigargin. This work suggests that, prolonged incubations with even low concentrations of 2-APB (5μM) would lead to the reduction in the [Ca 2+ ] ER that might explain the inhibitory effect of this compound on those signals that require Ca 2+ release from the ER store. Copyright © 2017 Elsevier Ltd. All rights reserved.

Muscle Non-shivering Thermogenesis and Its Role in the Evolution of Endothermy

PubMed Central

Nowack, Julia; Giroud, Sylvain; Arnold, Walter; Ruf, Thomas

2017-01-01

The development of sustained, long-term endothermy was one of the major transitions in the evolution of vertebrates. Thermogenesis in endotherms does not only occur via shivering or activity, but also via non-shivering thermogenesis (NST). Mammalian NST is mediated by the uncoupling protein 1 in the brown adipose tissue (BAT) and possibly involves an additional mechanism of NST in skeletal muscle. This alternative mechanism is based on Ca2+-slippage by a sarcoplasmatic reticulum Ca2+-ATPase (SERCA) and is controlled by the protein sarcolipin. The existence of muscle based NST has been discussed for a long time and is likely present in all mammals. However, its importance for thermoregulation was demonstrated only recently in mice. Interestingly, birds, which have evolved from a different reptilian lineage than mammals and lack UCP1-mediated NST, also exhibit muscle based NST under the involvement of SERCA, though likely without the participation of sarcolipin. In this review we summarize the current knowledge on muscle NST and discuss the efficiency of muscle NST and BAT in the context of the hypothesis that muscle NST could have been the earliest mechanism of heat generation during cold exposure in vertebrates that ultimately enabled the evolution of endothermy. We suggest that the evolution of BAT in addition to muscle NST was related to heterothermy being predominant among early endothermic mammals. Furthermore, we argue that, in contrast to small mammals, muscle NST is sufficient to maintain high body temperature in birds, which have enhanced capacities to fuel muscle NST by high rates of fatty acid import. PMID:29170642

The calcium–frequency response in the rat ventricular myocyte: an experimental and modelling study

PubMed Central

Gattoni, Sara; Røe, Åsmund Treu; Frisk, Michael; Louch, William E.; Niederer, Steven A.

2016-01-01

Key points In the majority of species, including humans, increased heart rate increases cardiac contractility. This change is known as the force–frequency response (FFR). The majority of mammals have a positive force–frequency relationship (FFR). In rat the FFR is controversial.We derive a species‐ and temperature‐specific data‐driven model of the rat ventricular myocyte.As a measure of the FFR, we test the effects of changes in frequency and extracellular calcium on the calcium–frequency response (CFR) in our model and three altered models.The results show a biphasic peak calcium–frequency response, due to biphasic behaviour of the ryanodine receptor and the combined effect of the rapid calmodulin buffer and the frequency‐dependent increase in diastolic calcium.Alterations to the model reveal that inclusion of Ca2+/calmodulin‐dependent protein kinase II (CAMKII)‐mediated L‐type channel and transient outward K+ current activity enhances the positive magnitude calcium–frequency response, and the absence of CAMKII‐mediated increase in activity of the sarco/endoplasmic reticulum Ca2+‐ATPase induces a negative magnitude calcium–frequency response. Abstract An increase in heart rate affects the strength of cardiac contraction by altering the Ca2+ transient as a response to physiological demands. This is described by the force–frequency response (FFR), a change in developed force with pacing frequency. The majority of mammals, including humans, have a positive FFR, and cardiac contraction strength increases with heart rate. However, the rat and mouse are exceptions, with the majority of studies reporting a negative FFR, while others report either a biphasic or a positive FFR. Understanding the differences in the FFR between humans and rats is fundamental to interpreting rat‐based experimental findings in the context of human physiology. We have developed a novel model of rat ventricular electrophysiology and calcium dynamics, derived predominantly from experimental data recorded under physiological conditions. As a measure of FFR, we tested the effects of changes in stimulation frequency and extracellular calcium concentration on the simulated Ca2+ transient characteristics and showed a biphasic peak calcium–frequency relationship, consistent with recent observations of a shift from negative to positive FFR when approaching the rat physiological frequency range. We tested the hypotheses that (1) inhibition of Ca2+/calmodulin‐dependent protein kinase II (CAMKII)‐mediated increase in sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) activity, (2) CAMKII modulation of SERCA, L‐type channel and transient outward K+ current activity and (3) Na+/K+ pump dynamics play a significant role in the rat FFR. The results reveal a major role for CAMKII modulation of SERCA in the peak Ca2+–frequency response, driven most significantly by the cytosolic calcium buffering system and changes in diastolic Ca2+. PMID:26916026

Increase in Cardiac Ischemia-Reperfusion Injuries in Opa1+/- Mouse Model

PubMed Central

Fauconnier, Jérémy; Cellier, Laura; Tamareille, Sophie; Gharib, Abdallah; Chevrollier, Arnaud; Loufrani, Laurent; Grenier, Céline; Kamel, Rima; Sarzi, Emmanuelle; Lacampagne, Alain; Ovize, Michel; Henrion, Daniel; Reynier, Pascal; Lenaers, Guy; Mirebeau-Prunier, Delphine

2016-01-01

Background Recent data suggests the involvement of mitochondrial dynamics in cardiac ischemia/reperfusion (I/R) injuries. Whilst excessive mitochondrial fission has been described as detrimental, the role of fusion proteins in this context remains uncertain. Objectives To investigate whether Opa1 (protein involved in mitochondrial inner-membrane fusion) deficiency affects I/R injuries. Methods and Results We examined mice exhibiting Opa1delTTAG mutations (Opa1+/-), showing 70% Opa1 protein expression in the myocardium as compared to their wild-type (WT) littermates. Cardiac left-ventricular systolic function assessed by means of echocardiography was observed to be similar in 3-month-old WT and Opa1+/- mice. After subjection to I/R, infarct size was significantly greater in Opa1+/- than in WTs both in vivo (43.2±4.1% vs. 28.4±3.5%, respectively; p<0.01) and ex vivo (71.1±3.2% vs. 59.6±8.5%, respectively; p<0.05). No difference was observed in the expression of other main fission/fusion protein, oxidative phosphorylation, apoptotic markers, or mitochondrial permeability transition pore (mPTP) function. Analysis of calcium transients in isolated ventricular cardiomyocytes demonstrated a lower sarcoplasmic reticulum Ca2+ uptake, whereas cytosolic Ca2+ removal from the Na+/Ca2+ exchanger (NCX) was increased, whilst SERCA2a, phospholamban, and NCX protein expression levels were unaffected in Opa1+/- compared to WT mice. Simultaneous whole-cell patch-clamp recordings of mitochondrial Ca2+ movements and ventricular action potential (AP) showed impairment of dynamic mitochondrial Ca2+ uptake and a marked increase in the AP late repolarization phase in conjunction with greater occurrence of arrhythmia in Opa1+/- mice. Conclusion Opa1 deficiency was associated with increased sensitivity to I/R, imbalance in dynamic mitochondrial Ca2+ uptake, and subsequent increase in NCX activity. PMID:27723783

Pharmacological inhibition of DNA methylation attenuates pressure overload-induced cardiac hypertrophy in rats.

PubMed

Stenzig, Justus; Schneeberger, Yvonne; Löser, Alexandra; Peters, Barbara S; Schaefer, Andreas; Zhao, Rong-Rong; Ng, Shi Ling; Höppner, Grit; Geertz, Birgit; Hirt, Marc N; Tan, Wilson; Wong, Eleanor; Reichenspurner, Hermann; Foo, Roger S-Y; Eschenhagen, Thomas

2018-07-01

Heart failure is associated with altered gene expression and DNA methylation. De novo DNA methylation is associated with gene silencing, but its role in cardiac pathology remains incompletely understood. We hypothesized that inhibition of DNA methyltransferases (DNMT) might prevent the deregulation of gene expression and the deterioration of cardiac function under pressure overload (PO). To test this hypothesis, we evaluated a DNMT inhibitor in PO in rats and analysed DNA methylation in cardiomyocytes. Young male Wistar rats were subjected to PO by transverse aortic constriction (TAC) or to sham surgery. Rats from both groups received solvent or 12.5 mg/kg body weight of the non-nucleosidic DNMT inhibitor RG108, initiated on the day of the intervention. After 4 weeks, we analysed cardiac function by MRI, fibrosis with Sirius Red staining, gene expression by RNA sequencing and qPCR, and DNA methylation by reduced representation bisulphite sequencing (RRBS). RG108 attenuated the ~70% increase in heart weight/body weight ratio of TAC over sham to 47% over sham, partially rescued reduced contractility, diminished the fibrotic response and the downregulation of a set of genes including Atp2a2 (SERCA2a) and Adrb1 (beta1-adrenoceptor). RG108 was associated with significantly lower global DNA methylation in cardiomyocytes by ~2%. The differentially methylated pathways were "cardiac hypertrophy", "cell death" and "xenobiotic metabolism signalling". Among these, "cardiac hypertrophy" was associated with significant methylation differences in the group comparison sham vs. TAC, but not significant between sham+RG108 and TAC+RG108 treatment, suggesting that RG108 partially prevented differential methylation. However, when comparing TAC and TAC+RG108, the pathway cardiac hypertrophy was not significantly differentially methylated. DNMT inhibitor treatment is associated with attenuation of cardiac hypertrophy and moderate changes in cardiomyocyte DNA methylation. The potential mechanistic link between these two effects and the role of non-myocytes need further clarification. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effect of 23-day muscle disuse on sarcoplasmic reticulum Ca2+ properties and contractility in human type I and type II skeletal muscle fibers.

PubMed

Lamboley, C R; Wyckelsma, V L; Perry, B D; McKenna, M J; Lamb, G D

2016-08-01

Inactivity negatively impacts on skeletal muscle function mainly through muscle atrophy. However, recent evidence suggests that the quality of individual muscle fibers is also altered. This study examined the effects of 23 days of unilateral lower limb suspension (ULLS) on specific force and sarcoplasmic reticulum (SR) Ca(2+) content in individual skinned muscle fibers. Muscle biopsies of the vastus lateralis were taken from six young healthy adults prior to and following ULLS. After disuse, the endogenous SR Ca(2+) content was ∼8% lower in type I fibers and maximal SR Ca(2+) capacity was lower in both type I and type II fibers (-11 and -5%, respectively). The specific force, measured in single skinned fibers from three subjects, decreased significantly after ULLS in type II fibers (-23%) but not in type I fibers (-9%). Western blot analyses showed no significant change in the amounts of myosin heavy chain (MHC) I and MHC IIa following the disuse, whereas the amounts of sarco(endo)plasmic reticulum Ca(2+)-ATPase 1 (SERCA1) and calsequestrin increased by ∼120 and ∼20%, respectively, and the amount of troponin I decreased by ∼21%. These findings suggest that the decline in force and power occurring with muscle disuse is likely to be exacerbated in part by reductions in maximum specific force in type II fibers, and in the amount of releasable SR Ca(2+) in both fiber types, the latter not being attributable to a reduced calsequestrin level. Furthermore, the ∼3-wk disuse in human elicits change in SR properties, in particular a more than twofold upregulation in SERCA1 density, before any fiber-type shift. Copyright © 2016 the American Physiological Society.

Antenatal/early postnatal hypothyroidism alters arterial tone regulation in 2-week-old rats.

PubMed

Sofronova, Svetlana I; Gaynullina, Dina K; Shvetsova, Anastasia A; Borzykh, Anna A; Selivanova, Ekaterina K; Kostyunina, Daria S; Sharova, Anna P; Martyanov, Andrey A; Tarasova, Olga S

2017-11-01

The mechanisms of vascular alterations resulting from early thyroid hormones deficiency are poorly understood. We tested the hypothesis that antenatal/early postnatal hypothyroidism would alter the activity of endothelial NO pathway and Rho-kinase pathway, which are specific for developing vasculature. Dams were treated with propylthiouracil (PTU, 7 ppm) in drinking water during gestation and 2 weeks after delivery, and their progeny had normal body weight but markedly reduced blood levels of thyroid hormones (ELISA). Small arteries from 2-week-old male pups were studied using wire myography, qPCR and Western blotting. Mesenteric arteries of PTU pups, compared to controls, demonstrated smaller maximum response to α 1 -adrenergic agonist methoxamine and reduced mRNA contents of smooth muscle differentiation markers α-actin and SERCA2A. Inhibition of basal NO synthesis by l-NNA led to tonic contraction of mesenteric arteries and augmented their contractile responses to methoxamine; both l-NNA effects were impaired in PTU pups. PTU pups demonstrated lower blood level of NO metabolites compared to control group (Griess reaction). Rho-kinase inhibitor Y27632 strongly reduced mesenteric arteries responses to methoxamine in PTU pups, that was accompanied by elevated Rho-kinase content in their arteries in comparison to control ones. Unlike mesenteric, saphenous arteries of PTU pups, compared to controls, had no changes in α-actin and SERCA2A contents and in responses to l-NNA and Y27632. In conclusion, thyroid hormones deficiency suppresses the anticontractile effect of NO and potentiates the procontractile Rho-kinase effects in mesenteric arteries of 2-week-old pups. Such alterations disturb perinatal cardiovascular homeostasis and might lead to cardiovascular pathologies in adulthood. © 2017 Society for Endocrinology.

Mitochondrial calcium handling within the interstitial cells of Cajal

PubMed Central

Cheng, Leo K.

2014-01-01

The interstitial cells of Cajal (ICC) drive rhythmic pacemaking contractions in the gastrointestinal system. The ICC generate pacemaking signals by membrane depolarizations associated with the release of intracellular calcium (Ca2+) in the endoplasmic reticulum (ER) through inositol-trisphosphate (IP3) receptors (IP3R) and uptake by mitochondria (MT). This Ca2+ dynamic is hypothesized to generate pacemaking signals by calibrating ER Ca2+ store depletions and membrane depolarization with ER store-operated Ca2+ entry mechanisms. Using a biophysically based spatio-temporal model of integrated Ca2+ transport in the ICC, we determined the feasibility of ER depletion timescale correspondence with experimentally observed pacemaking frequencies while considering the impact of IP3R Ca2+ release and MT uptake on bulk cytosolic Ca2+ levels because persistent elevations of free intracellular Ca2+ are toxic to the cell. MT densities and distributions are varied in the model geometry to observe MT influence on free cytosolic Ca2+ and the resulting frequencies of ER Ca2+ store depletions, as well as the sarco-endoplasmic reticulum Ca2+ ATP-ase (SERCA) and IP3 agonist concentrations. Our simulations show that high MT densities observed in the ICC are more relevant to ER establishing Ca2+ depletion frequencies than protection of the cytosol from elevated free Ca2+, whereas the SERCA pump is more relevant to containing cytosolic Ca2+ elevations. Our results further suggest that the level of IP3 agonist stimulating ER Ca2+ release, subsequent MT uptake, and eventual activation of ER store-operated Ca2+ entry may determine frequencies of rhythmic pacemaking exhibited by the ICC across species and tissue types. PMID:24789203

Abnormalities of Calcium Handling Proteins in Skeletal Muscle Mirror those of the Heart in Humans with Heart Failure: a Shared Mechanism?

PubMed Central

Middlekauff, Holly R.; Vigna, Chris; Verity, M. Anthony; Fonarow, Gregg C.; Horwich, Tamara B.; Hamilton, Michele A.; Shieh, Perry; Tupling, A. Russell

2012-01-01

Background In the failing human heart, abnormalities of Ca2+ cycling have been described, but there is scant knowledge about Ca2+ handling in the skeletal muscle of humans with HF. We tested the hypothesis that in humans with HF, Ca2+ cycling proteins in skeletal muscle are abnormal. Methods and Results Ten advanced HF patients (50.4±3.7 years), and 9 age matched controls underwent vastus lateralis biopsy. Western blot analysis showed that sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a, which is responsible for Ca2+ sequestration into the sarcoplasmic reticulum(SR), was lower in HF vs controls (4.8±0.5vs7.5±0.8AU, p=0.01). Although phospholamban (PLN), which inhibits SERCA2a, was not different in HF vs controls, phosphorylation (SER16 site) of PLN, which relieves this inhibition, was reduced (0.8±0.1vs3.9±0.9AU, p=0.004). Dihydropyridine receptors were reduced in HF, (2.1±0.4vs3.6±0.5AU, p=0.04). We tested the hypothesis that these abnormalities of Ca2+ handling protein content and regulation were due to increased oxidative stress, but oxygen radical scavenger proteins were not elevated in the skeletal muscle of HF patients. Conclusion In chronic HF, marked abnormalities of Ca2+ handling proteins are present in skeletal muscle, which mirror those in failing heart tissue. This suggests a common mechanism, such as chronic augmentation of sympathetic activity and autophosphorylation of Ca2+-calmodulin-dependent-protein kinase II. PMID:22939042

Myocardial Ca2+ handling and cell-to-cell coupling, key factors in prevention of sudden cardiac death.

PubMed

Tribulova, Narcis; Seki, Shingo; Radosinska, Jana; Kaplan, Peter; Babusikova, Eva; Knezl, Vladimir; Mochizuki, Seibu

2009-12-01

Using whole-heart preparations, we tested our hypothesis that Ca(2+) handling is closely related to cell-to-cell coupling at the gap junctions and that both are critical for the development and particularly the termination of ventricular fibrillation (VF) and hence the prevention of sudden arrhythmic death. Intracellular free calcium concentration ([Ca(2+)](i)), ECG, and left ventricular pressure were continuously monitored in isolated guinea pig hearts before and during development of low K(+)-induced sustained VF and during its conversion into sinus rhythm facilitated by stobadine. We also examined myocardial ultrastructure to detect cell-to-cell coupling alterations. We demonstrated that VF occurrence was preceded by a 55.9% +/- 6.2% increase in diastolic [Ca(2+)](i), which was associated with subcellular alterations indicating Ca(2+) overload of the cardiomyocytes and disorders in coupling among the cells. Moreover, VF itself further increased [Ca(2+)](i) by 58.2% +/- 3.4% and deteriorated subcellular and cell-to-cell coupling abnormalities that were heterogeneously distributed throughout the myocardium. In contrast, termination of VF and its conversion into sinus rhythm was marked by restoration of basal [Ca(2+)](i), resulting in recovery of intercellular coupling linked with synchronous contraction. Furthermore, we have shown that hearts exhibiting lower SERCA2a (sarcoplasmic reticulum Ca(2+)-ATPase) activity and abnormal intercellular coupling (as in older guinea pigs) are more prone to develop Ca(2+) overload associated with cell-to-cell uncoupling than hearts with higher SERCA2a activity (as in young guinea pigs). Consequently, young animals are better able to terminate VF spontaneously. These findings indicate the crucial role of Ca(2+) handling in relation to cell-to-cell coupling in both the occurrence and termination of malignant arrhythmia.

Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.

PubMed

Rincon, Melvin Y; VandenDriessche, Thierry; Chuah, Marinee K

2015-10-01

Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca(2+)-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Cardiology.

Long-term obesity promotes alterations in diastolic function induced by reduction of phospholamban phosphorylation at serine-16 without affecting calcium handling.

PubMed

Lima-Leopoldo, Ana Paula; Leopoldo, André S; da Silva, Danielle C T; do Nascimento, André F; de Campos, Dijon H S; Luvizotto, Renata A M; de Deus, Adriana F; Freire, Paula P; Medeiros, Alessandra; Okoshi, Katashi; Cicogna, Antonio C

2014-09-15

Few studies have evaluated the relationship between the duration of obesity, cardiac function, and the proteins involved in myocardial calcium (Ca(2+)) handling. We hypothesized that long-term obesity promotes cardiac dysfunction due to a reduction of expression and/or phosphorylation of myocardial Ca(2+)-handling proteins. Thirty-day-old male Wistar rats were distributed into two groups (n = 10 each): control (C; standard diet) and obese (Ob; high-fat diet) for 30 wk. Morphological and histological analyses were assessed. Left ventricular cardiac function was assessed in vivo by echocardiographic evaluation and in vitro by papillary muscle. Cardiac protein expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a), calsequestrin, L-type Ca(2+) channel, and phospholamban (PLB), as well as PLB serine-16 phosphorylation (pPLB Ser(16)) and PLB threonine-17 phosphorylation (pPLB Thr(17)) were determined by Western blot. The adiposity index was higher (82%) in Ob rats than in C rats. Obesity promoted cardiac hypertrophy without alterations in interstitial collagen levels. Ob rats had increased endocardial and midwall fractional shortening, posterior wall shortening velocity, and A-wave compared with C rats. Cardiac index, early-to-late diastolic mitral inflow ratio, and isovolumetric relaxation time were lower in Ob than in C. The Ob muscles developed similar baseline data and myocardial responsiveness to increased extracellular Ca(2+). Obesity caused a reduction in cardiac pPLB Ser(16) and the pPLB Ser(16)/PLB ratio in Ob rats. Long-term obesity promotes alterations in diastolic function, most likely due to the reduction of pPLB Ser(16), but does not impair the myocardial Ca(2+) entry and recapture to SR. Copyright © 2014 the American Physiological Society.

Active inhibitor-1 maintains protein hyper-phosphorylation in aging hearts and halts remodeling in failing hearts.

PubMed

Pritchard, Tracy J; Kawase, Yoshiaki; Haghighi, Kobra; Anjak, Ahmad; Cai, Wenfeng; Jiang, Min; Nicolaou, Persoulla; Pylar, George; Karakikes, Ioannis; Rapti, Kleopatra; Rubinstein, Jack; Hajjar, Roger J; Kranias, Evangelia G

2013-01-01

Impaired sarcoplasmic reticulum calcium cycling and depressed contractility are key characteristics in heart failure. Defects in sarcoplasmic reticulum function are characterized by decreased SERCA2a Ca-transport that is partially attributable to dephosphorylation of its regulator phospholamban by increased protein phosphatase 1 activity. Inhibition of protein phosphatase 1 through activation of its endogenous inhibitor-1 has been shown to enhance cardiac Ca-handling and contractility as well as protect from pathological stress remodeling in young mice. In this study, we assessed the long-term effects of inducible expression of constitutively active inhibitor-1 in the adult heart and followed function and remodeling through the aging process, up to 20 months. Mice with inhibitor-1 had normal survival and similar function to WTs. There was no overt remodeling as evidenced by measures of left ventricular end-systolic and diastolic diameters and posterior wall dimensions, heart weight to tibia length ratio, and histology. Higher phosphorylation of phospholamban at both Ser16 and Thr17 was maintained in aged hearts with active inhibitor-1, potentially offsetting the effects of elevated Ser2815-phosphorylation in ryanodine receptor, as there were no increases in arrhythmias under stress conditions in 20-month old mice. Furthermore, long-term expression of active inhibitor-1 via recombinant adeno-associated virus type 9 gene transfer in rats with pressure-overload induced heart failure improved function and prevented remodeling, associated with increased phosphorylation of phospholamban at Ser16 and Thr17. Thus, chronic inhibition of protein phosphatase 1, through increases in active inhibitor-1, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling in the long term.

Ca2+ handling remodeling and STIM1L/Orai1/TRPC1/TRPC4 upregulation in monocrotaline-induced right ventricular hypertrophy.

PubMed

Jessica, Sabourin; Angèle, Boet; Catherine, Rucker-Martin; Mélanie, Lambert; Ana-Maria, Gomez; Jean-Pierre, Benitah; Frédéric, Perros; Marc, Humbert; Fabrice, Antigny

2018-05-01

Right ventricular (RV) function is the most important prognostic factor for pulmonary arterial hypertension (PAH) patients. The progressive increase of pulmonary vascular resistance induces RV hypertrophy (RVH) and at term RV failure (RVF). However, the molecular mechanisms of RVH and RVF remain understudied. In this study, we gained insights into cytosolic Ca 2+ signaling remodeling in ventricular cardiomyocytes during the pathogenesis of severe pulmonary hypertension (PH) induced in rats by monocrotaline (MCT) exposure, and we further identified molecular candidates responsible for this Ca 2+ remodeling. After PH induction, hypertrophied RV myocytes presented longer action potential duration, higher and faster [Ca 2+ ] i transients and increased sarcoplasmic reticulum (SR) Ca 2+ content, whereas no changes in these parameters were detected in left ventricular (LV) myocytes. These modifications were associated with increased P-Ser 16 -phospholamban pentamer expression without altering SERCA2a (Sarco/Endoplasmic Reticulum Ca 2+ -ATPase) pump abundance. Moreover, after PH induction, Ca 2+ sparks frequency were higher in hypertrophied RV cells, while total RyR2 (Ryanodine Receptor) expression and phosphorylation were unaffected. Together with cellular hypertrophy, the T-tubules network was disorganized. Hypertrophied RV cardiomyocytes from MCT-exposed rats showed decreased expression of classical STIM1 (Stromal Interaction molecule) associated with increased expression of muscle-specific STIM1 Long isoform, glycosylated-Orai1 channel form, and TRPC1 and TRPC4 channels, which was correlated with an enhanced Ca 2+ -release-activated Ca 2+ (CRAC)-like current. Pharmacological inhibition of TRPCs/Orai1 channels in hypertrophied RV cardiomyocytes normalized [Ca 2+ ] i transients amplitude, the SR Ca 2+ content and cell contractility to control levels. Finally, we showed that most of these changes did not appear in LV cardiomyocytes. These new findings demonstrate RV-specific cellular Ca 2+ cycling remodeling in PH rats with maladaptive RVH and that the STIM1L/Orai1/TRPC1/C4-dependent Ca 2+ current participates in this Ca 2+ remodeling in RVH secondary to PH. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Ca2+ pump inhibitor, thapsigargin, inhibits root gravitropism in Arabidopsis thaliana.

PubMed

Urbina, Daniela C; Silva, Herman; Meisel, Lee A

2006-01-01

Thapsigargin, a specific inhibitor of most animal intracellular SERCA-type Ca2+ pumps present in the sarcoplasmic/endoplasmic reticulum, was originally isolated from the roots of the Mediterranean plant Thapsia gargancia L. Here, we demonstrate that this root-derived compound is capable of altering root gravitropism in Arabidopsis thaliana. Thapsigargin concentrations as low as 0.1 microM alter root gravitropism whereas under similar conditions cyclopiazonic acid does not. Furthermore, a fluorescently conjugated thapsigargin (BODIPY FL thapsigargin) suggests that target sites for thapsigargin are located in intracellular organelles in the root distal elongation zone and the root cap, regions known to regulate root gravitropism.

Vitamin D attenuates pressure overload-induced cardiac remodeling and dysfunction in mice.

PubMed

Zhang, Liang; Yan, Xiao; Zhang, Yun-Long; Bai, Jie; Hidru, Tesfaldet Habtemariam; Wang, Qing-Shan; Li, Hui-Hua

2018-04-01

Vitamin D (VD) and its analogues play critical roles in metabolic and cardiovascular diseases. Recent studies have demonstrated that VD exerts a protective role in cardiovascular diseases. However, the beneficial effect of VD on pressure overload-induced cardiac remodeling and dysfunction and its underlying mechanisms are not fully elucidated. In this study, cardiac dysfunction and hypertrophic remodeling in mice were induced by pressure overload. Cardiac function was evaluated by echocardiography, and myocardial histology was detected by H&E and Masson's trichrome staining. Cardiomyocyte size was detected by wheat germ agglutinin staining. The protein levels of signaling mediators were examined by western blotting while mRNA expression of hypertrophic and fibrotic markers was examined by qPCR analysis. Oxidative stress was detected by dihydroethidine staining. Our results showed that administration of VD3 significantly ameliorates pressure overload-induced contractile dysfunction, cardiac hypertrophy, fibrosis and inflammation in mice. In addition, VD3 treatment also markedly inhibited cardiac oxidative stress and apoptosis. Moreover, protein levels of calcineurin A, ERK1/2, AKT, TGF-β, GRP78, cATF6, and CHOP were significantly reduced whereas SERCA2 level was upregulated in the VD3-treated hearts compared with control. These results suggest that VD3 attenuates cardiac remodeling and dysfunction induced by pressure overload, and this protective effect is associated with inhibition of multiple signaling pathways. Copyright © 2018 Elsevier Ltd. All rights reserved.

Thermoregulatory and Cardiovascular Consequences of a Transient Thyrotoxicosis and Recovery in Male Mice.

PubMed

Hoefig, Carolin S; Harder, Lisbeth; Oelkrug, Rebecca; Meusel, Moritz; Vennström, Björn; Brabant, Georg; Mittag, Jens

2016-07-01

Thyroid hormones play a major role in body homeostasis, regulating energy expenditure and cardiovascular function. Given that obese people or athletes might consider rapid weight loss as beneficial, voluntary intoxication with T4 preparations is a growing cause for thyrotoxicosis. However, the long-lasting effects of transient thyrotoxicosis are poorly understood. Here we examined metabolic, thermoregulatory, and cardiovascular function upon induction and recovery from a 2-week thyrotoxicosis in male C57BL/6J mice. Our results showed that T4 treatment caused tachycardia, decreased hepatic glycogen stores, and higher body temperature as expected; however, we did not observe an increase in brown fat thermogenesis or decreased tail heat loss, suggesting that these tissues do not contribute to the hyperthermia induced by thyroid hormone. Most interestingly, when the T4 treatment was ended, a pronounced bradycardia was observed in the animals, which was likely caused by a rapid decline of T3 even below baseline levels. On the molecular level, this was accompanied by an overexpression of cardiac phospholamban and Serca2a mRNA, supporting the hypothesis that the heart depends more on T3 than T4. Our findings therefore demonstrate that a transient thyrotoxicosis can have pathological effects that even persist beyond the recovery of serum T4 levels, and in particular the observed bradycardia could be of clinical relevance when treating hyperthyroid patients.

The calcium-frequency response in the rat ventricular myocyte: an experimental and modelling study.

PubMed

Gattoni, Sara; Røe, Åsmund Treu; Frisk, Michael; Louch, William E; Niederer, Steven A; Smith, Nicolas P

2016-08-01

In the majority of species, including humans, increased heart rate increases cardiac contractility. This change is known as the force-frequency response (FFR). The majority of mammals have a positive force-frequency relationship (FFR). In rat the FFR is controversial. We derive a species- and temperature-specific data-driven model of the rat ventricular myocyte. As a measure of the FFR, we test the effects of changes in frequency and extracellular calcium on the calcium-frequency response (CFR) in our model and three altered models. The results show a biphasic peak calcium-frequency response, due to biphasic behaviour of the ryanodine receptor and the combined effect of the rapid calmodulin buffer and the frequency-dependent increase in diastolic calcium. Alterations to the model reveal that inclusion of Ca(2+) /calmodulin-dependent protein kinase II (CAMKII)-mediated L-type channel and transient outward K(+) current activity enhances the positive magnitude calcium-frequency response, and the absence of CAMKII-mediated increase in activity of the sarco/endoplasmic reticulum Ca(2+) -ATPase induces a negative magnitude calcium-frequency response. An increase in heart rate affects the strength of cardiac contraction by altering the Ca(2+) transient as a response to physiological demands. This is described by the force-frequency response (FFR), a change in developed force with pacing frequency. The majority of mammals, including humans, have a positive FFR, and cardiac contraction strength increases with heart rate. However, the rat and mouse are exceptions, with the majority of studies reporting a negative FFR, while others report either a biphasic or a positive FFR. Understanding the differences in the FFR between humans and rats is fundamental to interpreting rat-based experimental findings in the context of human physiology. We have developed a novel model of rat ventricular electrophysiology and calcium dynamics, derived predominantly from experimental data recorded under physiological conditions. As a measure of FFR, we tested the effects of changes in stimulation frequency and extracellular calcium concentration on the simulated Ca(2+) transient characteristics and showed a biphasic peak calcium-frequency relationship, consistent with recent observations of a shift from negative to positive FFR when approaching the rat physiological frequency range. We tested the hypotheses that (1) inhibition of Ca(2+) /calmodulin-dependent protein kinase II (CAMKII)-mediated increase in sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) activity, (2) CAMKII modulation of SERCA, L-type channel and transient outward K(+) current activity and (3) Na(+) /K(+) pump dynamics play a significant role in the rat FFR. The results reveal a major role for CAMKII modulation of SERCA in the peak Ca(2+) -frequency response, driven most significantly by the cytosolic calcium buffering system and changes in diastolic Ca(2+) . © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

The Endoplasmic Reticulum: A Central Player in Cell Signalling and Protein Synthesis

NASA Astrophysics Data System (ADS)

Llewelyn Roderick, H.; Berridge, Michael J.; Bootman, Martin D.

In addition to being the principle intracellular Ca 2+ store, the endoplasmic reticulum (ER) is the initial site of synthesis and folding of membrane and secretory proteins. These two roles of the ER are intimately linked. First, the function of many proteins involved in Ca 2+ handling are modulated by Ca 2+ and second, ER lumenal Ca 2+ modulates protein synthesis and folding. Within the ER, Ca 2+ is stored by low affinity high capacity Ca 2+ binding proteins and is maintained at a free concentration between 0.1 and 1 μM relative to 0.1 μM cytosolic Ca 2+. This concentration gradient is maintained by the action of the Sarco-Endoplasmic Reticulum ATPases (SERCa) which hydrolyse ATP to pump Ca 2+ into the ER. Following stimulation Ca 2+ is released from the ER through several classes of ligand gated channels. The most well characterized of these being the inositol 1,4,5-trisphosphate receptor ( IP 3 R) and the Ryanodine receptor (RyR) families of proteins. This release of Ca 2+ results in a drop of ER free Ca 2+ to levels as low as 10 μM. This decrease in lumenal Ca 2+ inhibits further release through the channels and increases the rate of re-sequestration of Ca 2+ into the ER by the SERCa pumps. Under these conditions, in addition to effects on Ca 2+ handling proteins, protein synthesis is inhibited, chaperones dissociate from their substrates, secondary modifications of proteins are inhibited and the retention of many proteins within the ER is lost. Furthermore, a signalling cascade resulting in the up-regulation of many proteins involved in protein folding and Ca 2+ homeostasis is initiated. This review will focus on the proteins involved in the regulation ER lumenal Ca 2+ and the role of ER lumenal Ca 2+ in cell signalling and protein synthesis.

Phasic Contractions of the Mouse Vagina and Cervix at Different Phases of the Estrus Cycle and during Late Pregnancy

PubMed Central

Gravina, Fernanda S.; van Helden, Dirk F.; Kerr, Karen P.; de Oliveira, Ramatis B.; Jobling, Phillip

2014-01-01

Background/Aims The pacemaker mechanisms activating phasic contractions of vaginal and cervical smooth muscle remain poorly understood. Here, we investigate properties of pacemaking in vaginal and cervical tissues by determining whether: 1) functional pacemaking is dependent on the phase of the estrus cycle or pregnancy; 2) pacemaking involves Ca2+ release from sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) -dependent intracellular Ca2+ stores; and 3) c-Kit and/or vimentin immunoreactive ICs have a role in pacemaking. Methodology/Principal Findings Vaginal and cervical contractions were measured in vitro, as was the distribution of c-Kit and vimentin positive interstitial cells (ICs). Cervical smooth muscle was spontaneously active in estrus and metestrus but quiescent during proestrus and diestrus. Vaginal smooth muscle was normally quiescent but exhibited phasic contractions in the presence of oxytocin or the K+ channel blocker tetraethylammonium (TEA) chloride. Spontaneous contractions in the cervix and TEA-induced phasic contractions in the vagina persisted in the presence of cyclopiazonic acid (CPA), a blocker of the SERCA that refills intracellular SR Ca2+ stores, but were inhibited in low Ca2+ solution or in the presence of nifedipine, an inhibitor of L-type Ca2+channels. ICs were found in small numbers in the mouse cervix but not in the vagina. Conclusions/Significance Cervical smooth muscle strips taken from mice in estrus, metestrus or late pregnancy were generally spontaneously active. Vaginal smooth muscle strips were normally quiescent but could be induced to exhibit phasic contractions independent on phase of the estrus cycle or late pregnancy. Spontaneous cervical or TEA-induced vaginal phasic contractions were not mediated by ICs or intracellular Ca2+ stores. Given that vaginal smooth muscle is normally quiescent then it is likely that increases in hormones such as oxytocin, as might occur through sexual stimulation, enhance the effectiveness of such pacemaking until phasic contractile activity emerges. PMID:25337931

Phasic contractions of the mouse vagina and cervix at different phases of the estrus cycle and during late pregnancy.

PubMed

Gravina, Fernanda S; van Helden, Dirk F; Kerr, Karen P; de Oliveira, Ramatis B; Jobling, Phillip

2014-01-01

The pacemaker mechanisms activating phasic contractions of vaginal and cervical smooth muscle remain poorly understood. Here, we investigate properties of pacemaking in vaginal and cervical tissues by determining whether: 1) functional pacemaking is dependent on the phase of the estrus cycle or pregnancy; 2) pacemaking involves Ca2+ release from sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) -dependent intracellular Ca2+ stores; and 3) c-Kit and/or vimentin immunoreactive ICs have a role in pacemaking. Vaginal and cervical contractions were measured in vitro, as was the distribution of c-Kit and vimentin positive interstitial cells (ICs). Cervical smooth muscle was spontaneously active in estrus and metestrus but quiescent during proestrus and diestrus. Vaginal smooth muscle was normally quiescent but exhibited phasic contractions in the presence of oxytocin or the K+ channel blocker tetraethylammonium (TEA) chloride. Spontaneous contractions in the cervix and TEA-induced phasic contractions in the vagina persisted in the presence of cyclopiazonic acid (CPA), a blocker of the SERCA that refills intracellular SR Ca2+ stores, but were inhibited in low Ca2+ solution or in the presence of nifedipine, an inhibitor of L-type Ca2+channels. ICs were found in small numbers in the mouse cervix but not in the vagina. Cervical smooth muscle strips taken from mice in estrus, metestrus or late pregnancy were generally spontaneously active. Vaginal smooth muscle strips were normally quiescent but could be induced to exhibit phasic contractions independent on phase of the estrus cycle or late pregnancy. Spontaneous cervical or TEA-induced vaginal phasic contractions were not mediated by ICs or intracellular Ca2+ stores. Given that vaginal smooth muscle is normally quiescent then it is likely that increases in hormones such as oxytocin, as might occur through sexual stimulation, enhance the effectiveness of such pacemaking until phasic contractile activity emerges.

Do microplastic particles affect Daphnia magna at the morphological, life history and molecular level?

PubMed Central

Rusek, Jakub; Thiel, Michaela; Wolinska, Justyna; Laforsch, Christian

2017-01-01

Microplastic particles are ubiquitous not only in marine but also in freshwater ecosystems. However, the impacts of microplastics, consisting of a large variety of synthetic polymers, on freshwater organisms remains poorly understood. We examined the effects of two polymer mixtures on the morphology, life history and on the molecular level of the waterflea Daphnia magna (three different clones). Microplastic particles of ~40 μm were supplied at a low concentration (1% of the food particles) leading to an average of ~30 particles in the digestive tract which reflects a high microplastic contamination but still resembles a natural situation. Neither increased mortality nor changes on the morphological (body length, width and tail spine length) or reproductive parameters were observed for adult Daphnia. The analyses of juvenile Daphnia revealed a variety of small and rather subtle responses of morphological traits (body length, width and tail spine length). For adult Daphnia, alterations in expression of genes related to stress responses (i.e. HSP60, HSP70 & GST) as well as of other genes involved in body function and body composition (i.e. SERCA) were observed already 48h after exposure. We anticipate that the adverse effects of microplastic might be influenced by many additional factors like size, shape, type and even age of the particles and that the rather weak effects, as detected in a laboratory, may lead to reduced fitness in a natural multi-stressor environment. PMID:29145427

Ion channel signaling influences cellular proliferation and phagocyte activity during axolotl tail regeneration.

PubMed

Franklin, Brandon M; Voss, S Randal; Osborn, Jeffrey L

2017-08-01

Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, K V 2.1, K V 2.2, L-type Ca V channels and H/K ATPases) or completely (GlyR, GABA A R, K V 1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K + channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H + pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair. Copyright © 2017 Elsevier B.V. All rights reserved.

High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction and upregulation of Foxo3a transcription factor independent of lipotoxicity and apoptosis.

PubMed

Relling, David P; Esberg, Lucy B; Fang, Cindy X; Johnson, W Thomas; Murphy, Eric J; Carlson, Edward C; Saari, Jack T; Ren, Jun

2006-03-01

Obesity is associated with dyslipidemia, which leads to elevated triglyceride and ceramide levels, apoptosis and compromised cardiac function. To determine the role of high-fat diet-induced obesity on cardiomyocyte function, weanling male Sprague-Dawley rats were fed diets incorporating 10% of kcal or 45% of kcal from fat. Mechanical function of ventricular myocytes was evaluated including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening and relengthening (+/- dl/dt). Intracellular Ca properties were assessed using fluorescent microscopy. High-fat diet induced hyperinsulinemic insulin-resistant obesity with depressed PS, +/- dl/dt, prolonged TPS/TR90 reduced intracellular Ca release and Ca clearing rate in the absence of hypertension, diabetes, lipotoxicity and apoptosis. Myocyte responsiveness to increased stimulus frequency and extracellular Ca was compromised. SERCA2a and phospholamban levels were increased, whereas phosphorylated phospholamban and potassium channel (Kv1,2) were reduced in high-fat diet group. High-fat diet upregulated the forkhead transcription factor Foxo3a, and suppressed mitochondrial aconitase activity without affecting expression of the caloric sensitive gene silent information regulator 2 (Sir2), protein nitrotyrosine formation, lipid peroxidation and apoptosis. Levels of endothelial nitric oxide synthase (NOS), inducible NOS, triglycerides and ceramide were similar between the two groups. Collectively, our data show that high-fat diet-induced obesity resulted in impaired cardiomyocyte function, upregulated Foxo3a transcription factor and mitochondrial damage without overt lipotoxicity or apoptosis.

Do microplastic particles affect Daphnia magna at the morphological, life history and molecular level?

PubMed

Imhof, Hannes K; Rusek, Jakub; Thiel, Michaela; Wolinska, Justyna; Laforsch, Christian

2017-01-01

Microplastic particles are ubiquitous not only in marine but also in freshwater ecosystems. However, the impacts of microplastics, consisting of a large variety of synthetic polymers, on freshwater organisms remains poorly understood. We examined the effects of two polymer mixtures on the morphology, life history and on the molecular level of the waterflea Daphnia magna (three different clones). Microplastic particles of ~40 μm were supplied at a low concentration (1% of the food particles) leading to an average of ~30 particles in the digestive tract which reflects a high microplastic contamination but still resembles a natural situation. Neither increased mortality nor changes on the morphological (body length, width and tail spine length) or reproductive parameters were observed for adult Daphnia. The analyses of juvenile Daphnia revealed a variety of small and rather subtle responses of morphological traits (body length, width and tail spine length). For adult Daphnia, alterations in expression of genes related to stress responses (i.e. HSP60, HSP70 & GST) as well as of other genes involved in body function and body composition (i.e. SERCA) were observed already 48h after exposure. We anticipate that the adverse effects of microplastic might be influenced by many additional factors like size, shape, type and even age of the particles and that the rather weak effects, as detected in a laboratory, may lead to reduced fitness in a natural multi-stressor environment.

Calcium Signaling enhancement during oocyte maturation

NASA Astrophysics Data System (ADS)

Jung, Peter; Ullah, Ghanim; Machaca, Khaled

2006-03-01

A Ca2+ signal with a special spatial and temporal characteristic universally removes cell-cycle arrest after fertilization of a mature egg cell. The Ca2+ signal is characterized by a fast rise of intracellular Ca2+ and a slow decay on the time scale of minutes. We use computational modeling of Ca2+ release on the microscale (Ca2+ puffs) and cell-scale in conjunction with experimental knowledge of the changes in the Ca2+ signaling apparatus during oocyte maturation and changing signaling patterns to explore the relationship between organization and sensitivity of IP3 receptors and SERCA pumps and the resulting signaling patterns. We hypothesize that potentiation of the IP3 receptors during oocyte maturation is the main cause for the differentiation in the signaling patterns.

Finite-element estimation of protein-ligand association rates with post-encounter effects: applications to calcium binding in troponin C and SERCA

NASA Astrophysics Data System (ADS)

Kekenes-Huskey, P. M.; Gillette, A.; Hake, J.; McCammon, J. A.

2012-01-01

We introduce a computational pipeline and suite of software tools for the approximation of diffusion-limited binding based on a recently developed theoretical framework. Our approach handles molecular geometries generated from high-resolution structural data and can account for active sites buried within the protein or behind gating mechanisms. Using tools from the FEniCS library and the APBS solver, we implement a numerical code for our method and study two Ca2+-binding proteins: troponin C and the sarcoplasmic reticulum Ca2+ ATPase. We find that a combination of diffusional encounter and internal ‘buried channel’ descriptions provides superior descriptions of association rates, improving estimates by orders of magnitude.

Increased passive stiffness promotes diastolic dysfunction despite improved Ca2+ handling during left ventricular concentric hypertrophy

PubMed Central

Røe, Åsmund T.; Aronsen, Jan Magnus; Skårdal, Kristine; Hamdani, Nazha; Linke, Wolfgang A.; Danielsen, Håvard E.; Sejersted, Ole M.; Sjaastad, Ivar; Louch, William E.

2017-01-01

Abstract Aims Concentric hypertrophy following pressure-overload is linked to preserved systolic function but impaired diastolic function, and is an important substrate for heart failure with preserved ejection fraction. While increased passive stiffness of the myocardium is a suggested mechanism underlying diastolic dysfunction in these hearts, the contribution of active diastolic Ca2+ cycling in cardiomyocytes remains unclear. In this study, we sought to dissect contributions of passive and active mechanisms to diastolic dysfunction in the concentrically hypertrophied heart following pressure-overload. Methods and results Rats were subjected to aortic banding (AB), and experiments were performed 6 weeks after surgery using sham-operated rats as controls. In vivo ejection fraction and fractional shortening were normal, confirming preservation of systolic function. Left ventricular concentric hypertrophy and diastolic dysfunction following AB were indicated by thickening of the ventricular wall, reduced peak early diastolic tissue velocity, and higher E/e’ values. Slowed relaxation was also observed in left ventricular muscle strips isolated from AB hearts, during both isometric and isotonic stimulation, and accompanied by increases in passive tension, viscosity, and extracellular collagen. An altered titin phosphorylation profile was observed with hypophosphorylation of the phosphosites S4080 and S3991 sites within the N2Bus, and S12884 within the PEVK region. Increased titin-based stiffness was confirmed by salt-extraction experiments. In contrast, isolated, unloaded cardiomyocytes exhibited accelerated relaxation in AB compared to sham, and less contracture at high pacing frequencies. Parallel enhancement of diastolic Ca2+ handling was observed, with augmented NCX and SERCA2 activity and lowered resting cytosolic [Ca2+]. Conclusion In the hypertrophied heart with preserved systolic function, in vivo diastolic dysfunction develops as cardiac fibrosis and alterations in titin phosphorylation compromise left ventricular compliance, and despite compensatory changes in cardiomyocyte Ca2+ homeostasis. PMID:28472418

A Separate Pool of Cardiac Phospholemman That Does Not Regulate or Associate with the Sodium Pump

PubMed Central

Wypijewski, Krzysztof J.; Howie, Jacqueline; Reilly, Louise; Tulloch, Lindsay B.; Aughton, Karen L.; McLatchie, Linda M.; Shattock, Michael J.; Calaghan, Sarah C.; Fuller, William

2013-01-01

Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser63 and one associated with the pump, both phosphorylated at Ser68 and unphosphorylated. Phosphorylation of PLM at Ser63 following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser63-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser63-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump. PMID:23532852

Nitrosative stress in human skeletal muscle attenuated by exercise countermeasure after chronic disuse.

PubMed

Salanova, Michele; Schiffl, Gudrun; Gutsmann, Martina; Felsenberg, Dieter; Furlan, Sandra; Volpe, Pompeo; Clarke, Andrew; Blottner, Dieter

2013-01-01

Activity-induced nitric oxide (NO) imbalance and "nitrosative stress" are proposed mechanisms of disrupted Ca(2+) homeostasis in atrophic skeletal muscle. We thus mapped S-nitrosylated (SNO) functional muscle proteins in healthy male subjects in a long-term bed rest study (BBR2-2 Study) without and with exercise as countermeasure in order to assess (i) the negative effects of chronic muscle disuse by nitrosative stress, (ii) to test for possible attenuation by exercise countermeasure in bed rest and (iii) to identify new NO target proteins. Muscle biopsies from calf soleus and hip vastus lateralis were harvested at start (Pre) and at end (End) from a bed rest disuse control group (CTR, n=9) and two bed rest resistive exercise groups either without (RE, n=7) or with superimposed vibration stimuli (RVE, n=7). At subcellular compartments, strong anti-SNO-Cys immunofluorescence patterns in control muscle fibers after bed rest returned to baseline following vibration exercise. Total SNO-protein levels, Nrf-2 gene expression and nucleocytoplasmic shuttling were changed to varying degrees in all groups. Excess SNO-protein levels of specific calcium release/uptake proteins (SNO-RyR1, -SERCA1 and -PMCA) and of contractile myosin heavy chains seen in biopsy samples of chronically disused skeletal muscle were largely reduced by vibration exercise. We also identified NOS1 as a novel NO target in human skeletal muscle controlled by activity driven auto-nitrosylation mechanisms. Our findings suggest that aberrant levels of functional SNO-proteins represent signatures of uncontrolled nitrosative stress management in disused human skeletal muscle that can be offset by exercise as countermeasure.

The benefits of endurance training in cardiomyocyte function in hypertensive rats are reversed within four weeks of detraining.

PubMed

Carneiro-Júnior, Miguel Araujo; Quintão-Júnior, Judson Fonseca; Drummond, Lucas Rios; Lavorato, Victor Neiva; Drummond, Filipe Rios; da Cunha, Daise Nunes Queiroz; Amadeu, Marco Aurélio; Felix, Leonardo Bonato; de Oliveira, Edilamar Menezes; Cruz, Jader Santos; Prímola-Gomes, Thales Nicolau; Mill, José Geraldo; Natali, Antonio José

2013-04-01

The aim of the present study was to verify the effects of low-intensity endurance training and detraining on the mechanical and molecular properties of cardiomyocytes from spontaneously hypertensive rats (SHRs). Male SHRs and normotensive control Wistar rats at 16-weeks of age were randomly divided into eight groups of eight animals: NC8 and HC8 (normotensive and hypertensive control for 8weeks); NT8 and HT8 (normotensive and hypertensive trained at 50-60% of maximal exercise capacity for 8weeks); NC12 and HC12 (normotensive and hypertensive control for 12weeks); NDT and HDT (normotensive and hypertensive trained for 8weeks and detrained for 4weeks). The total exercise time until fatigue (TTF) was determined by a maximal exercise capacity test. Resting heart rate (RHR) and systolic arterial pressure (SAP) were measured. After the treatments, animals were killed by cervical dislocation and left ventricular myocytes were isolated by enzymatic dispersion. Isolated cells were used to determine intracellular global Ca(2+) ([Ca(2+)]i) transient and cardiomyocyte contractility (1Hz; ~25°C). [Ca(2+)]i regulatory proteins were measured by Western blot, and the markers of pathologic cardiac hypertrophy by quantitative real-time polymerase chain reaction (q-RT-PCR). Exercise training augmented the TTF (NC8, 11.4±1.5min vs. NT8, 22.5±1.4min; HC8, 11.7±1.4min vs. HT8, 24.5±1.3min; P<0.05), reduced RHR (NT8initial, 340±8bpm vs. NT8final, 322±10bpm; HT8initial, 369±8bpm vs. HT8final, 344±10bpm; P<0.05), and SBP in SHR animals (HC8, 178±3mmHg vs. HT8, 161±4mmHg; P<0.05). HC8 rats showed a slower [Ca(2+)]i transient (Tpeak, 83.7±1.8ms vs. 71.7±2.4ms; T50%decay, 284.0±4.3ms vs. 264.0±4.1ms; P<0.05) and cell contractility (Vshortening, 86.1±6.7μm/s vs. 118.6±6.7μm/s; Vrelengthening, 57.5±7.4μm/s vs. 101.3±7.4μm/s; P<0.05), and higher expression of ANF (300%; P<0.05), skeletal α-actin (250%; P<0.05) and a decreased α/β-MHC ratio (70%; P<0.05) compared to NC8. Exercise training increased [Ca(2+)]i transient (NC8, 2.39±0.06F/F0 vs. NT8, 2.72±0.06F/F0; HC8, 2.28±0.05F/F0 vs. HT8, 2.82±0.05F/F0; P<0.05), and cell contractility (NC8, 7.4±0.3% vs. NT8, 8.4±0.3%; HC8, 6.8±0.3% vs. HT8, 7.8±0.3%; P<0.05). Furthermore, exercise normalized the expression of ANF, skeletal α-actin, and the α/β-MHC ratio in HT8 rats, augmented the expression of SERCA2a (NC8, 0.93±0.15 vs. NT8, 1.49±0.14; HC8, 0.83±0.13 vs. HT8, 1.32±0.14; P<0.05) and PLBser16 (NC8, 0.89±0.18 vs. NT8, 1.23±0.17; HC8, 0.77±0.17 vs. HT8, 1.32±0.16; P<0.05), and reduced PLBt/SERCA2a (NC8, 1.21±0.19 vs. NT8, 0.50±0.21; HC8, 1.38±0.17 vs. HT8, 0.66±0.21; P<0.05). However, all these adaptations returned to control values within 4weeks of detraining in both SHR and normotensive control animals. In conclusion, low-intensity endurance training induces positive benefits to left ventricular myocyte mechanical and molecular properties, which are reversed within 4weeks of detraining. Copyright © 2013 Elsevier Ltd. All rights reserved.

David Triggle: Research collaborations and scientific exchanges with the China Pharmaceutical University, Nanjing, China.

PubMed

Dai, De-Zai

2015-11-15

Over the period 1995-2012, David Triggle was a frequent visitor to the China Pharmaceutical University in Nanjing, China making many important contributions that enhanced the activities of the Research Division of Pharmacology at the University. In addition to providing collegial advice and facilitating interactions with the international pharmacological community, Professor Triggle's international reputation as a thought leader in the field of ion channel research and drug discovery provided important insights into the potential pathophysiological and therapeutic effects of targeting ion channels. This included the L-type calcium channel and the outward delayed rectified potassium currents of rapid (IKr) and slow (IKs) components in the myocardium. The Nanjing research team had been particularly interested in ion channel dysfunction in the context of cardiac arrhythmias, remodeling and drug discovery. With Professor Triggle's assistance, the relationship between an increase in ICa.L and other biological events including an enhancement of IKr and IKr currents, NADPH oxidase and endothelin receptor activation, down regulation of calcium modulating protein FKBP12.6, sarco/endoplasmic reticulum Ca(2+)ATPse (SERCA2A) and calsequens 2 (CASQ2), calcium leak at the diastole and endoplasmic reticulum stress, were evaluated and are discussed. Additionally, the organization of several international symposia was greatly enhanced by input from Professor Triggle as were the published research manuscripts in international pharmacology journals. During his association with the China Pharmaceutical University, Professor Triggle aided in enhancing the scientific standing of the Pharmacology department and was a highly effective ambassador for international research cooperation. Copyright © 2015. Published by Elsevier Inc.

Fatiguing contractions increase protein S-glutathionylation occupancy in mouse skeletal muscle.

PubMed

Kramer, Philip A; Duan, Jicheng; Gaffrey, Matthew J; Shukla, Anil K; Wang, Lu; Bammler, Theo K; Qian, Wei-Jun; Marcinek, David J

2018-05-23

Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 min after the last stimulation and processed for redox proteomics assay of S-glutathionylation. Using selective reduction with a glutaredoxin enzyme cocktail and resin-assisted enrichment technique, we quantified the levels of site-specific protein S-glutathionylation at rest and following fatiguing contractions. Redox proteomics revealed over 2200 sites of S-glutathionylation modifications, of which 1290 were significantly increased after fatiguing contractions. Muscle contraction leads to the greatest increase in S-glutathionylation in the mitochondria (1.03%) and the smallest increase in the nucleus (0.47%). Regulatory cysteines were significantly S-glutathionylated on mitochondrial complex I and II, GAPDH, MDH1, ACO2, and mitochondrial complex V among others. Similarly, S-glutathionylation of RYR1, SERCA1, titin, and troponin I2 are known to regulate muscle contractility and were significantly S-glutathionylated after just 15 min of fatiguing contractions. The largest fold changes (> 1.6) in the S-glutathionylated proteome after fatigue occurred on signaling proteins such as 14-3-3 protein gamma and MAP2K4, as well as proteins like SERCA1, and NDUV2 of mitochondrial complex I, at previously unknown glutathionylation sites. These findings highlight the important role of redox control over muscle physiology, metabolism, and the exercise adaptive response. This study lays the groundwork for future investigation into the altered exercise adaptation associated with chronic conditions, such as sarcopenia. Copyright © 2018. Published by Elsevier B.V.

Silica nanoparticles induce cardiotoxicity interfering with energetic status and Ca2+ handling in adult rat cardiomyocytes

PubMed Central

Bernal-Ramírez, Judith; Lozano, Omar; Oropeza-Almazán, Yuriana; Castillo, Elena Cristina; Garza, Jesús Roberto; García, Noemí; Vela, Jorge; Ortega, Eduardo; Torre-Amione, Guillermo; Ornelas-Soto, Nancy

2017-01-01

Recent evidence has shown that nanoparticles that have been used to improve or create new functional properties for common products may pose potential risks to human health. Silicon dioxide (SiO2) has emerged as a promising therapy vector for the heart. However, its potential toxicity and mechanisms of damage remain poorly understood. This study provides the first exploration of SiO2-induced toxicity in cultured cardiomyocytes exposed to 7- or 670-nm SiO2 particles. We evaluated the mechanism of cell death in isolated adult cardiomyocytes exposed to 24-h incubation. The SiO2 cell membrane association and internalization were analyzed. SiO2 showed a dose-dependent cytotoxic effect with a half-maximal inhibitory concentration for the 7 nm (99.5 ± 12.4 µg/ml) and 670 nm (>1,500 µg/ml) particles, which indicates size-dependent toxicity. We evaluated cardiomyocyte shortening and intracellular Ca2+ handling, which showed impaired contractility and intracellular Ca2+ transient amplitude during β-adrenergic stimulation in SiO2 treatment. The time to 50% Ca2+ decay increased 39%, and the Ca2+ spark frequency and amplitude decreased by 35 and 21%, respectively, which suggest a reduction in sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity. Moreover, SiO2 treatment depolarized the mitochondrial membrane potential and decreased ATP production by 55%. Notable glutathione depletion and H2O2 generation were also observed. These data indicate that SiO2 increases oxidative stress, which leads to mitochondrial dysfunction and low energy status; these underlie reduced SERCA activity, shortened Ca2+ release, and reduced cell shortening. This mechanism of SiO2 cardiotoxicity potentially plays an important role in the pathophysiology mechanism of heart failure, arrhythmias, and sudden death. NEW & NOTEWORTHY Silica particles are used as novel nanotechnology-based vehicles for diagnostics and therapeutics for the heart. However, their potential hazardous effects remain unknown. Here, the cardiotoxicity of silica nanoparticles in rat myocytes has been described for the first time, showing an impairment of mitochondrial function that interfered directly with Ca2+ handling. PMID:28130337

Surfactant bilayers maintain transmembrane protein activity.

PubMed

Rayan, Gamal; Adrien, Vladimir; Reffay, Myriam; Picard, Martin; Ducruix, Arnaud; Schmutz, Marc; Urbach, Wladimir; Taulier, Nicolas

2014-09-02

In vitro studies of membrane proteins are of interest only if their structure and function are significantly preserved. One approach is to insert them into the lipid bilayers of highly viscous cubic phases rendering the insertion and manipulation of proteins difficult. Less viscous lipid sponge phases are sometimes used, but their relatively narrow domain of existence can be easily disrupted by protein insertion. We present here a sponge phase consisting of nonionic surfactant bilayers. Its extended domain of existence and its low viscosity allow easy insertion and manipulation of membrane proteins. We show for the first time, to our knowledge, that transmembrane proteins, such as bacteriorhodopsin, sarcoplasmic reticulum Ca(2+)ATPase (SERCA1a), and its associated enzymes, are fully active in a surfactant phase. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Chronic N(G)-nitro-L-arginine methyl ester-induced hypertension : novel molecular adaptation to systolic load in absence of hypertrophy

NASA Technical Reports Server (NTRS)

Bartunek, J.; Weinberg, E. O.; Tajima, M.; Rohrbach, S.; Katz, S. E.; Douglas, P. S.; Lorell, B. H.; Schneider, M. (Principal Investigator)

2000-01-01

BACKGROUND: Chronic N(G)-nitro-L-arginine methyl ester (L-NAME), which inhibits nitric oxide synthesis, causes hypertension and would therefore be expected to induce robust cardiac hypertrophy. However, L-NAME has negative metabolic effects on protein synthesis that suppress the increase in left ventricular (LV) mass in response to sustained pressure overload. In the present study, we used L-NAME-induced hypertension to test the hypothesis that adaptation to pressure overload occurs even when hypertrophy is suppressed. METHODS AND RESULTS: Male rats received L-NAME (50 mg. kg(-1). d(-1)) or no drug for 6 weeks. Rats with L-NAME-induced hypertension had levels of systolic wall stress similar to those of rats with aortic stenosis (85+/-19 versus 92+/-16 kdyne/cm). Rats with aortic stenosis developed a nearly 2-fold increase in LV mass compared with controls. In contrast, in the L-NAME rats, no increase in LV mass (1. 00+/-0.03 versus 1.04+/-0.04 g) or hypertrophy of isolated myocytes occurred (3586+/-129 versus 3756+/-135 microm(2)) compared with controls. Nevertheless, chronic pressure overload was not accompanied by the development of heart failure. LV systolic performance was maintained by mechanisms of concentric remodeling (decrease of in vivo LV chamber dimension relative to wall thickness) and augmented myocardial calcium-dependent contractile reserve associated with preserved expression of alpha- and beta-myosin heavy chain isoforms and sarcoplasmic reticulum Ca(2+) ATPase (SERCA-2). CONCLUSIONS: When the expected compensatory hypertrophic response is suppressed during L-NAME-induced hypertension, severe chronic pressure overload is associated with a successful adaptation to maintain systolic performance; this adaptation depends on both LV remodeling and enhanced contractility in response to calcium.

Ca2+ regulatory mechanisms of exercise protection against coronary artery disease in metabolic syndrome and diabetes.

PubMed

Sturek, Michael

2011-08-01

Chronic exercise attenuates coronary artery disease (CAD) in humans largely independent of reductions in risk factors; thus major protective mechanisms of exercise are directly within the coronary vasculature. Further, tight control of diabetes, e.g., blood glucose, can be detrimental. Accordingly, knowledge of mechanisms by which exercise attenuates diabetic CAD could catalyze development of molecular therapies. Exercise attenuates CAD (atherosclerosis) and restenosis in miniature swine models, which enable precise control of exercise parameters (intensity, duration, and frequency) and characterization of the metabolic syndrome (MetS) and diabetic milieu. Intracellular Ca(2+) is a pivotal second messenger for coronary smooth muscle (CSM) excitation-contraction and excitation-transcription coupling that modulates CSM proliferation, migration, and calcification. CSM of diabetic dyslipidemic Yucatan swine have impaired Ca(2+) extrusion via the plasmalemma Ca(2+) ATPase (PMCA), downregulation of L-type voltage-gated Ca(2+) channels (VGCC), increased Ca(2+) sequestration by the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA), increased nuclear Ca(2+) localization, and greater activation of K channels by Ca(2+) release from the SR. Endurance exercise training prevents Ca(2+) transport changes with virtually no effect on the diabetic milieu (glucose, lipids). In MetS Ossabaw swine transient receptor potential canonical (TRPC) channels are upregulated and exercise training reverses expression and TRPC-mediated Ca(2+) influx with almost no change in the MetS milieu. Overall, exercise effects on Ca(2+) signaling modulate CSM phenotype. Future studies should 1) selectively target key Ca(2+) transporters to determine definitively their causal role in atherosclerosis and 2) combine mechanistic studies with clinical outcomes, e.g., reduction of myocardial infarction.

Females Are Protected From Iron-Overload Cardiomyopathy Independent of Iron Metabolism: Key Role of Oxidative Stress.

PubMed

Das, Subhash K; Patel, Vaibhav B; Basu, Ratnadeep; Wang, Wang; DesAulniers, Jessica; Kassiri, Zamaneh; Oudit, Gavin Y

2017-01-23

Sex-related differences in cardiac function and iron metabolism exist in humans and experimental animals. Male patients and preclinical animal models are more susceptible to cardiomyopathies and heart failure. However, whether similar differences are seen in iron-overload cardiomyopathy is poorly understood. Male and female wild-type and hemojuvelin-null mice were injected and fed with a high-iron diet, respectively, to develop secondary iron overload and genetic hemochromatosis. Female mice were completely protected from iron-overload cardiomyopathy, whereas iron overload resulted in marked diastolic dysfunction in male iron-overloaded mice based on echocardiographic and invasive pressure-volume analyses. Female mice demonstrated a marked suppression of iron-mediated oxidative stress and a lack of myocardial fibrosis despite an equivalent degree of myocardial iron deposition. Ovariectomized female mice with iron overload exhibited essential pathophysiological features of iron-overload cardiomyopathy showing distinct diastolic and systolic dysfunction, severe myocardial fibrosis, increased myocardial oxidative stress, and increased expression of cardiac disease markers. Ovariectomy prevented iron-induced upregulation of ferritin, decreased myocardial SERCA2a levels, and increased NCX1 levels. 17β-Estradiol therapy rescued the iron-overload cardiomyopathy in male wild-type mice. The responses in wild-type and hemojuvelin-null female mice were remarkably similar, highlighting a conserved mechanism of sex-dependent protection from iron-overload-mediated cardiac injury. Male and female mice respond differently to iron-overload-mediated effects on heart structure and function, and females are markedly protected from iron-overload cardiomyopathy. Ovariectomy in female mice exacerbated iron-induced myocardial injury and precipitated severe cardiac dysfunction during iron-overload conditions, whereas 17β-estradiol therapy was protective in male iron-overloaded mice. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Akt2 Knockout Alleviates Prolonged Caloric Restriction-Induced Change in Cardiac Contractile Function through Regulation of Autophagy

PubMed Central

Zhang, Yingmei; Han, Xuefeng; Hu, Nan; Huff, Anna F.; Gao, Feng; Ren, Jun

2014-01-01

Caloric restriction leads to changes in heart geometry and function although the underlying mechanism remains elusive. Autophagy, a conserved pathway for degradation of intracellular proteins and organelles, preserves energy and nutrient in the face of caloric insufficiency. This study was designed to examine the role of Akt2 in prolonged caloric restriction-induced change in cardiac homeostasis and the underlying mechanism(s) involved. Wild-type (WT) and Akt2 knockout mice were caloric restricted (by 40%) for 30 weeks. Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties, autophagy and its regulatory proteins were evaluated. Caloric restriction compromised echocardiographic indices (decreased left ventricular mass, left ventricular diameters and cardiac output), cardiomyocyte contractile and intracellular Ca2+ properties associated with dampened SERCA2a phosphorylation, upregulated phospholamban and autophagy (Beclin-1, Atg7, LC3BII-to-LC3BI ratio), increased autophagy adaptor protein p62, elevated phosphorylation of AMPK, Akt2 and the Akt downstream signal molecule TSC2, the effects of which with the exception of autophagy protein markers (Beclin-1, Atg7, LC3B) and AMPK were mitigated or significantly alleviated by Akt2 knockout. Lysosomal inhibition using bafilomycin A1 negated Akt2 knockout-induced protective effect on p62. Evaluation of downstream signaling molecules of Akt and AMPK including mTOR and ULK1 revealed that caloric restriction suppressed and promoted phosphorylation of mTOR and ULK1, respectively, without affecting total mTOR and ULK1 expression. Akt2 knockout significantly augmented caloric restriction-induced responses on mTOR and ULK1. Taken together, these data suggest a beneficial role of Akt2 knockout in preservation of cardiac homeostasis against prolonged caloric restriction-induced pathological changes possibly through facilitating autophagy. PMID:24368095

Chronic Cardiac-Targeted RNA Interference for the Treatment of Heart Failure Restores Cardiac Function and Reduces Pathological Hypertrophy

PubMed Central

Suckau, Lennart; Fechner, Henry; Chemaly, Elie; Krohn, Stefanie; Hadri, Lahouaria; Kockskämper, Jens; Westermann, Dirk; Bisping, Egbert; Ly, Hung; Wang, Xiaomin; Kawase, Yoshiaki; Chen, Jiqiu; Liang, Lifan; Sipo, Isaac; Vetter, Roland; Weger, Stefan; Kurreck, Jens; Erdmann, Volker; Tschope, Carsten; Pieske, Burkert; Lebeche, Djamel; Schultheiss, Heinz-Peter; Hajjar, Roger J.; Poller, Wolfgang Ch.

2009-01-01

Background RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. We report on targeted RNAi for the treatment of heart failure (HF), an important disorder in humans resulting from multiple etiologies. Successful treatment of HF is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban (PLB), a key regulator of cardiac Ca2+ homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy employs regulatory RNAs to achieve its effect. Methods and Results We describe structural requirements to obtain high RNAi activity from adenoviral (AdV) and adeno-associated virus (AAV9) vectors and show that an AdV short hairpin RNA vector (AdV-shRNA) silenced PLB in cardiomyocytes (NRCMs) and improved hemodynamics in HF rats 1 month after aortic root injection. For simplified long-term therapy we developed a dimeric cardiotropic AAV vector (rAAV9-shPLB) delivering RNAi activity to the heart via intravenous injection. Cardiac PLB protein was reduced to 25% and SERCA2a suppression in the HF groups was rescued. In contrast to traditional vectors rAAV9 shows high affinity for myocardium, but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (LVEDP, dp/dtmin, Tau) and systolic (fractional shortening) functional parameters to normal range. The massive cardiac dilation was normalized and the cardiac hypertrophy, cardiomyocyte diameter and cardiac fibrosis significantly reduced. Importantly, there was no evidence of microRNA deregulation or hepatotoxicity during these RNAi therapies. Conclusion Our data show, for the first time, high efficacy of an RNAi therapeutic strategy in a cardiac disease. PMID:19237664

The plasma membrane calcium pumps: focus on the role in (neuro)pathology.

PubMed

Brini, Marisa; Carafoli, Ernesto; Calì, Tito

2017-02-19

The plasma membrane Ca 2+ ATPase (PMCA pump) is a member of the superfamily of P-type pumps. It is organized in the plasma membrane with ten transmembrane helices and two main cytosolic loops, one of which contains the catalytic center. It also contains a long C-terminal tail that houses the binding site for calmodulin, the main regulator of the activity of the pump. The pump also contains a number of other regulators, among them acidic phospholipids, kinases, and numerous protein interactors. Separate genes code for 4 basic pump isoforms in mammals, additional isoform complexity being generated by the alternative splicing of primary transcripts. Pumps 1 and 4 are expressed ubiquitously, pumps 2 and 3 are tissue restricted, with preference for the nervous system. In essentially all cells, the pump coexists with much more powerful systems that clear Ca 2+ from the cytosol, e.g. the SERCA pump and the Na + /Ca 2+ exchanger. Its role in the global regulation of cellular Ca 2+ homeostasis is thus quantitatively marginal: its main function is the regulation of Ca 2+ signaling in selected sub-plasma membrane microdomains where Ca 2+ modulated interactors also reside. Malfunctions of the pump linked to genetic mutations are now described with increasing frequency, the disease phenotypes being especially severe in the nervous system where isoforms 2 and 3 predominate. The analysis of the pump defects suggests that the disease phenotypes are likely to be related to the imperfect modulation of Ca 2+ signaling in selected sub-plasma membrane microdomains, leading to the defective control of the activity of important Ca 2+ dependent interactors. Copyright © 2016 Elsevier Inc. All rights reserved.

Re-evaluation of how artemisinins work in light of emerging evidence of in vitro resistance

PubMed Central

Krishna, Sanjeev; Woodrow, Charles J.; Staines, Henry M.; Haynes, Richard K.; Mercereau-Puijalon, Odile

2006-01-01

There are more than half a billion cases of malaria every year. Combinations of an artemisinin with other antimalarial drugs are now recommended treatments for Plasmodium falciparum malaria in most endemic areas. These treatment regimens act rapidly to relieve symptoms and effect cure. There is considerable controversy on how artemisinins work and over emerging indications of resistance to this class of antimalarial drugs. Several individual molecules have been proposed as targets for artemisinins, in addition to the idea that artemisinins might have many targets at the same time. Our suggestion that artemisinins inhibit the parasite-encoded sarco–endoplasmic reticulum Ca2+-ATPase (SERCA) PfATP6 has gained support from recent observations that a polymorphism in the gene encoding PfATP6 is associated with in vitro resistance to artemether in field isolates of P. falciparum. PMID:16616639

Untangling the Contributions of Sex-Specific Gene Regulation and X-Chromosome Dosage to Sex-Biased Gene Expression in Caenorhabditis elegans

PubMed Central

Kramer, Maxwell; Rao, Prashant; Ercan, Sevinc

2016-01-01

Dosage compensation mechanisms equalize the level of X chromosome expression between sexes. Yet the X chromosome is often enriched for genes exhibiting sex-biased, i.e., imbalanced expression. The relationship between X chromosome dosage compensation and sex-biased gene expression remains largely unexplored. Most studies determine sex-biased gene expression without distinguishing between contributions from X chromosome copy number (dose) and the animal’s sex. Here, we uncoupled X chromosome dose from sex-specific gene regulation in Caenorhabditis elegans to determine the effect of each on X expression. In early embryogenesis, when dosage compensation is not yet fully active, X chromosome dose drives the hermaphrodite-biased expression of many X-linked genes, including several genes that were shown to be responsible for hermaphrodite fate. A similar effect is seen in the C. elegans germline, where X chromosome dose contributes to higher hermaphrodite X expression, suggesting that lack of dosage compensation in the germline may have a role in supporting higher expression of X chromosomal genes with female-biased functions in the gonad. In the soma, dosage compensation effectively balances X expression between the sexes. As a result, somatic sex-biased expression is almost entirely due to sex-specific gene regulation. These results suggest that lack of dosage compensation in different tissues and developmental stages allow X chromosome copy number to contribute to sex-biased gene expression and function. PMID:27356611

Modeling CICR in rat ventricular myocytes: voltage clamp studies

PubMed Central

2010-01-01

Background The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect Ca2+ loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca2+ release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic Ca2+ concentration ([Ca2+]myo). Methods The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed Ca2+ channels (trigger-channel and release-channel). It releases Ca2+ flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[Ca2+]myo. Results Our model reproduces measured VC data published by several laboratories, and generates graded Ca2+ release at high Ca2+ gain in a homeostatically-controlled environment where [Ca2+]myo is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR Ca2+ release, its activation by trigger Ca2+, and its refractory characteristics mediated by the luminal SR Ca2+ sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence Ca2+ homeostasis. Conclusions We examine the role of the above Ca2+ regulating mechanisms in handling various types of induced disturbances in Ca2+ levels by quantifying cellular Ca2+ balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses. PMID:21062495

Examining the contribution of handwriting and spelling to written expression in kindergarten children

PubMed Central

AlOtaiba, Stephanie

2011-01-01

In this study, we examined the development of beginning writing skills in kindergarten children and the contribution of spelling and handwriting to these writing skills after accounting for early language, literacy, cognitive skills, and student characteristics. Two hundred and forty two children were given a battery of cognitive, oral language, reading, and writing measures. They exhibited a range of competency in spelling, handwriting, written expression, and in their ability to express ideas. Handwriting and spelling made statistically significant contributions to written expression, demonstrating the importance of these lower-order transcription skills to higher order text-generation skills from a very early age. The contributions of oral language and reading skills were not significant. Implications of these findings for writing development and instruction are addressed. PMID:23087544

Contributions of feature shapes and surface cues to the recognition of facial expressions.

PubMed

Sormaz, Mladen; Young, Andrew W; Andrews, Timothy J

2016-10-01

Theoretical accounts of face processing often emphasise feature shapes as the primary visual cue to the recognition of facial expressions. However, changes in facial expression also affect the surface properties of the face. In this study, we investigated whether this surface information can also be used in the recognition of facial expression. First, participants identified facial expressions (fear, anger, disgust, sadness, happiness) from images that were manipulated such that they varied mainly in shape or mainly in surface properties. We found that the categorization of facial expression is possible in either type of image, but that different expressions are relatively dependent on surface or shape properties. Next, we investigated the relative contributions of shape and surface information to the categorization of facial expressions. This employed a complementary method that involved combining the surface properties of one expression with the shape properties from a different expression. Our results showed that the categorization of facial expressions in these hybrid images was equally dependent on the surface and shape properties of the image. Together, these findings provide a direct demonstration that both feature shape and surface information make significant contributions to the recognition of facial expressions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modes of gene duplication contribute differently to genetic novelty and redundancy, but show parallels across divergent angiosperms.

PubMed

Wang, Yupeng; Wang, Xiyin; Tang, Haibao; Tan, Xu; Ficklin, Stephen P; Feltus, F Alex; Paterson, Andrew H

2011-01-01

Both single gene and whole genome duplications (WGD) have recurred in angiosperm evolution. However, the evolutionary effects of different modes of gene duplication, especially regarding their contributions to genetic novelty or redundancy, have been inadequately explored. In Arabidopsis thaliana and Oryza sativa (rice), species that deeply sample botanical diversity and for which expression data are available from a wide range of tissues and physiological conditions, we have compared expression divergence between genes duplicated by six different mechanisms (WGD, tandem, proximal, DNA based transposed, retrotransposed and dispersed), and between positional orthologs. Both neo-functionalization and genetic redundancy appear to contribute to retention of duplicate genes. Genes resulting from WGD and tandem duplications diverge slowest in both coding sequences and gene expression, and contribute most to genetic redundancy, while other duplication modes contribute more to evolutionary novelty. WGD duplicates may more frequently be retained due to dosage amplification, while inferred transposon mediated gene duplications tend to reduce gene expression levels. The extent of expression divergence between duplicates is discernibly related to duplication modes, different WGD events, amino acid divergence, and putatively neutral divergence (time), but the contribution of each factor is heterogeneous among duplication modes. Gene loss may retard inter-species expression divergence. Members of different gene families may have non-random patterns of origin that are similar in Arabidopsis and rice, suggesting the action of pan-taxon principles of molecular evolution. Gene duplication modes differ in contribution to genetic novelty and redundancy, but show some parallels in taxa separated by hundreds of millions of years of evolution.

Modes of Gene Duplication Contribute Differently to Genetic Novelty and Redundancy, but Show Parallels across Divergent Angiosperms

PubMed Central

Wang, Yupeng; Wang, Xiyin; Tang, Haibao; Tan, Xu; Ficklin, Stephen P.; Feltus, F. Alex; Paterson, Andrew H.

2011-01-01

Background Both single gene and whole genome duplications (WGD) have recurred in angiosperm evolution. However, the evolutionary effects of different modes of gene duplication, especially regarding their contributions to genetic novelty or redundancy, have been inadequately explored. Results In Arabidopsis thaliana and Oryza sativa (rice), species that deeply sample botanical diversity and for which expression data are available from a wide range of tissues and physiological conditions, we have compared expression divergence between genes duplicated by six different mechanisms (WGD, tandem, proximal, DNA based transposed, retrotransposed and dispersed), and between positional orthologs. Both neo-functionalization and genetic redundancy appear to contribute to retention of duplicate genes. Genes resulting from WGD and tandem duplications diverge slowest in both coding sequences and gene expression, and contribute most to genetic redundancy, while other duplication modes contribute more to evolutionary novelty. WGD duplicates may more frequently be retained due to dosage amplification, while inferred transposon mediated gene duplications tend to reduce gene expression levels. The extent of expression divergence between duplicates is discernibly related to duplication modes, different WGD events, amino acid divergence, and putatively neutral divergence (time), but the contribution of each factor is heterogeneous among duplication modes. Gene loss may retard inter-species expression divergence. Members of different gene families may have non-random patterns of origin that are similar in Arabidopsis and rice, suggesting the action of pan-taxon principles of molecular evolution. Conclusion Gene duplication modes differ in contribution to genetic novelty and redundancy, but show some parallels in taxa separated by hundreds of millions of years of evolution. PMID:22164235

Orthogonal control of expression mean and variance by epigenetic features at different genomic loci

DOE PAGES

Dey, Siddharth S.; Foley, Jonathan E.; Limsirichai, Prajit; ...

2015-05-05

While gene expression noise has been shown to drive dramatic phenotypic variations, the molecular basis for this variability in mammalian systems is not well understood. Gene expression has been shown to be regulated by promoter architecture and the associated chromatin environment. However, the exact contribution of these two factors in regulating expression noise has not been explored. Using a dual-reporter lentiviral model system, we deconvolved the influence of the promoter sequence to systematically study the contribution of the chromatin environment at different genomic locations in regulating expression noise. By integrating a large-scale analysis to quantify mRNA levels by smFISH andmore » protein levels by flow cytometry in single cells, we found that mean expression and noise are uncorrelated across genomic locations. Furthermore, we showed that this independence could be explained by the orthogonal control of mean expression by the transcript burst size and noise by the burst frequency. Finally, we showed that genomic locations displaying higher expression noise are associated with more repressed chromatin, thereby indicating the contribution of the chromatin environment in regulating expression noise.« less

Skeletal muscle cells express ICAM-1 after muscle overload and ICAM-1 contributes to the ensuing hypertrophic response.

PubMed

Dearth, Christopher L; Goh, Qingnian; Marino, Joseph S; Cicinelli, Peter A; Torres-Palsa, Maria J; Pierre, Philippe; Worth, Randall G; Pizza, Francis X

2013-01-01

We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells.

Skeletal Muscle Cells Express ICAM-1 after Muscle Overload and ICAM-1 Contributes to the Ensuing Hypertrophic Response

PubMed Central

Dearth, Christopher L.; Goh, Qingnian; Marino, Joseph S.; Cicinelli, Peter A.; Torres-Palsa, Maria J.; Pierre, Philippe; Worth, Randall G.; Pizza, Francis X.

2013-01-01

We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells. PMID:23505517

Darwin's contributions to our understanding of emotional expressions

PubMed Central

Ekman, Paul

2009-01-01

Darwin charted the field of emotional expressions with five major contributions. Possible explanations of why he was able to make such important and lasting contributions are proposed. A few of the important questions that he did not consider are described. Two of those questions have been answered at least in part; one remains a major gap in our understanding of emotion. PMID:19884139

Cardiogenic Genes Expressed in Cardiac Fibroblasts Contribute to Heart Development and Repair

PubMed Central

Furtado, Milena B.; Costa, Mauro W.; Pranoto, Edward Adi; Salimova, Ekaterina; Pinto, Alex; Lam, Nicholas T.; Park, Anthony; Snider, Paige; Chandran, Anjana; Harvey, Richard P.; Boyd, Richard; Conway, Simon J.; Pearson, James; Kaye, David M.; Rosenthal, Nadia A.

2014-01-01

Rationale Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers. Objective To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration. Methods and Results High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical MSC and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. Whilst genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, Tbx20, caused marked myocardial dysmorphology and perturbations in scar formation upon myocardial infarction. Conclusions The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs and direct contribution to cardiac development and repair provokes alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies. PMID:24650916

Skeletal Muscle Thermogenesis and Its Role in Whole Body Energy Metabolism

PubMed Central

Herrera, Jose Luis; Reis, Felipe C. G.

2017-01-01

Obesity and diabetes has become a major epidemic across the globe. Controlling obesity has been a challenge since this would require either increased physical activity or reduced caloric intake; both are difficult to enforce. There has been renewed interest in exploiting pathways such as uncoupling protein 1 (UCP1)-mediated uncoupling in brown adipose tissue (BAT) and white adipose tissue to increase energy expenditure to control weight gain. However, relying on UCP1-based thermogenesis alone may not be sufficient to control obesity in humans. On the other hand, skeletal muscle is the largest organ and a major contributor to basal metabolic rate and increasing energy expenditure in muscle through nonshivering thermogenic mechanisms, which can substantially affect whole body metabolism and weight gain. In this review we will describe the role of Sarcolipin-mediated uncoupling of Sarcoplasmic Reticulum Calcium ATPase (SERCA) as a potential mechanism for increased energy expenditure both during cold and diet-induced thermogenesis. PMID:29086530

A robust method to screen detergents for membrane protein stabilization, revisited.

PubMed

Champeil, Philippe; Orlowski, Stéphane; Babin, Simon; Lund, Sten; le Maire, Marc; Møller, Jesper; Lenoir, Guillaume; Montigny, Cédric

2016-10-15

This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907-4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of β-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains useful in pre-screening assays testing various detergents. Copyright © 2016 Elsevier Inc. All rights reserved.

Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

NASA Astrophysics Data System (ADS)

Restrepo, Simon; Basler, Konrad

2016-08-01

Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. We probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Here we discover that wing discs display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. Our results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies.

Current perspectives on the mechanism of action of artemisinins.

PubMed

Golenser, Jacob; Waknine, Judith H; Krugliak, Miriam; Hunt, Nicholas H; Grau, Georges E

2006-12-01

Artemisinin derivatives are the most recent single drugs approved and introduced for public antimalarial treatment. Although their recommended use is for treatment of Plasmodium falciparum infection, these drugs also act against other parasites, as well as against tumor cells. The mechanisms of action attributed to artemisinin include interference with parasite transport proteins, disruption of parasite mitochondrial function, modulation of host immune function and inhibition of angiogenesis. Artemisinin combination therapies are currently the preferred treatment for malaria. These combinations may prevent the induction of parasite drug resistance. However, in view of the multiple mechanisms involved, especially when additional drugs are used, the combined therapy should be carefully examined for antagonistic effects. It is now a general theory that the crucial mechanism is interference with plasmodial SERCA. Therefore, future development of resistance may be associated with overproduction or mutations of this transporter. However, a general mechanism, such as alterations in general drug transport pathways, is feasible. In this article, we review the evidence for each mechanism of action suggested.

Advances in gene therapy for heart failure.

PubMed

Fish, Kenneth M; Ishikawa, Kiyotake

2015-04-01

Chronic heart failure is expected to increase its social and economic burden as a consequence of improved survival in patients with acute cardiac events. Cardiac gene therapy holds significant promise in heart failure treatment for patients with currently very limited or no treatment options. The introduction of adeno-associated virus (AAV) gene vector changed the paradigm of cardiac gene therapy, and now it is the primary vector of choice for chronic heart failure gene therapy in clinical and preclinical studies. Recently, there has been significant progress towards clinical translation in this field spearheaded by AAV-1 mediated sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) gene therapy targeting chronic advanced heart failure patients. Meanwhile, several independent laboratories are reporting successful gene therapy approaches in clinically relevant large animal models of heart failure and some of these approaches are expected to enter clinical trials in the near future. This review will focus on gene therapy approaches targeting heart failure that is in clinical trials and those close to its initial clinical trial application.

Curcumin induces endoplasmic reticulum stress-associated apoptosis in human papillary thyroid carcinoma BCPAP cells via disruption of intracellular calcium homeostasis.

PubMed

Zhang, Li; Cheng, Xian; Xu, Shichen; Bao, Jiandong; Yu, Huixin

2018-06-01

Thyroid cancer is the most common endocrine tumor. Our previous studies have demonstrated that curcumin can induce apoptosis in human papillary thyroid carcinoma BCPAP cells. However, the underlined mechanism has not been clearly elucidated. Endoplasmic reticulum (ER) is a major organelle for synthesis, maturation, and folding proteins as well as a large store for Ca. Overcoming chronically activated ER stress by triggering pro-apoptotic pathways of the unfolded protein response (UPR) is a novel strategy for cancer therapeutics. Our study aimed to uncover the ER stress pathway involved in the apoptosis caused by curcumin. BCPAP cells were treated with different doses of curcumin (12.5-50 μM). Annexin V/PI double staining was used to determine cell apoptosis. Rhod-2/AM calcium fluorescence probe assay was performed to measure the calcium level of endoplasmic reticulum. Western blot was used to examine the expression of ER stress marker C/EBP homologous protein 10 (CHOP) and glucose-regulated protein 78 (GRP78). X-box binding protein1 (XBP-1) spliced form was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Curcumin significantly inhibited anchorage-independent cell growth and induced apoptosis in BCPAP cells. Curcumin induced ER stress and UPR responses in a dose- and time-dependent manner, and the chemical chaperone 4-phenylbutyrate (4-PBA) partially reversed the antigrowth activity of curcumin. Moreover, curcumin significantly increased inositol-requiring enzyme 1α (IRE1α) phosphorylation and XBP-1 mRNA splicing to induce a subsets of ER chaperones. Increased cleavage of activating transcription factor 6 (ATF6), which enhances expression of its downstream target CHOP was also observed. Furthermore, curcumin induced intracellular Ca influx through inhibition of the sarco-endoplasmic reticulum ATPase 2A (SERCA2) pump. The increased cytosolic Ca then bound to calmodulin to activate calcium/calmodulin-dependent protein kinase II (CaMKII) signaling, leading to mitochondrial apoptosis pathway activation. Ca chelator BAPTA partially reversed curcumin-induced ER stress and growth suppression, confirming the possible involvement of calcium homeostasis disruption in this response. Curcumin inhibits thyroid cancer cell growth, at least partially, through ER stress-associated apoptosis. Our observations provoked that ER stress activation may be a promising therapeutic target for thyroid cancer treatment.(Figure is included in full-text article.).

Regulation of SFRP-1 expression in the rat dental follicle.

PubMed

Liu, Dawen; Yao, Shaomian; Wise, Gary E

2012-01-01

Tooth eruption requires osteoclastogenesis and subsequent bone resorption. Secreted frizzled-related protein-1 (SFRP-1) negatively regulates osteoclastogenesis. Our previous studies indicated that SFRP-1 is expressed in the rat dental follicle (DF), with reduced expression at days 3 and 9 close to the times for the major and minor bursts of osteoclastogenesis, respectively; but it remains unclear as to what molecules contribute to its reduced expression at these critical times. Thus, it was the aim of this study to determine which molecules regulate the expression of SFRP-1 in the DF. To that end, the DF cells were treated with cytokines that are maximally expressed at days 3 or 9, and SFRP-1 expression was determined. Our study indicated that colony-stimulating factor-1 (CSF-1), a molecule maximally expressed in the DF at day 3, down-regulated SFRP-1 expression. As to endothelial monocyte-activating polypeptide II (EMAP-II), a highly expressed molecule in the DF at day 3, it had no effect on the expression of SFRP-1. However, when EMAP-II was knocked down by siRNA, the expression of SFRP-1 was elevated, and this elevated SFRP-1 expression could be reduced by adding recombinant EMAP-II protein. This suggests that EMAP-II maintained a lower level of SFRP-1 in the DF. TNF-α is a molecule maximally expressed at day 9, and this study indicated that it also down-regulated the expression of SFRP-1 in the DF cells. In conclusion, CSF-1 and EMAP-II may contribute to the reduced SFRP-1 expression seen on day 3, while TNF-α may contribute to the reduced SFRP-1 expression at day 9.

In vivo Ca2+ buffering capacity and microvascular oxygen pressures following muscle contractions in diabetic rat skeletal muscles: fiber-type specific effects.

PubMed

Eshima, Hiroaki; Poole, David C; Kano, Yutaka

2015-07-15

In Type 1 diabetes, skeletal muscle resting intracellular Ca(2+) concentration ([Ca(2+)]i) homeostasis is impaired following muscle contractions. It is unclear to what degree this behavior is contingent upon fiber type and muscle oxygenation conditions. We tested the hypotheses that: 1) the rise in resting [Ca(2+)]i evident in diabetic rat slow-twitch (type I) muscle would be exacerbated in fast-twitch (type II) muscle following contraction; and 2) these elevated [Ca(2+)]i levels would relate to derangement of microvascular partial pressure of oxygen (PmvO2 ) rather than sarcoplasmic reticulum dysfunction per se. Adult male Wistar rats were divided randomly into diabetic (DIA: streptozotocin ip) and healthy (CONT) groups. Four weeks later extensor digitorum longus (EDL, predominately type II fibers) and soleus (SOL, predominately type I fibers) muscle contractions were elicited by continuous electrical stimulation (120 s, 100 Hz). Ca(2+) imaging was achieved using fura 2-AM in vivo (i.e., circulation intact). DIA increased fatigability in EDL (P < 0.05) but not SOL. In recovery, SOL [Ca(2+)]i either returned to its resting baseline within 150 s (CONT 1.00 ± 0.02 at 600 s) or was not elevated in recovery at all (DIA 1.03 ± 0.02 at 600 s, P > 0.05). In recovery, EDL CONT [Ca(2+)]i also decreased to values not different from baseline (1.06 ± 0.01, P > 0.05) at 600 s. In marked contrast, EDL DIA [Ca(2+)]i remained elevated for the entire recovery period (i.e., 1.23 ± 0.03 at 600 s, P < 0.05). The inability of [Ca(2+)]i to return to baseline in EDL DIA was not associated with any reduction of SR Ca(2+)-ATPase (SERCA) 1 or SERCA2 protein levels (both increased 30-40%, P < 0.05). However, Pmv(O2) recovery kinetics were markedly slowed in EDL such that mean Pmv(O2) was substantially depressed (CONT 27.9 ± 2.0 vs. DIA 18.4 ± 2.0 Torr, P < 0.05), and this behavior was associated with the elevated [Ca(2+)]i. In contrast, this was not the case for SOL (P > 0.05) in that neither [Ca(2+)]i nor Pmv(O2) were deranged in recovery with DIA. In conclusion, recovery of [Ca(2+)]i homeostasis is impaired in diabetic rat fast-twitch but not slow-twitch muscle in concert with reduced Pmv(O2) pressures. Copyright © 2015 the American Physiological Society.

Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity

USDA-ARS?s Scientific Manuscript database

The etiology of type 2 diabetes often involves diet-induced obesity (DIO), which is associated with elevated plasma fatty acids and lipoprotein associated triglycerides. Since aberrant hepatic fatty acid uptake may contribute to this, we investigated whether increased expression of a fatty acid tran...

Sources of Variance in Baseline Gene Expression in the Rodent Liver

PubMed Central

Corton, J. Christopher; Bushel, Pierre R.; Fostel, Jennifer; O'Lone, Raegan B.

2012-01-01

The use of gene expression profiling in both clinical and laboratory settings would be enhanced by better characterization of variation due to individual, environmental, and technical factors. Analysis of microarray data from untreated or vehicle-treated animals within the control arm of toxicogenomics studies has yielded useful information on baseline fluctuations in liver gene expression in the rodent. Here, studies which highlight contributions of different factors to gene expression variability in the rodent liver are discussed including a large meta-analysis of rat liver, which identified genes that vary in control animals in the absence of chemical treatment. Genes and their pathways that are the most and least variable were identified in a number of these studies. Life stage, fasting, sex, diet, circadian rhythm and liver lobe source can profoundly influence gene expression in the liver. Recognition of biological and technical factors that contribute to variability of background gene expression can help the investigator in the design of an experiment that maximizes sensitivity and reduces the influence of confounders that may lead to misinterpretation of genomic changes. The factors that contribute to variability in liver gene expression in rodents are likely analogous to those contributing to human interindividual variability in drug response and chemical toxicity. Identification of batteries of genes that are altered in a variety of background conditions could be used to predict responses to drugs and chemicals in appropriate models of the human liver. PMID:22230429

Testosterone deficiency prevents left ventricular contractility dysfunction after myocardial infarction.

PubMed

Ribeiro Júnior, R F; Ronconi, K S; Jesus, I C G; Almeida, P W M; Forechi, L; Vassallo, D V; Guatimosim, S; Stefanon, I; Fernandes, A A

2018-01-15

Testosterone may affect myocardial contractility since its deficiency decreases the contraction and relaxation of the heart. Meanwhile, testosterone replacement therapy has raised concerns because it may worsen cardiac dysfunction and remodeling after myocardial infarction (MI). In this study, we evaluate cardiac contractility 60 days after MI in rats with suppressed testosterone. Male Wistar rats underwent bilateral orchidectomy one week before the ligation of the anterior descending left coronary artery. The animals were divided into orchidectomized (OCT); MI; orchidectomized + MI (OCT + MI); orchidectomized + MI + testosterone (OCT + MI + T) and control (Sham) groups. Eight weeks after MI, papillary muscle contractility was analyzed under increasing calcium (0.62, 1.25, 2.5 and 3.75 mM) and isoproterenol (10 -8 to 10 -2  M) concentrations. Ventricular myocytes were isolated for intracellular calcium measurements and assessment of Ca 2+ handling proteins. Contractility was preserved in the orchidectomized animals after myocardial infarction and was reduced when testosterone was replaced (Ca 2+ 3.75 mM: Sham: 608 ± 70 (n = 11); OCT: 590 ± 37 (n = 16); MI: 311 ± 33* (n = 9); OCT + MI: 594 ± 76 (n = 7); OCT + MI + T: 433 ± 38* (n=4), g/g *p < 0.05 vs Sham). Orchidectomy also increased the Ca 2+ transient amplitude of the ventricular myocytes and SERCA-2a protein expression levels. PLB phosphorylation levels at Thr 17 were not different in the orchidectomized animals compared to the Sham animals but were reduced after testosterone replacement. CAMKII phosphorylation and protein nitrosylation increased in the orchidectomized animals. Our results support the view that testosterone deficiency prevents MI contractility dysfunction by altering the key proteins involved in Ca 2+ handling. Copyright © 2017 Elsevier B.V. All rights reserved.

AMP-Dependent Kinase and Autophagic Flux are Involved in Aldehyde Dehydrogenase 2-Offered Protection against Cardiac Toxicity of Ethanol

PubMed Central

Ge, Wei; Guo, Rui; Ren, Jun

2011-01-01

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) alleviates ethanol toxicity although the precise mechanism is unclear. This study was designed to evaluate the effect of ALDH2 on ethanol-induced myocardial damage with a focus on autophagy. Wild-type FVB and transgenic mice overexpressing ALDH2 were challenged with ethanol (3 g/kg/d, i.p.) for 3 days and cardiac mechanical function was assessed using the echocardiographic and IonOptix systems. Western blot analysis was used to evaluate essential autophagy markers, Akt and AMPK and their downstream signaling mTOR. Ethanol challenge altered cardiac geometry and function evidenced by enlarged ventricular end systolic and diastolic diameters, decreased cell shortening and intracellular Ca2+ rise, prolonged relengthening and intracellular Ca2+ decay, as well as reduced SERCA Ca2+ uptake, the effects of which were mitigated by ALDH2. Ethanol challenge facilitated myocardial autophagy as evidenced by enhanced expression of Beclin, ATG7 and LC3B II, as well as mTOR dephosphorylation, which was alleviated by ALDH2. Ethanol challenge-induced cardiac defect and apoptosis were reversed by the ALDH-2 agonist Alda-1, the autophagy inhibitor 3-MA, and the AMPK inhibitor compound C whereas the autophagy inducer rapamycin and the AMPK activator AICAR mimicked or exacerbated ethanol-induced cell injury. Ethanol promoted or suppressed phosphorylation of AMPK and Akt, respectively, in FVB but not ALDH2 murine hearts. Moreover, AICAR nullified Alda-1-induced protection against ethanol-triggered autophagic and functional changes. Ethanol increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by Alda-1 and 3-MA. Lysosomal inhibition using bafilomycin A1, E64D and pepstatin A obliterated Alda-1- but not ethanol-induced responses in GFP-LC3 puncta. Our results suggested that ALDH2 protects against ethanol toxicity through altered Akt and AMPK signaling and regulation of autophagic flux. PMID:21871561

Beta1-adrenoceptor antagonist, metoprolol attenuates cardiac myocyte Ca2+ handling dysfunction in rats with pulmonary artery hypertension.

PubMed

Fowler, Ewan D; Drinkhill, Mark J; Norman, Ruth; Pervolaraki, Eleftheria; Stones, Rachel; Steer, Emma; Benoist, David; Steele, Derek S; Calaghan, Sarah C; White, Ed

2018-07-01

Right heart failure is the major cause of death in Pulmonary Artery Hypertension (PAH) patients but is not a current, specific therapeutic target. Pre-clinical studies have shown that adrenoceptor blockade can improve cardiac function but the mechanisms of action within right ventricular (RV) myocytes are unknown. We tested whether the β 1 -adrenoceptor blocker metoprolol could improve RV myocyte function in an animal model of PAH, by attenuating adverse excitation-contraction coupling remodeling. PAH with RV failure was induced in rats by monocrotaline injection. When PAH was established, animals were given 10 mg/kg/day metoprolol (MCT + BB) or vehicle (MCT). The median time to the onset of heart failure signs was delayed from 23 days (MCT), to 31 days (MCT + BB). At 23 ± 1 days post-injection, MCT + BB showed improved in vivo cardiac function, measured by echocardiography. RV hypertrophy was reduced despite persistent elevated afterload. RV myocyte contractility during field stimulation was improved at higher pacing frequencies in MCT + BB. Preserved t-tubule structure, more uniform evoked Ca 2+ release, increased SERCA2a expression and faster ventricular repolarization (measured in vivo by telemetry) may account for the improved contractile function. Sarcoplasmic reticulum Ca 2+ overload was prevented in MCT + BB myocytes resulting in fewer spontaneous Ca 2+ waves, with a lower pro-arrhythmic potential. Our novel finding of attenuation of defects in excitation contraction coupling by β 1 -adrenoceptor blockade with delays in the onset of HF, identifies the RV as a promising therapeutic target in PAH. Moreover, our data suggest existing therapies for left ventricular failure may also be beneficial in PAH induced RV failure. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Akt2 knockout alleviates prolonged caloric restriction-induced change in cardiac contractile function through regulation of autophagy.

PubMed

Zhang, Yingmei; Han, Xuefeng; Hu, Nan; Huff, Anna F; Gao, Feng; Ren, Jun

2014-06-01

Caloric restriction leads to changes in heart geometry and function although the underlying mechanism remains elusive. Autophagy, a conserved pathway for degradation of intracellular proteins and organelles, preserves energy and nutrient in the face of caloric insufficiency. This study was designed to examine the role of Akt2 in prolonged caloric restriction-induced change in cardiac homeostasis and the underlying mechanism(s) involved. Wild-type (WT) and Akt2 knockout mice were calorie restricted (by 40%) for 30weeks. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, autophagy and its regulatory proteins were evaluated. Caloric restriction compromised echocardiographic indices (decreased left ventricular mass, left ventricular diameters and cardiac output), cardiomyocyte contractile and intracellular Ca(2+) properties associated with dampened SERCA2a phosphorylation, upregulated phospholamban and autophagy (Beclin-1, Atg7, LC3BII-to-LC3BI ratio), increased autophagy adaptor protein p62, elevated phosphorylation of AMPK, Akt2 and the Akt downstream signal molecule TSC2, the effects of which with the exception of autophagy protein markers (Beclin-1, Atg7, LC3B) and AMPK were mitigated or significantly alleviated by Akt2 knockout. Lysosomal inhibition using bafilomycin A1 negated Akt2 knockout-induced protective effect on p62. Evaluation of downstream signaling molecules of Akt and AMPK including mTOR and ULK1 revealed that caloric restriction suppressed and promoted phosphorylation of mTOR and ULK1, respectively, without affecting total mTOR and ULK1 expression. Akt2 knockout significantly augmented caloric restriction-induced responses on mTOR and ULK1. Taken together, these data suggest a beneficial role of Akt2 knockout in preservation of cardiac homeostasis against prolonged caloric restriction-induced pathological changes possibly through facilitating autophagy. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy." Copyright © 2013 Elsevier Ltd. All rights reserved.

Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I

PubMed Central

Frantz, Stefan; Klaiber, Michael; Baba, Hideo A.; Oberwinkler, Heike; Völker, Katharina; Gaβner, Birgit; Bayer, Barbara; Abeβer, Marco; Schuh, Kai; Feil, Robert; Hofmann, Franz; Kuhn, Michaela

2013-01-01

Aims Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism. Methods and results To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to β-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca2+]i-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte Ca2+i homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca2+i-handling, and contractility via cGKI. Conclusion These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca2+i handling and contractility. PMID:22199120

Cellular heterogeneity contributes to subtype-specific expression of ZEB1 in human glioblastoma.

PubMed

Euskirchen, Philipp; Radke, Josefine; Schmidt, Marc Sören; Schulze Heuling, Eva; Kadikowski, Eric; Maricos, Meron; Knab, Felix; Grittner, Ulrike; Zerbe, Norman; Czabanka, Marcus; Dieterich, Christoph; Miletic, Hrvoje; Mørk, Sverre; Koch, Arend; Endres, Matthias; Harms, Christoph

2017-01-01

The transcription factor ZEB1 has gained attention in tumor biology of epithelial cancers because of its function in epithelial-mesenchymal transition, DNA repair, stem cell biology and tumor-induced immunosuppression, but its role in gliomas with respect to invasion and prognostic value is controversial. We characterized ZEB1 expression at single cell level in 266 primary brain tumors and present a comprehensive dataset of high grade gliomas with Ki67, p53, IDH1, and EGFR immunohistochemistry, as well as EGFR FISH. ZEB1 protein expression in glioma stem cell lines was compared to their parental tumors with respect to gene expression subtypes based on RNA-seq transcriptomic profiles. ZEB1 is widely expressed in glial tumors, but in a highly variable fraction of cells. In glioblastoma, ZEB1 labeling index is higher in tumors with EGFR amplification or IDH1 mutation. Co-labeling studies showed that tumor cells and reactive astroglia, but not immune cells contribute to the ZEB1 positive population. In contrast, glioma cell lines constitutively express ZEB1 irrespective of gene expression subtype. In conclusion, our data indicate that immune infiltration likely contributes to differential labelling of ZEB1 and confounds interpretation of bulk ZEB1 expression data.

Meconium increases type 1 angiotensin II receptor expression and alveolar cell death.

PubMed

Rosenfeld, Charles R; Zagariya, Alexander M; Liu, Xiao-Tie; Willis, Brigham C; Fluharty, Steven; Vidyasagar, Dharmapuri

2008-03-01

The pulmonary renin-angiotensin system (RAS) contributes to inflammation and epithelial apoptosis in meconium aspiration. It is unclear if both angiotensin II receptors (ATR) contribute, where they are expressed and if meconium modifies subtype expression. We examined ATR subtypes in 2 wk rabbit pup lungs before and after meconium exposure and with and without captopril pretreatment or type 1 receptor (AT1R) inhibition with losartan, determining expression and cellular localization with immunoblots, RT-PCR and immunohistochemistry, respectively. Responses of cultured rat alveolar type II pneumocytes were also examined. Type 2 ATR were undetected in newborn lung before and after meconium instillation. AT1R were expressed in pulmonary vascular and bronchial smooth muscle and alveolar and bronchial epithelium. Meconium increased total lung AT1R protein approximately 3-fold (p = 0.006), mRNA 29% (p = 0.006) and immunostaining in bronchial and alveolar epithelium and smooth muscle, which were unaffected by captopril and losartan. Meconium also increased AT1R expression >3-fold in cultured type II pneumocytes and caused concentration-dependent cell death inhibited by losartan. Meconium increases AT1R expression in newborn rabbit lung and cultured type II pneumocytes and induces AT1R-mediated cell death. The pulmonary RAS contributes to the pathogenesis of meconium aspiration through increased receptor expression.

DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons

PubMed Central

Zhao, Jian-Yuan; Liang, Lingli; Gu, Xiyao; Li, Zhisong; Wu, Shaogen; Sun, Linlin; Atianjoh, Fidelis E.; Feng, Jian; Mo, Kai; Jia, Shushan; Lutz, Brianna Marie; Bekker, Alex; Nestler, Eric J.; Tao, Yuan-Xiang

2017-01-01

Nerve injury induces changes in gene transcription in dorsal root ganglion (DRG) neurons, which may contribute to nerve injury-induced neuropathic pain. DNA methylation represses gene expression. Here, we report that peripheral nerve injury increases expression of the DNA methyltransferase DNMT3a in the injured DRG neurons via the activation of the transcription factor octamer transcription factor 1. Blocking this increase prevents nerve injury-induced methylation of the voltage-dependent potassium (Kv) channel subunit Kcna2 promoter region and rescues Kcna2 expression in the injured DRG and attenuates neuropathic pain. Conversely, in the absence of nerve injury, mimicking this increase reduces the Kcna2 promoter activity, diminishes Kcna2 expression, decreases Kv current, increases excitability in DRG neurons and leads to spinal cord central sensitization and neuropathic pain symptoms. These findings suggest that DNMT3a may contribute to neuropathic pain by repressing Kcna2 expression in the DRG. PMID:28270689

Subunit-dependent postsynaptic expression of kainate receptors on hippocampal interneurons in area CA1

PubMed Central

Wondolowski, Joyce; Frerking, Matthew

2009-01-01

Kainate receptors (KARs) contribute to postsynaptic excitation in only a select subset of neurons. To define the parameters that specify the postsynaptic expression of KARs, we examined the contribution of KARs to EPSCs on hippocampal interneurons in area CA1. Interneurons in stratum radiatum/lacunosum-moleculare (SR/SLM) express KARs both with and without the GluR5 subunit, but KAR-mediated EPSCs are generated mainly, if not entirely, by GluR5-containing KARs. Extrasynaptic glutamate spillover profoundly recruits AMPARs with little effect on KARs, indicating that KARs are targeted at the synapse more precisely than AMPARs. However, spontaneous EPSCs with a conventional AMPAR component did not have a resolvable contribution of KARs, suggesting that the KARs that contribute to the evoked EPSCs are at a distinct set of synapses. GluR5-containing KARs on interneurons in stratum oriens do not contribute substantially to the EPSC. We conclude that KARs are localized to synapses by cell type-, synapse-, and subunit-selective mechanisms. PMID:19144856

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

PubMed Central

Bardy, G; Virsolvy, A; Quignard, J F; Ravier, M A; Bertrand, G; Dalle, S; Cros, G; Magous, R; Richard, S; Oiry, C

2013-01-01

Background and Purpose Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca2+]i) in beta cells, in the absence of any co-stimulating factor. Experimental Approach Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca2+]i were measured using the ratiometric fluorescent Ca2+ indicator Fura-2. Ca2+ channel currents were recorded with the whole-cell patch-clamp technique. Key Results Quercetin concentration-dependently increased insulin secretion and elevated [Ca2+]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L−1), but were nearly abolished by the L-type Ca2+ channel antagonist nifedipine (1 μmol·L−1). Similar to the L-type Ca2+ channel agonist Bay K 8644, quercetin enhanced the L-type Ca2+ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca2+]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L−1), with the two drugs having cumulative effects on [Ca2+]i. Conclusions and Implications Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca2+ influx through an interaction with L-type Ca2+ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion. PMID:23530660

Genomic and transcriptomic analysis of aroma synthesis in two hybrids between Saccharomyces cerevisiae and S. kudriavzevii in winemaking conditions.

PubMed

Gamero, Amparo; Belloch, Carmela; Querol, Amparo

2015-09-04

Aroma is one of the most important attributes defining wine quality in which yeasts play a crucial role, synthesizing aromatic compounds or releasing odourless conjugates. A present-day trend in winemaking consists of lowering fermentation temperature to achieve higher aroma production and retention. S. cerevisiae × S. kudriavzevii hybrids seem to have inherited beneficial traits from their parental species, like fermenting efficiently at low temperature or producing higher amounts of certain aromatic compounds. In this study, allelic composition and gene expression of the genes related to aroma synthesis in two genetically and phenotypically different S. cerevisiae × S. kudriavzevii hybrids, Lalvin W27 and VIN7, were compared and related to aroma production in microvinifications at 12 and 28 °C. In addition, the contribution of the allele coming from each parental to the overall expression was explored by RT-PCR. The results indicated large differences in allele composition, gene expression and the contribution of each parental to the overall expression at the fermentation temperatures tested. Results obtained by RT-PCR showed that in ARO1 and ATF2 genes the S. kudriavzevii allele was more expressed than that of S. cerevisiae particularly at 12 °C. This study revealed high differences regarding allele composition and gene expression in two S. cerevisiae × S. kudriavzevii hybrids, which may have led to different aroma profiles in winemaking conditions. The contribution of the alleles coming from each parental to the overall expression has proved to differently influence aroma synthesis. Besides, the quantitative contribution to the overall gene expression of the alleles coming from one parental strain or the other was clearly determined by the fermentation temperature for some genes.

Sex Bias and Maternal Contribution to Gene Expression Divergence in Drosophila Blastoderm Embryos

PubMed Central

Paris, Mathilde; Villalta, Jacqueline E.; Eisen, Michael B.; Lott, Susan E.

2015-01-01

Early embryogenesis is a unique developmental stage where genetic control of development is handed off from mother to zygote. Yet the contribution of this transition to the evolution of gene expression is poorly understood. Here we study two aspects of gene expression specific to early embryogenesis in Drosophila: sex-biased gene expression prior to the onset of canonical X chromosomal dosage compensation, and the contribution of maternally supplied mRNAs. We sequenced mRNAs from individual unfertilized eggs and precisely staged and sexed blastoderm embryos, and compared levels between D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. First, we find that mRNA content is highly conserved for a given stage and that studies relying on pooled embryos likely systematically overstate the degree of gene expression divergence. Unlike studies done on larvae and adults where most species show a larger proportion of genes with male-biased expression, we find that transcripts in Drosophila embryos are largely female-biased in all species, likely due to incomplete dosage compensation prior to the activation of the canonical dosage compensation mechanism. The divergence of sex-biased gene expression across species is observed to be often due to lineage-specific decrease of expression; the most drastic example of which is the overall reduction of male expression from the neo-X chromosome in D. pseudoobscura, leading to a pervasive female-bias on this chromosome. We see no evidence for a faster evolution of expression on the X chromosome in embryos (no “faster-X” effect), unlike in adults, and contrary to a previous study on pooled non-sexed embryos. Finally, we find that most genes are conserved in regard to their maternal or zygotic origin of transcription, and present evidence that differences in maternal contribution to the blastoderm transcript pool may be due to species-specific divergence of transcript degradation rates. PMID:26485701

Altering sensorimotor feedback disrupts visual discrimination of facial expressions.

PubMed

Wood, Adrienne; Lupyan, Gary; Sherrin, Steven; Niedenthal, Paula

2016-08-01

Looking at another person's facial expression of emotion can trigger the same neural processes involved in producing the expression, and such responses play a functional role in emotion recognition. Disrupting individuals' facial action, for example, interferes with verbal emotion recognition tasks. We tested the hypothesis that facial responses also play a functional role in the perceptual processing of emotional expressions. We altered the facial action of participants with a gel facemask while they performed a task that involved distinguishing target expressions from highly similar distractors. Relative to control participants, participants in the facemask condition demonstrated inferior perceptual discrimination of facial expressions, but not of nonface stimuli. The findings suggest that somatosensory/motor processes involving the face contribute to the visual perceptual-and not just conceptual-processing of facial expressions. More broadly, our study contributes to growing evidence for the fundamentally interactive nature of the perceptual inputs from different sensory modalities.

Somatostatin and its receptors contribute in a tissue-specific manner to the sex-dependent metabolic (fed/fasting) control of growth hormone axis in mice

PubMed Central

Córdoba-Chacón, José; Gahete, Manuel D.; Castaño, Justo P.; Kineman, Rhonda D.

2011-01-01

Somatostatin (SST) inhibits growth hormone (GH) secretion and regulates multiple processes by signaling through its receptors sst1–5. Differential expression of SST/ssts may contribute to sex-specific GH pattern and fasting-induced GH rise. To further delineate the tissue-specific roles of SST and sst1–5 in these processes, their expression patterns were evaluated in hypothalamus, pituitary, and stomach of male and female mice under fed/fasted conditions in the presence (wild type) or absence (SST-knockout) of endogenous SST. Under fed conditions, hypothalamic/stomach SST/ssts expression did not differ between sexes, whereas male pituitary expressed more SST and sst2A/2B/3/5A/5TMD2/5TMD1 and less sst1, and male pituitary cell cultures were more responsive to SST inhibitory actions on GH release compared with females. This suggests that local pituitary SST/ssts can contribute to the sexually dimorphic pattern of GH release. Fasting (48 h) reduced stomach sst2A/B and hypothalamic SST/sst2A expression in both sexes, whereas it caused a generalized downregulation of pituitary sst subtypes in male and of sst2A only in females. Thus, fasting can reduce SST sensitivity across tissues and SST input to the pituitary, thereby jointly contributing to enhance GH release. In SST-knockout mice, lack of SST differentially altered sst subtype expression levels in both sexes, supporting an important role for SST in sex-dependent control of GH axis. Evaluation of SST, IGF-I, and glucocorticoid effects on hypothalamic and pituitary cell cultures revealed that these hormones could directly account for alterations in sst2/5 expression in the physiological states examined. Taken together, these results indicate that changes in SST output and sensitivity can contribute critically to precisely define, in a tissue-dependent manner, the sex-specific metabolic regulation of the GH axis. PMID:20943754

Dissociable roles of internal feelings and face recognition ability in facial expression decoding.

PubMed

Zhang, Lin; Song, Yiying; Liu, Ling; Liu, Jia

2016-05-15

The problem of emotion recognition has been tackled by researchers in both affective computing and cognitive neuroscience. While affective computing relies on analyzing visual features from facial expressions, it has been proposed that humans recognize emotions by internally simulating the emotional states conveyed by others' expressions, in addition to perceptual analysis of facial features. Here we investigated whether and how our internal feelings contributed to the ability to decode facial expressions. In two independent large samples of participants, we observed that individuals who generally experienced richer internal feelings exhibited a higher ability to decode facial expressions, and the contribution of internal feelings was independent of face recognition ability. Further, using voxel-based morphometry, we found that the gray matter volume (GMV) of bilateral superior temporal sulcus (STS) and the right inferior parietal lobule was associated with facial expression decoding through the mediating effect of internal feelings, while the GMV of bilateral STS, precuneus, and the right central opercular cortex contributed to facial expression decoding through the mediating effect of face recognition ability. In addition, the clusters in bilateral STS involved in the two components were neighboring yet separate. Our results may provide clues about the mechanism by which internal feelings, in addition to face recognition ability, serve as an important instrument for humans in facial expression decoding. Copyright © 2016 Elsevier Inc. All rights reserved.

The expression of long-term potentiation: reconciling the preists and the postivists

PubMed Central

MacDougall, Matthew J.; Fine, Alan

2014-01-01

Long-term potentiation (LTP) of excitatory synaptic transmission in the hippocampus has been investigated in great detail over the past 40 years. Where and how LTP is actually expressed, however, remain controversial issues. Considerable evidence has been offered to support both pre- and postsynaptic contributions to LTP expression. Though it is widely held that postsynaptic expression mechanisms are the primary contributors to LTP expression, evidence for that conclusion is amenable to alternative explanations. Here, we briefly review some key contributions to the ‘locus’ debate and describe data that support a dominant role for presynaptic mechanisms. Recognition of the state-dependency of expression mechanisms, and consideration of the consequences of the spatial relationship between postsynaptic glutamate receptors and presynaptic vesicular release sites, lead to a model that may reconcile views from both sides of the synapse. PMID:24298138

Protein kinase Cε regulates nuclear translocation of extracellular signal-regulated kinase, which contributes to bradykinin-induced cyclooxygenase-2 expression.

PubMed

Nakano, Rei; Kitanaka, Taku; Namba, Shinichi; Kitanaka, Nanako; Sugiya, Hiroshi

2018-06-04

The proinflammatory mediator bradykinin stimulated cyclooxygenase-2 (COX-2) expression and subsequently prostaglandin E 2 synthesis in dermal fibroblasts. The involvement of B2 receptors and Gαq in the role of bradykinin was suggested by using pharmacological inhibitors. The PKC activator PMA stimulated COX-2 mRNA expression. Bradykinin failed to induce COX-2 mRNA expression in the presence of PKC inhibitors, whereas the effect of bradykinin was observed in the absence of extracellular Ca 2+ . Bradykinin-induced COX-2 mRNA expression was inhibited in cells transfected with PKCε siRNA. These observations suggest that the novel PKCε is concerned with bradykinin-induced COX-2 expression. Bradykinin-induced PKCε phosphorylation and COX-2 mRNA expression were inhibited by an inhibitor of 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and bradykinin-induced PDK-1 phosphorylation was inhibited by phospholipase D (PLD) inhibitors, suggesting that PLD/PDK-1 pathway contributes to bradykinin-induced PKCε activation. Pharmacological and knockdown studies suggest that the extracellular signal-regulated kinase 1 (ERK1) MAPK signaling is involved in bradykinin-induced COX-2 expression. Bradykinin-induced ERK phosphorylation was attenuated in the cells pretreated with PKC inhibitors or transfected with PKCε siRNA. We observed the interaction between PKCε and ERK by co-immunoprecipitation experiments. These observations suggest that PKCε activation contributes to the regulation of ERK1 activation. Bradykinin stimulated the accumulation of phosphorylated ERK in the nuclear fraction, that was inhibited in the cells treated with PKC inhibitors or transfected with PKCε siRNA. Consequently, we concluded that bradykinin activates PKCε via the PLD/PDK-1 pathway, which subsequently induces activation and translocation of ERK1 into the nucleus, and contributes to COX-2 expression for prostaglandin E 2 synthesis in dermal fibroblasts.

Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos.

PubMed

Masaki, Hideki; Kato-Itoh, Megumi; Takahashi, Yusuke; Umino, Ayumi; Sato, Hideyuki; Ito, Keiichi; Yanagida, Ayaka; Nishimura, Toshinobu; Yamaguchi, Tomoyuki; Hirabayashi, Masumi; Era, Takumi; Loh, Kyle M; Wu, Sean M; Weissman, Irving L; Nakauchi, Hiromitsu

2016-11-03

Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17 + endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17 + endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine. Copyright © 2016 Elsevier Inc. All rights reserved.

Functionally Selective Signaling for Morphine and Fentanyl Antinociception and Tolerance Mediated by the Rat Periaqueductal Gray

PubMed Central

Morgan, Michael M.; Reid, Rachel A.; Saville, Kimber A.

2014-01-01

Functionally selective signaling appears to contribute to the variability in mechanisms that underlie tolerance to the antinociceptive effects of opioids. The present study tested this hypothesis by examining the contribution of G protein-coupled receptor kinase (GRK)/Protein kinase C (PKC) and C-Jun N-terminal kinase (JNK) activation on both the expression and development of tolerance to morphine and fentanyl microinjected into the ventrolateral periaqueductal gray of the rat. Microinjection of morphine or fentanyl into the periaqueductal gray produced a dose-dependent increase in hot plate latency. Microinjection of the non-specific GRK/PKC inhibitor Ro 32-0432 into the periaqueductal gray to block mu-opioid receptor phosphorylation enhanced the antinociceptive effect of morphine but had no effect on fentanyl antinociception. Microinjection of the JNK inhibitor SP600125 had no effect on morphine or fentanyl antinociception, but blocked the expression of tolerance to repeated morphine microinjections. In contrast, a microinjection of Ro 32-0432 blocked the expression of fentanyl, but not morphine tolerance. Repeated microinjections of Ro 32-0432 blocked the development of morphine tolerance and inhibited fentanyl antinociception whether rats were tolerant or not. Repeated microinjections of SP600125 into the periaqueductal gray blocked the development of tolerance to both morphine and fentanyl microinjections. These data demonstrate that the signaling molecules that contribute to tolerance vary depending on the opioid and methodology used to assess tolerance (expression vs. development of tolerance). This signaling difference is especially clear for the expression of tolerance in which JNK contributes to morphine tolerance and GRK/PKC contributes to fentanyl tolerance. PMID:25503060

Oxidative stress and protein aggregation during biological aging.

PubMed

Squier, T C

2001-09-01

Biological aging is a fundamental process that represents the major risk factor with respect to the development of cancer, neurodegenerative, and cardiovascular diseases in vertebrates. It is, therefore, evident that the molecular mechanisms of aging are fundamental to understand many disease processes. In this regard, the oxidation and nitration of intracellular proteins and the formation of protein aggregates have been suggested to underlie the loss of cellular function and the reduced ability of senescent animals to withstand physiological stresses. Since oxidatively modified proteins are thermodynamically unstable and assume partially unfolded tertiary structures that readily form aggregates, it is likely that oxidized proteins are intermediates in the formation of amyloid fibrils. It is, therefore, of interest to identify oxidatively sensitive protein targets that may play a protective role through their ability to down-regulate energy metabolism and the consequent generation of reactive oxygen species (ROS). In this respect, the maintenance of cellular calcium gradients represents a major energetic expense, which links alterations in intracellular calcium levels to ATP utilization and the associated generation of ROS through respiratory control mechanisms. The selective oxidation or nitration of the calcium regulatory proteins calmodulin and Ca-ATPase that occurs in vivo during aging and under conditions of oxidative stress may represent an adaptive response to oxidative stress that functions to down-regulate energy metabolism and the associated generation of ROS. Since these calcium regulatory proteins are also preferentially oxidized or nitrated under in vitro conditions, these results suggest an enhanced sensitivity of these critical calcium regulatory proteins, which modulate signal transduction processes and intracellular energy metabolism, to conditions of oxidative stress. Thus, the selective oxidation of critical signal transduction proteins probably represents a regulatory mechanism that functions to minimize the generation of ROS through respiratory control mechanisms. The reduction of the rate of ROS generation, in turn, will promote cellular survival under conditions of oxidative stress, when reactive oxygen and nitrogen species overwhelm cellular antioxidant defense systems, by minimizing the non-selective oxidation of a range of biomolecules. Since protein aggregation occurs if protein repair and degradative systems are unable to act upon oxidized proteins and restore cellular function, the reduction of the oxidative load on the cell by the down-regulation of the electron transport chain functions to minimize protein aggregation. Thus, ROS function as signaling molecules that fine-tune cellular metabolism through the selective oxidation or nitration of calcium regulatory proteins in order to minimize wide-spread oxidative damage and protein aggregation. Oxidative damage to cellular proteins, the loss of calcium homeostasis and protein aggregation contribute to the formation of amyloid deposits that accumulate during biological aging. Critical to understand the relationship between these processes and biological aging is the identification of oxidatively sensitive proteins that modulate energy utilization and the associated generation of ROS. In this latter respect, oxidative modifications to the calcium regulatory proteins calmodulin (CaM) and the sarco/endoplasmic reticulum Ca-ATPase (SERCA) function to down-regulate ATP utilization and the associated generation of ROS associated with replenishing intracellular ATP through oxidative phosphorylation. Reductions in the rate of ROS generation, in turn, will minimize protein oxidation and facilitate intracellular repair and degradative systems that function to eliminate damaged and partially unfolded proteins. Since the rates of protein repair or degradation compete with the rate of protein aggregation, the modulation of intracellular calcium concentrations and energy metabolism through the selective oxidation or nitration of critical signal transduction proteins (i.e. CaM or SERCA) is thought to maintain cellular function by minimizing protein aggregation and amyloid formation. Age-dependent increases in the rate of ROS generation or declines in cellular repair or degradation mechanisms will increase the oxidative load on the cell, resulting in corresponding increases in the concentrations of oxidized proteins and the associated formation of amyloid.

Contribution of HIF-1alpha or HIF-2alpha to erythropoietin expression: in vivo evidence based on chromatin immunoprecipitation.

PubMed

Yeo, Eun-Jin; Cho, Young-Suk; Kim, Myung-Suk; Park, Jong-Wan

2008-01-01

Circulating erythropoietin (EPO) is mainly produced by the kidneys and mediates erythrogenesis in bone marrow and nonhematopoietic cell survival. EPO is also produced in other tissues where it functions as a paracrine. Moreover, the hypoxic induction of EPO is known to be mediated by HIF-1alpha and HIF-2alpha, but it remains obscure as to which of these two mediators mainly contributes to EPO expression. Thus, we designed in vivo experiments to evaluate the contributions made by HIF-1alpha and HIF-2alpha to EPO expression. In mice exposed to mild whole body hypoxia, HIF-1alpha and HIF-2alpha were both induced in all tissues examined. However, EPO mRNA was expressed in kidney and brain, but not in liver and lung. Likewise, chromatin immunoprecipitation (CHIP) analyses demonstrated that HIF-1alpha or HIF-2alpha binding to the EPO gene increased under hypoxic conditions only in kidney and brain. A comparison of CHIP data and EPO mRNA levels suggested that, during mild hypoxia, renal EPO transcription is induced equally by HIF-1alpha and HIF-2alpha, but that brain EPO is mainly induced during hypoxia by HIF-2alpha. Thus, HIF-1alpha and HIF-2alpha appear to contribute to EPO expression tissue specifically.

A High-Throughput Data Mining of Single Nucleotide Polymorphisms in Coffea Species Expressed Sequence Tags Suggests Differential Homeologous Gene Expression in the Allotetraploid Coffea arabica1[W

PubMed Central

Vidal, Ramon Oliveira; Mondego, Jorge Maurício Costa; Pot, David; Ambrósio, Alinne Batista; Andrade, Alan Carvalho; Pereira, Luiz Filipe Protasio; Colombo, Carlos Augusto; Vieira, Luiz Gonzaga Esteves; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães

2010-01-01

Polyploidization constitutes a common mode of evolution in flowering plants. This event provides the raw material for the divergence of function in homeologous genes, leading to phenotypic novelty that can contribute to the success of polyploids in nature or their selection for use in agriculture. Mounting evidence underlined the existence of homeologous expression biases in polyploid genomes; however, strategies to analyze such transcriptome regulation remained scarce. Important factors regarding homeologous expression biases remain to be explored, such as whether this phenomenon influences specific genes, how paralogs are affected by genome doubling, and what is the importance of the variability of homeologous expression bias to genotype differences. This study reports the expressed sequence tag assembly of the allopolyploid Coffea arabica and one of its direct ancestors, Coffea canephora. The assembly was used for the discovery of single nucleotide polymorphisms through the identification of high-quality discrepancies in overlapped expressed sequence tags and for gene expression information indirectly estimated by the transcript redundancy. Sequence diversity profiles were evaluated within C. arabica (Ca) and C. canephora (Cc) and used to deduce the transcript contribution of the Coffea eugenioides (Ce) ancestor. The assignment of the C. arabica haplotypes to the C. canephora (CaCc) or C. eugenioides (CaCe) ancestral genomes allowed us to analyze gene expression contributions of each subgenome in C. arabica. In silico data were validated by the quantitative polymerase chain reaction and allele-specific combination TaqMAMA-based method. The presence of differential expression of C. arabica homeologous genes and its implications in coffee gene expression, ontology, and physiology are discussed. PMID:20864545

Calcium regulation in crustaceans during the molt cycle: a review and update.

PubMed

Ahearn, Gregory A; Mandal, Prabir K; Mandal, Anita

2004-02-01

Epithelial cells of the gut, gills, antennal glands and integument regulate calcium concentrations in crustaceans during the molt cycle. A cellular calcium transport model has been proposed suggesting the presence of calcium pumps, cation antiporters and calcium channels in transporting epithelial membranes that regulate the movements of this cation across the cell layer. Basolateral calcium transport during postmolt appears mainly regulated by the low affinity NCX antiporter, while calcium regulating 'housekeeping' activities of these cells in intermolt are controlled by the high affinity calcium ATPase (PMCA). A model is proposed for the involvement of the epithelial ER in the massive transepithelial calcium fluxes that occur during premolt and postmolt. This model involves the endoplasmic reticulum SERCA and RyR proteins and proposed cytoplasmic unstirred layers adjacent to apical and basolateral plasma membranes where calcium activities may largely exceed those in the bulk cytoplasmic phase. A result of the proposed transepithelial calcium transport model is that large quantities of calcium can be moved through these cells by these processes without affecting the low, and carefully controlled, bulk cytoplasmic calcium activities.

Perceptual and affective mechanisms in facial expression recognition: An integrative review.

PubMed

Calvo, Manuel G; Nummenmaa, Lauri

2016-09-01

Facial expressions of emotion involve a physical component of morphological changes in a face and an affective component conveying information about the expresser's internal feelings. It remains unresolved how much recognition and discrimination of expressions rely on the perception of morphological patterns or the processing of affective content. This review of research on the role of visual and emotional factors in expression recognition reached three major conclusions. First, behavioral, neurophysiological, and computational measures indicate that basic expressions are reliably recognized and discriminated from one another, albeit the effect may be inflated by the use of prototypical expression stimuli and forced-choice responses. Second, affective content along the dimensions of valence and arousal is extracted early from facial expressions, although this coarse affective representation contributes minimally to categorical recognition of specific expressions. Third, the physical configuration and visual saliency of facial features contribute significantly to expression recognition, with "emotionless" computational models being able to reproduce some of the basic phenomena demonstrated in human observers. We conclude that facial expression recognition, as it has been investigated in conventional laboratory tasks, depends to a greater extent on perceptual than affective information and mechanisms.

CD30 expression by bone marrow mast cells from different diagnostic variants of systemic mastocytosis.

PubMed

Morgado, José M; Perbellini, Omar; Johnson, Ryan C; Teodósio, Cristina; Matito, Almudena; Álvarez-Twose, Iván; Bonadonna, Patrizia; Zamò, Alberto; Jara-Acevedo, Maria; Mayado, Andrea; Garcia-Montero, Andrés; Mollejo, Manuela; George, Tracy I; Zanotti, Roberta; Orfao, Alberto; Escribano, Luis; Sánchez-Muñoz, Laura

2013-12-01

CD30 expression by bone marrow (BM) mast cells (MC) has been reported recently in systemic mastocytosis (SM) patients. The aim of this study was to investigate the potential diagnostic and prognostic value of CD30 expression in SM as assessed by multiparameter flow cytometry. A total of 163 consecutive BM samples corresponding to 142 SM patients and 21 non-mastocytosis cases were studied. CD30 was positive in most SM patients (80%), but in only one non-mastocytosis case (4.8%). When combined with CD25, CD30 contributed to an improved accuracy over that of CD25 alone (98% versus 93%) mainly because most (eight of nine) of the well-differentiated SM (WDSM), who lacked CD25, were CD30(+). Similar levels of expression of CD30 were observed among all different subgroups of SM except mast cell leukaemia; among indolent SM (ISM) patients, no significant association was observed between the levels of CD30 expression and other clinical and biological features of the disease. The increased expression of CD30 associated with absence of CD25 contributes to the diagnosis of WDSM and its distinction from other subtypes of SM. By contrast, CD30 expression did not contribute either to prognostic stratification of ISM or to the differential diagnosis between ISM and aggressive SM cases. © 2013 John Wiley & Sons Ltd.

Reduced microRNA-188-3p expression contributes to apoptosis of spermatogenic cells in patients with azoospermia.

PubMed

Song, Wen-Yan; Meng, Hui; Wang, Xue-Gai; Jin, Hai-Xia; Yao, Gui-Dong; Shi, Sen-Lin; Wu, Liang; Zhang, Xiang-Yang; Sun, Ying-Pu

2017-02-01

Human mutL homologl (MLH1) works coordinately in sequential steps to initiate repair of DNA mismatches, and aberrant MLH1 expression is related to spermatogenetic malfunction. In the present study, MLH1 expression in patients with azoospermia was investigated, and moderating effects of miR-188-3p on MLH1 expression and spermatogenesis were identified. Testicular tissues from 16 patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA), and tissues of eight healthy patients were collected. Real-time PCR, Western blotting and immunohistochemical staining were used to detect MLH1 expression. Chromatin immunoprecipitation assay and luciferase reporter assay were performed to evaluate histone acetylation level of miR-188-3p and relationships between miR-188-3p and MLH1. Testicular MLH1 expression at mRNA and protein levels was significantly increased, while miR-188-3p expression was lower in patients with OA and NOA than that in controls. Reduced histone acetylation level of miR-188-3p promoter was observed in patients with azoospermia. Overexpression/inhibition of HDAC1, but not HDAC2, contributed to the significant reduction/increase of miR-188-3p expression. miR-188-3p targeted 3' UTR of MLH1 and regulated MLH1 expression. miR-188-3p inhibitor led to elevation of apoptotic level of spermatogenic cells in mice, while this effect was reversed by si-MLH1. Down-regulation of miR-188-3p by reducing histone acetylation up-regulated MLH1 expression and contributed to promotion of apoptosis in spermatogenic cells, in patients with azoospermia. © 2016 John Wiley & Sons Ltd.

Tumor cells versus host immune cells: whose PD-L1 contributes to PD-1/PD-L1 blockade mediated cancer immunotherapy?

PubMed

Tang, Fei; Zheng, Pan

2018-01-01

Antibody blockade of the PD-1/PD-L1 pathway has elicited durable antitumor responses in the therapy of a broad spectrum of cancers. PD-L1 is constitutively expressed in certain tumors and host immune cells, and its expression can be induced or maintained by many factors. The expression of PD-L1 on tumor tissues has been reported to be positively correlated with the efficacy of anti-PD-1/PD-L1 therapy in patients. However, multiple clinical trials indicate that patients with PD-L1-negative tumors also respond to this blockade therapy, which suggests the potential contribution of PD-L1 from host immune cells. Recently, six articles independently evaluated and verified the contributions of PD-L1 from tumor versus non-tumor cells in various mouse tumor models. These studies confirmed that PD-L1 on either tumor cells or host immune cells contributes to tumor escape, and the relative contributions of PD-L1 on these cells seem to be context-dependent. While both tumor- and host-derived PD-L1 can play critical roles in immune suppression, differences in tumor immunogenicity appear to underlie their relative importance. Notably, these reports highlight the essential roles of PD-L1 from host myeloid cells in negatively regulating T cell activation and limiting T cell trafficking. Therefore, comprehensive evaluating the global PD-L1 expression, rather than monitoring PD-L1 expression on tumor cells alone, should be a more accurate way for predicting responses in PD-1/PD-L1 blockade therapy in cancer patients.

Why do we need three levels to understand the molecular optical response?

NASA Astrophysics Data System (ADS)

Perez-Moreno, Javier; Clays, Koen; Kuzyk, Mark G.

2011-10-01

Traditionally, the nonlinear optical response at the molecular level has been modeled using the two-level approximation, under the assumption that the behavior of the exact sum-over-states (SOS) expressions for the molecular polarizabilities is well represented by the contribution of only two levels. We show how, a rigorous application of the Thomas-Kuhn sum-rules over the SOS expression for the diagonal component of the first-hyperpolarziability proves that the two-level approximation is unphysical. In addition, we indicate how the contributions of potentially infinite number of states to the SOS expressions for the first-hyperpolarizability are well represented by the contributions of a generic three-level system. This explains why the analysis of the three-level model in conjugation with the sum rules has lead to successful paradigms for the optimization of organic chromophores.

TGFβ1-induced down-regulation of microRNA-138 contributes to epithelial-mesenchymal transition in primary lung cancer cells.

PubMed

Zhang, Fang; Li, Tiepeng; Han, Lu; Qin, Peng; Wu, Zhao; Xu, Benling; Gao, Quanli; Song, Yongping

2018-02-19

The existence of cancer stem cells within the tumor could lead to cancer therapy resistance. TGFβ1 is considered as one of the most powerful players in the generation of CSCs through induction of epithelial-mesenchymal transition in different types of cancer including lung cancer, however, the detailed mechanisms by which TGFβ1 contribute to EMT induction and CSC maintenance remains unclear. Here, we showed primary lung cancer cells treated by TGFβ1 exhibit mesenchymal features, including morphology and expression of mesenchymal marker in a time-dependent manner. We also observed long-term TGFβ1 exposure leads to an enrichment of a sub-population of CD44 + CD90 + cells which represent CSCs in lung cancer cells. Moreover, the differential expression microRNAs between CSCs and non-CSCs were identified using next-generation sequencing to screen key miRNAs which might contribute to TGFβ1-induced EMT and CSCs generation. Among those differentially expressed miRNAs, the expression of microRNA-138 was time-dependently down-regulated by TGFβ1 treatment. We further demonstrated primary lung cancer cells, in which we knockdown the expression of miR-138, exhibit mesenchymal phenotypes and stem cell properties. Taken together, these findings indicate TGFβ1-induced down-regulation of microRNA-138 contributes to EMT in primary lung cancer cells, and suggest that miR-138 might serve as a potential therapeutic target. Copyright © 2018 Elsevier Inc. All rights reserved.

Intra-tubular hydrodynamic forces influence tubulo-interstitial fibrosis in the kidney

PubMed Central

Rohatgi, Rajeev; Flores, Daniel

2010-01-01

Purpose of review Renal epithelial cells respond to mechanical stimuli with immediate transduction events (e.g., activation of ion channels), intermediate biological responses (e.g., changes in gene expression), and long term cellular adaptation (e.g., protein expression). Progressive renal disease is characterized by disturbed glomerular hydrodynamics that contributes to glomerulosclerosis, but, how intra-tubular biomechanical forces contribute to tubulo-interstital inflammation and fibrosis is poorly understood. Recent findings In vivo and in vitro models of obstructive uropathy demonstrate that tubular stretch induces robust expression of transforming growth factor β-1 (TGFβ-1), activation of tubular apoptosis, and induction of NF-κB signaling which contribute to the inflammatory and fibrotic milieu. Non-obstructive structural kidney diseases associated with nephron loss follow a course characterized by compensatory increases of single nephron glomerular filtration rate and tubular flow rate. Resulting increases in tubular fluid shear stress (FSS) reduce tissue-plasminogen activator and urokinase enzymatic activity which diminishes breakdown of extracellular matrix. In models of high dietary Na intake, which increase tubular flow, urinary TGFβ-1 concentrations and renal mitogen activated protein kinase activity are increased. Summary In conclusion, intra-tubular biomechanical forces, stretch and FSS, generate changes in intracellular signaling and gene expression that contribute to the pathobiology of obstructive, and non-obstructive kidney disease. PMID:19851105

Effect of speed endurance training and reduced training volume on running economy and single muscle fiber adaptations in trained runners.

PubMed

Skovgaard, Casper; Christiansen, Danny; Christensen, Peter M; Almquist, Nicki W; Thomassen, Martin; Bangsbo, Jens

2018-02-01

The aim of the present study was to examine whether improved running economy with a period of speed endurance training and reduced training volume could be related to adaptations in specific muscle fibers. Twenty trained male (n = 14) and female (n = 6) runners (maximum oxygen consumption (VO 2 -max): 56.4 ± 4.6 mL/min/kg) completed a 40-day intervention with 10 sessions of speed endurance training (5-10 × 30-sec maximal running) and a reduced (36%) volume of training. Before and after the intervention, a muscle biopsy was obtained at rest, and an incremental running test to exhaustion was performed. In addition, running at 60% vVO 2 -max, and a 10-km run was performed in a normal and a muscle slow twitch (ST) glycogen-depleted condition. After compared to before the intervention, expression of mitochondrial uncoupling protein 3 (UCP3) was lower (P < 0.05) and dystrophin was higher (P < 0.05) in ST muscle fibers, and sarcoplasmic reticulum calcium ATPase 1 (SERCA1) was lower (P < 0.05) in fast twitch muscle fibers. Running economy at 60% vVO 2 -max (11.6 ± 0.2 km/h) and at v10-km (13.7 ± 0.3 km/h) was ~2% better (P < 0.05) after the intervention in the normal condition, but unchanged in the ST glycogen-depleted condition. Ten kilometer performance was improved (P < 0.01) by 3.2% (43.7 ± 1.0 vs. 45.2 ± 1.2 min) and 3.9% (45.8 ± 1.2 vs. 47.7 ± 1.3 min) in the normal and the ST glycogen-depleted condition, respectively. VO 2 -max was the same, but vVO 2 -max was 2.0% higher (P < 0.05; 19.3 ± 0.3 vs. 18.9 ± 0.3 km/h) after than before the intervention. Thus, improved running economy with intense training may be related to changes in expression of proteins linked to energy consuming processes in primarily ST muscle fibers. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Quantitative gene expression deregulation in mantle-cell lymphoma: correlation with clinical and biologic factors.

PubMed

Kienle, Dirk; Katzenberger, Tiemo; Ott, German; Saupe, Doreen; Benner, Axel; Kohlhammer, Holger; Barth, Thomas F E; Höller, Sylvia; Kalla, Jörg; Rosenwald, Andreas; Müller-Hermelink, Hans Konrad; Möller, Peter; Lichter, Peter; Döhner, Hartmut; Stilgenbauer, Stephan

2007-07-01

There is evidence for a direct role of quantitative gene expression deregulation in mantle-cell lymphoma (MCL) pathogenesis. Our aim was to investigate gene expression associations with other pathogenic factors and the significance of gene expression in a multivariate survival analysis. Quantitative expression of 20 genes of potential relevance for MCL prognosis and pathogenesis were analyzed using real-time reverse transcriptase polymerase chain reaction and correlated with clinical and genetic factors, tumor morphology, and Ki-67 index in 65 MCL samples. Genomic losses at the loci of TP53, RB1, and P16 were associated with reduced transcript levels of the respective genes, indicating a gene-dosage effect as the pathomechanism. Analysis of gene expression correlations between the candidate genes revealed a separation into two clusters, one dominated by proliferation activators, another by proliferation inhibitors and regulators of apoptosis. Whereas only weak associations were identified between gene expression and clinical parameters or blastoid morphology, several genes were correlated closely with the Ki-67 index, including the short CCND1 variant (positive correlation) and RB1, ATM, P27, and BMI (negative correlation). In multivariate survival analysis, expression levels of MYC, MDM2, EZH2, and CCND1 were the strongest prognostic factors independently of tumor proliferation and clinical factors. These results indicate a pathogenic contribution of several gene transcript levels to the biology and clinical course of MCL. Genes can be differentiated into factors contributing to proliferation deregulation, either by enhancement or loss of inhibition, and proliferation-independent factors potentially contributing to MCL pathogenesis by apoptosis impairment.

Epigenetic silencing of miR-124 prevents spermine oxidase regulation: implications for Helicobacter pylori-induced gastric cancer.

PubMed

Murray-Stewart, T; Sierra, J C; Piazuelo, M B; Mera, R M; Chaturvedi, R; Bravo, L E; Correa, P; Schneider, B G; Wilson, K T; Casero, R A

2016-10-20

Chronic inflammation contributes to the development of various forms of cancer. The polyamine catabolic enzyme spermine oxidase (SMOX) is induced in chronic inflammatory conditions, including Helicobacter pylori-associated gastritis, where its production of hydrogen peroxide contributes to DNA damage and subsequent tumorigenesis. MicroRNA expression levels are also altered in inflammatory conditions; specifically, the tumor suppressor miR-124 becomes silenced by DNA methylation. We sought to determine if this repression of miR-124 is associated with elevated SMOX activity and concluded that miR-124 is indeed a negative regulator of SMOX. In gastric adenocarcinoma cells harboring highly methylated and silenced mir-124 gene loci, 5-azacytidine treatment allowed miR-124 re-expression and decreased SMOX expression. Overexpression of an exogenous miR-124-3p mimic repressed SMOX mRNA and protein expression as well as H 2 O 2 production by >50% within 24 h. Reporter assays indicated that direct interaction of miR-124 with the 3'-untranslated region of SMOX mRNA contributes to this negative regulation. Importantly, overexpression of miR-124 before infection with H. pylori prevented the induction of SMOX believed to contribute to inflammation-associated tumorigenesis. Compelling human in vivo data from H. pylori-positive gastritis tissues indicated that the mir-124 gene loci are more heavily methylated in a Colombian population characterized by elevated SMOX expression and a high risk for gastric cancer. Furthermore, the degree of mir-124 methylation significantly correlated with SMOX expression throughout the population. These results indicate a protective role for miR-124 through the inhibition of SMOX-mediated DNA damage in the etiology of H. pylori-associated gastric cancer.

Epigenetic silencing of miR-124 prevents spermine oxidase regulation: Implications for Helicobacter pylori-induced gastric cancer

PubMed Central

Murray-Stewart, Tracy; Sierra, Johanna C.; Piazuelo, M. Blanca; Mera, Robertino M.; Chaturvedi, Rupesh; Bravo, Luis E.; Correa, Pelayo; Schneider, Barbara G.; Wilson, Keith T.; Casero, Robert A.

2016-01-01

Chronic inflammation contributes to the development of various forms of cancer. The polyamine catabolic enzyme spermine oxidase (SMOX) is induced in chronic inflammatory conditions, including Helicobacter pylori-associated gastritis, where its production of hydrogen peroxide contributes to DNA damage and subsequent tumorigenesis. MicroRNA expression levels are also altered in inflammatory conditions; specifically, the tumor suppressor miR-124 becomes silenced by DNA methylation. We sought to determine if this repression of miR-124 is associated with elevated SMOX activity and concluded that miR-124 is indeed a negative regulator of SMOX. In gastric adenocarcinoma cells harboring highly methylated and silenced mir-124 gene loci, 5-azacytidine treatment allowed miR-124 re-expression and decreased SMOX expression. Overexpression of an exogenous miR-124-3p mimic repressed SMOX mRNA and protein expression as well as H2O2 production by >50% within 24 hours. Reporter assays indicated that direct interaction of miR-124 with the 3′-untranslated region of SMOX mRNA contributes to this negative regulation. Importantly, overexpression of miR-124 prior to infection with H. pylori prevented the induction of SMOX believed to contribute to inflammation-associated tumorigenesis. Compelling human in vivo data from H. pylori-positive gastritis tissues indicated that the mir-124 gene loci are more heavily methylated in a Colombian population characterized by elevated SMOX expression and a high risk for gastric cancer. Furthermore, the degree of mir-124 methylation significantly correlated with SMOX expression throughout the population. These results indicate a protective role for miR-124 through the inhibition of SMOX-mediated DNA damage in the etiology of H. pylori-associated gastric cancer. PMID:27041578

HIV promoter integration site primarily modulates transcriptional burst size rather than frequency.

PubMed

Skupsky, Ron; Burnett, John C; Foley, Jonathan E; Schaffer, David V; Arkin, Adam P

2010-09-30

Mammalian gene expression patterns, and their variability across populations of cells, are regulated by factors specific to each gene in concert with its surrounding cellular and genomic environment. Lentiviruses such as HIV integrate their genomes into semi-random genomic locations in the cells they infect, and the resulting viral gene expression provides a natural system to dissect the contributions of genomic environment to transcriptional regulation. Previously, we showed that expression heterogeneity and its modulation by specific host factors at HIV integration sites are key determinants of infected-cell fate and a possible source of latent infections. Here, we assess the integration context dependence of expression heterogeneity from diverse single integrations of a HIV-promoter/GFP-reporter cassette in Jurkat T-cells. Systematically fitting a stochastic model of gene expression to our data reveals an underlying transcriptional dynamic, by which multiple transcripts are produced during short, infrequent bursts, that quantitatively accounts for the wide, highly skewed protein expression distributions observed in each of our clonal cell populations. Interestingly, we find that the size of transcriptional bursts is the primary systematic covariate over integration sites, varying from a few to tens of transcripts across integration sites, and correlating well with mean expression. In contrast, burst frequencies are scattered about a typical value of several per cell-division time and demonstrate little correlation with the clonal means. This pattern of modulation generates consistently noisy distributions over the sampled integration positions, with large expression variability relative to the mean maintained even for the most productive integrations, and could contribute to specifying heterogeneous, integration-site-dependent viral production patterns in HIV-infected cells. Genomic environment thus emerges as a significant control parameter for gene expression variation that may contribute to structuring mammalian genomes, as well as be exploited for survival by integrating viruses.

Notch-1 induces Epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells

PubMed Central

Bao, Bin; Wang, Zhiwei; Ali, Shadan; Kong, Dejuan; Li, Yiwei; Ahmad, Aamir; Banerjee, Sanjeev; Azmi, Asfar S.; Miele, Lucio; Sarkar, Fazlul H.

2011-01-01

Activation of Notch-1 is known to be associated with the development and progression of human malignancies including pancreatic cancer. Emerging evidence suggest that the acquisition of epithelial-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotype are interrelated and contributes to tumor recurrence and drug resistance. The molecular mechanism(s) by which Notch-1 contributes to the acquisition of EMT phenotype and CSC self-renewal capacity has not been fully elucidated. Here we show that forced over-expression of Notch-1 leads to increased cell growth, clonogenicity, migration and invasion of AsPC-1 cells. Moreover, over-expression of Notch-1 led to the induction of EMT phenotype by activation of mesenchymal cell markers such as ZEB1, CD44, EpCAM, and Hes 1. Here we also report, for the first time, that over-expression of Notch-1 leads to increased expression of miR-21, and decreased expression of miR-200b, miR-200c, let-7a, let-7b, and let-7c. Re-expression of miR-200b led to decreased expression of ZEB1, and vimentin, and increased expression of E-cadherin. Over-expression of Notch-1 also increased the formation of pancreatospheres consistent with expression of CSC surface markers CD44 and EpCAM. Finally, we found that genistein, a known natural anti-tumor agent inhibited cell growth, clonogenicity, migration, invasion, EMT phenotype, formation of pancreatospheres and expression of CD44 and EpCAM. These results suggest that the activation of Notch-1 signaling contributes to the acquisition of EMT phenotype, which is in part mediated through the regulation of miR-200b and CSC self-renewal capacity, and these processes could be attenuated by genistein treatment. PMID:21463919

Contribution of Fetal, but Not Adult, Pulmonary Mesothelium to Mesenchymal Lineages in Lung Homeostasis and Fibrosis.

PubMed

von Gise, Alexander; Stevens, Sean M; Honor, Leah B; Oh, Jin Hee; Gao, Chi; Zhou, Bin; Pu, William T

2016-02-01

The lung is enveloped by a layer of specialized epithelium, the pulmonary mesothelium. In other organs, mesothelial cells undergo epithelial-mesenchymal transition and contribute to organ stromal cells. The contribution of pulmonary mesothelial cells (PMCs) to the developing lung has been evaluated with differing conclusions. PMCs have also been indirectly implicated in lung fibrosis in the progressive, fatal lung disease idiopathic pulmonary fibrosis. We used fetal or postnatal genetic pulse labeling of PMCs to assess their fate in murine development, normal lung homeostasis, and models of pulmonary fibrosis. We found that most fetal PMC-derived mesenchymal cells (PMCDCs) expressed markers of pericytes and fibroblasts, only a small minority expressed smooth muscle markers, and none expressed endothelial cell markers. Postnatal PMCs did not contribute to lung mesenchyme during normal lung homeostasis or in models of lung fibrosis. However, fetal PMCDCs were abundant and actively proliferating within fibrotic regions in lung fibrosis models, suggesting that they actively participate in the fibrotic process. These data clarify the role of fetal and postnatal PMCDCs in lung development and disease.

Commensal bacteria-dependent select expression of CXCL2 contributes to periodontal tissue homeostasis.

PubMed

Zenobia, Camille; Luo, Xiao Long; Hashim, Ahmed; Abe, Toshiharu; Jin, Lijian; Chang, Yucheng; Jin, Zhi Chao; Sun, Jian Xun; Hajishengallis, George; Curtis, Mike A; Darveau, Richard P

2013-08-01

The oral and intestinal host tissues both carry a heavy microbial burden. Although commensal bacteria contribute to healthy intestinal tissue structure and function, their contribution to oral health is poorly understood. A crucial component of periodontal health is the recruitment of neutrophils to periodontal tissue. To elucidate this process, gingival tissues of specific-pathogen-free and germ-free wild-type mice and CXCR2KO and MyD88KO mice were examined for quantitative analysis of neutrophils and CXCR2 chemoattractants (CXCL1, CXCL2). We show that the recruitment of neutrophils to the gingival tissue does not require commensal bacterial colonization but is entirely dependent on CXCR2 expression. Strikingly, however, commensal bacteria selectively upregulate the expression of CXCL2, but not CXCL1, in a MyD88-dependent way that correlates with increased neutrophil recruitment as compared with germ-free conditions. This is the first evidence that the selective use of chemokine receptor ligands contributes to neutrophil homing to healthy periodontal tissue. © 2013 John Wiley & Sons Ltd.

Induction of type 1 iodothyronine deiodinase expression inhibits proliferation and migration of renal cancer cells.

PubMed

Poplawski, Piotr; Rybicka, Beata; Boguslawska, Joanna; Rodzik, Katarzyna; Visser, Theo J; Nauman, Alicja; Piekielko-Witkowska, Agnieszka

2017-02-15

Type 1 iodothyronine deiodinase (DIO1) regulates peripheral metabolism of thyroid hormones that control cellular proliferation, differentiation and metabolism. The significance of DIO1 in cancer is unknown. In this study we hypothesized that diminished expression of DIO1, observed in renal cancer, contributes to the carcinogenic process in the kidney. Here, we demonstrate that ectopic expression of DIO1 in renal cancer cells changes the expression of genes controlling cell cycle, including cyclin E1 and E2F5, and results in inhibition of proliferation. The expression of genes encoding collagens (COL1A1, COL4A2, COL5A1), integrins (ITGA4, ITGA5, ITGB3) and transforming growth factor-β-induced (TGFBI) is significantly altered in renal cancer cells with induced expression of DIO1. Finally, we show that overexpression of DIO1 inhibits migration of renal cancer cells. In conclusion, we demonstrate for the first time that loss of DIO1 contributes to renal carcinogenesis and that its induced expression protects cells against cancerous proliferation and migration. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Peripheral inflammation induces up-regulation of TRPV2 expression in rat DRG.

PubMed

Shimosato, Goshun; Amaya, Fumimasa; Ueda, Masashi; Tanaka, Yoshifumi; Decosterd, Isabelle; Tanaka, Masaki

2005-12-15

The transient receptor potential vanilloid subfamily member 2 (TRPV2) is a cation channel activated by temperatures above 52 degrees C. To analyze the contribution of TRPV2 to the development of inflammation-induced hyperalgesia, the expression of TRPV2 in primary sensory neurons was analyzed after intraplantar injection of complete Freund's adjuvant (CFA). Using specific antibodies, an increase in TRPV2-expressing neurons was identified after inflammation. TRPV2 expression is concentrated in a subset of medium-sized dorsal root ganglion neurons, independent of transient receptor potential vanilloid subfamily member 1 (TRPV1) expression. A similar distribution of TRPV2 was observed after inflammation. Intraplantar injection of nerve growth factor increased TRPV1 expression but not TRPV2, suggesting that induction of TRPV2 expression is driven by a mechanism distinct from that for TRPV1. Heat hyperalgesia assessment after chemical desensitization of TRPV1 by resiniferatoxin demonstrates a possible role for TRPV2 in inflammation at high temperatures (>56 degrees C). These results suggest that TRPV2 upregulation contributes to peripheral sensitization during inflammation and is responsible for pain hypersensitivity to noxious high temperature stimuli.

Increased expression of Interleukin-13 and connective tissue growth factor, and their potential roles during foreign body encapsulation of subcutaneous implants.

PubMed

Ward, W Kenneth; Li, Allen G; Siddiqui, Yasmin; Federiuk, Isaac F; Wang, Xiao-Jing

2008-01-01

The purpose of this study was to better understand whether interleukin-13 (IL-13) and connective tissue growth factor (CTGF) are highly expressed during foreign body encapsulation of subcutaneous devices. Mock biosensors were implanted into rats for three lengths of time (7-, 21- and 48-55 days) to address different stages of the foreign body response. Using quantitative real-time PCR and immunofluorescence, the expression of IL13, CTGF, collagen 1, decorin and fibronectin were measured in this tissue. IL-13, a product of Th2 cells, was highly expressed at all time points, with greatest expression at day 21. The IL-13 expression was paralleled by increased presence of T-cells at all time points. CTGF was also found to be more highly expressed in foreign body tissue than in controls. Collagen and decorin were highly expressed at the middle and later stages. Given the increased expression of IL-13 and CTGF in foreign body tissue, and their roles in other fibrotic disorders, these cytokines may well contribute to the formation of the foreign body capsule. Since the peak gene expression of IL-13 occurred later than the previously-reported TGFbeta expression peak, IL-13 is probably not the major stimulus to TGFbeta expression during foreign body encapsulation and may contribute to fibrosis independently.

Multivariate Pattern Classification of Facial Expressions Based on Large-Scale Functional Connectivity.

PubMed

Liang, Yin; Liu, Baolin; Li, Xianglin; Wang, Peiyuan

2018-01-01

It is an important question how human beings achieve efficient recognition of others' facial expressions in cognitive neuroscience, and it has been identified that specific cortical regions show preferential activation to facial expressions in previous studies. However, the potential contributions of the connectivity patterns in the processing of facial expressions remained unclear. The present functional magnetic resonance imaging (fMRI) study explored whether facial expressions could be decoded from the functional connectivity (FC) patterns using multivariate pattern analysis combined with machine learning algorithms (fcMVPA). We employed a block design experiment and collected neural activities while participants viewed facial expressions of six basic emotions (anger, disgust, fear, joy, sadness, and surprise). Both static and dynamic expression stimuli were included in our study. A behavioral experiment after scanning confirmed the validity of the facial stimuli presented during the fMRI experiment with classification accuracies and emotional intensities. We obtained whole-brain FC patterns for each facial expression and found that both static and dynamic facial expressions could be successfully decoded from the FC patterns. Moreover, we identified the expression-discriminative networks for the static and dynamic facial expressions, which span beyond the conventional face-selective areas. Overall, these results reveal that large-scale FC patterns may also contain rich expression information to accurately decode facial expressions, suggesting a novel mechanism, which includes general interactions between distributed brain regions, and that contributes to the human facial expression recognition.

Multivariate Pattern Classification of Facial Expressions Based on Large-Scale Functional Connectivity

PubMed Central

Liang, Yin; Liu, Baolin; Li, Xianglin; Wang, Peiyuan

2018-01-01

It is an important question how human beings achieve efficient recognition of others’ facial expressions in cognitive neuroscience, and it has been identified that specific cortical regions show preferential activation to facial expressions in previous studies. However, the potential contributions of the connectivity patterns in the processing of facial expressions remained unclear. The present functional magnetic resonance imaging (fMRI) study explored whether facial expressions could be decoded from the functional connectivity (FC) patterns using multivariate pattern analysis combined with machine learning algorithms (fcMVPA). We employed a block design experiment and collected neural activities while participants viewed facial expressions of six basic emotions (anger, disgust, fear, joy, sadness, and surprise). Both static and dynamic expression stimuli were included in our study. A behavioral experiment after scanning confirmed the validity of the facial stimuli presented during the fMRI experiment with classification accuracies and emotional intensities. We obtained whole-brain FC patterns for each facial expression and found that both static and dynamic facial expressions could be successfully decoded from the FC patterns. Moreover, we identified the expression-discriminative networks for the static and dynamic facial expressions, which span beyond the conventional face-selective areas. Overall, these results reveal that large-scale FC patterns may also contain rich expression information to accurately decode facial expressions, suggesting a novel mechanism, which includes general interactions between distributed brain regions, and that contributes to the human facial expression recognition. PMID:29615882

Expression profiling of snoRNAs in normal hematopoiesis and AML

PubMed Central

Warner, Wayne A.; Spencer, David H.; Trissal, Maria; White, Brian S.; Helton, Nichole; Ley, Timothy J.

2018-01-01

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosome RNAs, respectively. We optimized a next-generation sequencing approach and a custom analysis pipeline to identify and quantify expression of snoRNAs in acute myeloid leukemia (AML) and normal hematopoietic cell populations. We show that snoRNAs are expressed in a lineage- and development-specific fashion during hematopoiesis. The most striking examples involve snoRNAs located in 2 imprinted loci, which are highly expressed in hematopoietic progenitors and downregulated during myeloid differentiation. Although most snoRNAs are expressed at similar levels in AML cells compared with CD34+, a subset of snoRNAs showed consistent differential expression, with the great majority of these being decreased in the AML samples. Analysis of host gene expression, splicing patterns, and whole-genome sequence data for mutational events did not identify transcriptional patterns or genetic alterations that account for these expression differences. These data provide a comprehensive analysis of the snoRNA transcriptome in normal and leukemic cells and should be helpful in the design of studies to define the contribution of snoRNAs to normal and malignant hematopoiesis. PMID:29365324

MDM2 is an important prognostic and predictive factor for platin-pemetrexed therapy in malignant pleural mesotheliomas and deregulation of P14/ARF (encoded by CDKN2A) seems to contribute to an MDM2-driven inactivation of P53.

PubMed

Walter, R F H; Mairinger, F D; Ting, S; Vollbrecht, C; Mairinger, T; Theegarten, D; Christoph, D C; Schmid, K W; Wohlschlaeger, J

2015-03-03

Malignant pleural mesothelioma (MPM) is a highly aggressive tumour that is first-line treated with a combination of cisplatin and pemetrexed. Until now, predictive and prognostic biomarkers are lacking, making it a non-tailored therapy regimen with unknown outcome. P53 is frequently inactivated in MPM, but mutations are extremely rare. MDM2 and P14/ARF are upstream regulators of P53 that may contribute to P53 inactivation. A total of 72 MPM patients were investigated. MDM2 immunoexpression was assessed in 65 patients. MDM2 and P14/ARF mRNA expression was analysed in 48 patients of the overall collective. The expression results were correlated to overall survival (OS) and progression-free survival (PFS). OS and PFS correlated highly significantly with MDM2 mRNA and protein expression, showing a dismal prognosis for patients with elevated MDM2 expression (for OS: Score (logrank) test: P⩽0.002, and for PFS: Score (logrank) test; P<0.007). MDM2 was identified as robust prognostic and predictive biomarker for MPM on the mRNA and protein level. P14/ARF mRNA expression reached no statistical significance, but Kaplan-Meier curves distinguished patients with low P14/ARF expression and hence shorter survival from patients with higher expression and prolonged survival. MDM2 is a prognostic and predictive marker for a platin-pemetrexed therapy of patients with MPMs. Downregulation of P14/ARF expression seems to contribute to MDM2-overexpression-mediated P53 inactivation in MPM patients.

The paratenon contributes to scleraxis-expressing cells during patellar tendon healing.

PubMed

Dyment, Nathaniel A; Liu, Chia-Feng; Kazemi, Namdar; Aschbacher-Smith, Lindsey E; Kenter, Keith; Breidenbach, Andrew P; Shearn, Jason T; Wylie, Christopher; Rowe, David W; Butler, David L

2013-01-01

The origin of cells that contribute to tendon healing, specifically extrinsic epitenon/paratenon cells vs. internal tendon fibroblasts, is still debated. The purpose of this study is to determine the location and phenotype of cells that contribute to healing of a central patellar tendon defect injury in the mouse. Normal adult patellar tendon consists of scleraxis-expressing (Scx) tendon fibroblasts situated among aligned collagen fibrils. The tendon body is surrounded by paratenon, which consists of a thin layer of cells that do not express Scx and collagen fibers oriented circumferentially around the tendon. At 3 days following injury, the paratenon thickens as cells within the paratenon proliferate and begin producing tenascin-C and fibromodulin. These cells migrate toward the defect site and express scleraxis and smooth muscle actin alpha by day 7. The thickened paratenon tissue eventually bridges the tendon defect by day 14. Similarly, cells within the periphery of the adjacent tendon struts express these markers and become disorganized. Cells within the defect region show increased expression of fibrillar collagens (Col1a1 and Col3a1) but decreased expression of tenogenic transcription factors (scleraxis and mohawk homeobox) and collagen assembly genes (fibromodulin and decorin). By contrast, early growth response 1 and 2 are upregulated in these tissues along with tenascin-C. These results suggest that paratenon cells, which normally do not express Scx, respond to injury by turning on Scx and assembling matrix to bridge the defect. Future studies are needed to determine the signaling pathways that drive these cells and whether they are capable of producing a functional tendon matrix. Understanding this process may guide tissue engineering strategies in the future by stimulating these cells to improve tendon repair.

Taking Another Look: Expression through Form.

ERIC Educational Resources Information Center

Townley, Mary Ross

1984-01-01

Comparing the works of two artists whose subject is similar but whose formal qualities are different helps students understand how the use of formal qualities contributes to expression. Having students compare the works of different composers as well as different visual artists helps students understand how expression is achieved. (IS)

26 CFR 1.401(l)-1 - Permitted disparity in employer-provided contributions or benefits.

Code of Federal Regulations, 2014 CFR

2014-04-01

... with respect to an employee's average annual compensation at or below the integration level (expressed... or below the integration level (expressed as a percentage of such plan year compensation). (5... plan with respect to an employee's average annual compensation above the integration level (expressed...

A simple expression for the cold compression curve.

NASA Astrophysics Data System (ADS)

Čelebonović, V.

1996-10-01

The aim of this contribution is to present expressions for the bulk modulus of a material and its pressure derivative obtained by using the semi-classical theory of dense matter proposed by P. Savić and R. Kašanin. Some possibilities for the application of these expressions are briefly discussed.

Using differential gene expression to study Entamoeba histolytica pathogenesis

PubMed Central

Gilchrist, Carol A.; Petri, William A.

2010-01-01

The release of the Entamoeba histolytica genome has facilitated the development of techniques to survey rapidly and to relate gene expression with biology. The association and potential contribution of differential gene expression to the life cycle and the virulence of this protozoan parasite of humans are reviewed here. PMID:19217826

Protein-disulfide Isomerase Regulates the Thyroid Hormone Receptor-mediated Gene Expression via Redox Factor-1 through Thiol Reduction-Oxidation*

PubMed Central

Hashimoto, Shoko; Imaoka, Susumu

2013-01-01

Protein-disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase that regulates the redox state of proteins. We previously found that overexpression of PDI in rat pituitary tumor (GH3) cells suppresses 3,3′,5-triiodothyronine (T3)-stimulated growth hormone (GH) expression, suggesting the contribution of PDI to the T3-mediated gene expression via thyroid hormone receptor (TR). In the present study, we have clarified the mechanism of regulation by which TR function is regulated by PDI. Overexpression of wild-type but not redox-inactive mutant PDI suppressed the T3-induced GH expression, suggesting that the redox activity of PDI contributes to the suppression of GH. We considered that PDI regulates the redox state of the TR and focused on redox factor-1 (Ref-1) as a mediator of the redox regulation of TR by PDI. Interaction between Ref-1 and TRβ1 was detected. Overexpression of wild-type but not C64S Ref-1 facilitated the GH expression, suggesting that redox activity of Cys-64 in Ref-1 is involved in the TR-mediated gene expression. Moreover, PDI interacted with Ref-1 and changed the redox state of Ref-1, suggesting that PDI controls the redox state of Ref-1. Our studies suggested that Ref-1 contributes to TR-mediated gene expression and that the redox state of Ref-1 is regulated by PDI. Redox regulation of PDI via Ref-1 is a new aspect of PDI function. PMID:23148211

Linear enamel hypoplasia as an indicator of physiological stress in great apes: reviewing the evidence in light of enamel growth variation.

PubMed

Guatelli-Steinberg, Debbie; Ferrell, Rebecca J; Spence, Jennifer

2012-06-01

Physiological stress, such as malnutrition or illness, can disrupt normal enamel growth, resulting in linear enamel hypoplasias (LEHs). Although ecological factors may contribute to LEH expression, other factors, such as surface abrasion and enamel growth variables, are also likely to be involved. Attention to these other factors is necessary before we can begin to understand what LEH might signify in terms of ecological sources of physiological stress in non-human primates. This study focuses on assessing the contribution of these other factors to variation in LEH expression within and across great ape taxa. Here, we present LEH data from unabraded crown regions in samples of seven great ape species. We analyze these data with respect to lateral enamel formation time and the angles that striae of Retzius make with the enamel surface, as these variables are expected to affect variation in LEH expression. We find that although the duration of enamel formation is associated with sex differences in LEH expression, it is not clearly related to taxonomic variation in LEH expression, and does not explain the low frequency of LEH in mountain gorillas found in this and a previous study. Our data on striae of Retzius angles suggest that these influence LEH expression along the tooth crown and may contribute to the consistently high frequencies of LEH seen in Pongo in this and previous studies. We suggest that future work aimed at understanding species variation in these angles is crucial to evaluating taxonomic patterns of LEH expression in great apes. Copyright © 2012 Wiley Periodicals, Inc.

EGR-1 Expression in Catecholamine-synthesizing Neurons Reflects Auditory Learning and Correlates with Responses in Auditory Processing Areas.

PubMed

Dai, Jennifer B; Chen, Yining; Sakata, Jon T

2018-05-21

Distinguishing between familiar and unfamiliar individuals is an important task that shapes the expression of social behavior. As such, identifying the neural populations involved in processing and learning the sensory attributes of individuals is important for understanding mechanisms of behavior. Catecholamine-synthesizing neurons have been implicated in sensory processing, but relatively little is known about their contribution to auditory learning and processing across various vertebrate taxa. Here we investigated the extent to which immediate early gene expression in catecholaminergic circuitry reflects information about the familiarity of social signals and predicts immediate early gene expression in sensory processing areas in songbirds. We found that male zebra finches readily learned to differentiate between familiar and unfamiliar acoustic signals ('songs') and that playback of familiar songs led to fewer catecholaminergic neurons in the locus coeruleus (but not in the ventral tegmental area, substantia nigra, or periaqueductal gray) expressing the immediate early gene, EGR-1, than playback of unfamiliar songs. The pattern of EGR-1 expression in the locus coeruleus was similar to that observed in two auditory processing areas implicated in auditory learning and memory, namely the caudomedial nidopallium (NCM) and the caudal medial mesopallium (CMM), suggesting a contribution of catecholamines to sensory processing. Consistent with this, the pattern of catecholaminergic innervation onto auditory neurons co-varied with the degree to which song playback affected the relative intensity of EGR-1 expression. Together, our data support the contention that catecholamines like norepinephrine contribute to social recognition and the processing of social information. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Gene expression changes in human small airway epithelial cells exposed to Delta9-tetrahydrocannabinol.

PubMed

Sarafian, Theodore; Habib, Nancy; Mao, Jenny T; Tsu, I-Hsien; Yamamoto, Mitsuko L; Hsu, Erin; Tashkin, Donald P; Roth, Michael D

2005-08-14

Marijuana smoking is associated with inflammation, cellular atypia, and molecular dysregulation of the tracheobronchial epithelium. While marijuana smoke shares many components in common with tobacco, it also contains a high concentration of Delta9-tetrahydrocannabinol (THC). The potential contribution of THC to airway injury was assessed by exposing primary cultures of human small airway epithelial (SAE) cells to THC (0.1-10.0 microg/ml) for either 1 day or 7 days. THC induced a time- and concentration-dependent decrease in cell viability, ATP level, and mitochondrial membrane potential. Using a targeted gene expression array, we observed acute changes (24 h) in the expression of mRNA for caspase-8, catalase, Bax, early growth response-1, cytochrome P4501A1 (CYP1A1), metallothionein 1A, PLAB, and heat shock factor 1 (HSF1). After 7 days of exposure, decrease in expression of mRNA for heat shock proteins (HSPs) and the pro-apoptotic protein Bax was observed, while expression of GADD45A, IL-1A, CYP1A1, and PTGS-2 increased significantly. These findings suggest a contribution of THC to DNA damage, inflammation, and alterations in apoptosis. Treatment with selected prototypical toxicants, 2,3,7,8-tetrachlorodibenznzo-p-dioxin (TCDD) and carbonyl cyanide-p-(trifluoramethoxy)-phenyl hydrazone (FCCP), produced partially overlapping gene expression profiles suggesting some similarity in mechanism of action with THC. THC, delivered as a component of marijuana smoke, may induce a profile of gene expression that contributes to the pulmonary pathology associated with marijuana use.

Claudin 5 Expression in Mouse Seminiferous Epithelium Is Dependent upon the Transcription Factor Ets Variant 5 and Contributes to Blood-Testis Barrier Function1

PubMed Central

Morrow, Carla M.K.; Tyagi, Gaurav; Simon, Liz; Carnes, Kay; Murphy, Kenneth M.; Cooke, Paul S.; Hofmann, Marie-Claude C.; Hess, Rex A.

2009-01-01

The blood-testis barrier (BTB) is formed by tight junctions between Sertoli cells. Results of previous studies suggested that the barrier is deficient in ets variant 5 (ETV5) gene-deleted mice; therefore, microarray data were examined for changes in tight junction-associated genes. The tight junctional protein claudin 5 (CLDN5) was decreased in testes of 8-day-old Etv5−/− pups. The study reported herein examined the expression of CLDN5 in wild-type (WT) and Etv5−/− mice and evaluated its contribution to BTB function. CLDN5 protein expression was evaluated in 8-day-old WT and Etv5−/− and adult WT, Etv5−/−, and W/Wv testes by immunohistochemistry and in 8-day-old WT Sertoli cell-enriched and germ cell-enriched fractions by immunocytochemistry. Cldn5 mRNA expression was evaluated in 0- to 20-day-old and adult WT mice and in 8-day-old and adult Etv5−/− mice via quantitative PCR. Tracer studies were performed in adult WT, Etv5−/−, and W/Wv mice. The results indicate the following: 1) CLDN5 was expressed in Sertoli cells, spermatogonia, and preleptotene spermatocytes. 2) Seminiferous epithelial CLDN5 expression depended upon both the presence of germ cells and ETV5. 3) CLDN5 expression in testicular vascular endothelium and rete testis epithelium was ETV5 independent. 4) Cldn5 mRNA expression increased in the testes of juvenile mice at the time of BTB formation. 5) Testes of Etv5−/− and W/Wv mice, which are both deficient in seminiferous epithelial CLDN5 expression, had biotin tracer leakage from the interstitial space into the seminiferous tubule lumen. In conclusion, CLDN5 is expressed in the seminiferous epithelium, appears to be regulated by multiple influences, and contributes to BTB function. PMID:19571261

Sequences 5' to translation start regulate expression of petunia rbcS genes.

PubMed Central

Dean, C; Favreau, M; Bedbrook, J; Dunsmuir, P

1989-01-01

The promoter sequences that contribute to quantitative differences in expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase have been characterized. The promoter regions of the two most abundantly expressed petunia rbcS genes, SSU301 and SSU611, show sequence similarity not present in other rbcS genes. We investigated the significance of these and other sequences by adding specific regions from the SSU301 promoter (the most strongly expressed gene) to equivalent regions in the SSU911 promoter (the least strongly expressed gene) and assaying the expression of the fusions in transgenic tobacco plants. In this way, we characterized an SSU301 promoter region (either from -285 to -178 or -291 to -204) which, when added to SSU911, in either orientation, increased SSU911 expression 25-fold. This increase was equivalent to that caused by addition of the entire SSU301 5'-flanking region. Replacement of SSU911 promoter sequences between -198 and the start codon with sequences from the equivalent region of SSU301 did not increase SSU911 expression significantly. The -291 to -204 SSU301 promoter fragment contributes significantly to quantitative differences in expression between the petunia rbcS genes. PMID:2535543

Effect of Body Composition Methodology on Heritability Estimation of Body Fatness

PubMed Central

Elder, Sonya J.; Roberts, Susan B.; McCrory, Megan A.; Das, Sai Krupa; Fuss, Paul J.; Pittas, Anastassios G.; Greenberg, Andrew S.; Heymsfield, Steven B.; Dawson-Hughes, Bess; Bouchard, Thomas J.; Saltzman, Edward; Neale, Michael C.

2014-01-01

Heritability estimates of human body fatness vary widely and the contribution of body composition methodology to this variability is unknown. The effect of body composition methodology on estimations of genetic and environmental contributions to body fatness variation was examined in 78 adult male and female monozygotic twin pairs reared apart or together. Body composition was assessed by six methods – body mass index (BMI), dual energy x-ray absorptiometry (DXA), underwater weighing (UWW), total body water (TBW), bioelectric impedance (BIA), and skinfold thickness. Body fatness was expressed as percent body fat, fat mass, and fat mass/height2 to assess the effect of body fatness expression on heritability estimates. Model-fitting multivariate analyses were used to assess the genetic and environmental components of variance. Mean BMI was 24.5 kg/m2 (range of 17.8–43.4 kg/m2). There was a significant effect of body composition methodology (p<0.001) on heritability estimates, with UWW giving the highest estimate (69%) and BIA giving the lowest estimate (47%) for fat mass/height2. Expression of body fatness as percent body fat resulted in significantly higher heritability estimates (on average 10.3% higher) compared to expression as fat mass/height2 (p=0.015). DXA and TBW methods expressing body fatness as fat mass/height2 gave the least biased heritability assessments, based on the small contribution of specific genetic factors to their genetic variance. A model combining DXA and TBW methods resulted in a relatively low FM/ht2 heritability estimate of 60%, and significant contributions of common and unique environmental factors (22% and 18%, respectively). The body fatness heritability estimate of 60% indicates a smaller contribution of genetic variance to total variance than many previous studies using less powerful research designs have indicated. The results also highlight the importance of environmental factors and possibly genotype by environmental interactions in the etiology of weight gain and the obesity epidemic. PMID:25067962

Maintenance and Loss of Duplicated Genes by Dosage Subfunctionalization.

PubMed

Gout, Jean-Francois; Lynch, Michael

2015-08-01

Whole-genome duplications (WGDs) have contributed to gene-repertoire enrichment in many eukaryotic lineages. However, most duplicated genes are eventually lost and it is still unclear why some duplicated genes are evolutionary successful whereas others quickly turn to pseudogenes. Here, we show that dosage constraints are major factors opposing post-WGD gene loss in several Paramecium species that share a common ancestral WGD. We propose a model where a majority of WGD-derived duplicates preserve their ancestral function and are retained to produce enough of the proteins performing this same ancestral function. Under this model, the expression level of individual duplicated genes can evolve neutrally as long as they maintain a roughly constant summed expression, and this allows random genetic drift toward uneven contributions of the two copies to total expression. Our analysis suggests that once a high level of imbalance is reached, which can require substantial lengths of time, the copy with the lowest expression level contributes a small enough fraction of the total expression that selection no longer opposes its loss. Extension of our analysis to yeast species sharing a common ancestral WGD yields similar results, suggesting that duplicated-gene retention for dosage constraints followed by divergence in expression level and eventual deterministic gene loss might be a universal feature of post-WGD evolution. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

COX2 Inhibition Reduces Aortic Valve Calcification In Vivo

PubMed Central

Wirrig, Elaine E.; Gomez, M. Victoria; Hinton, Robert B.; Yutzey, Katherine E.

2016-01-01

Objective Calcific aortic valve disease (CAVD) is a significant cause of morbidity and mortality, which affects approximately 1% of the US population and is characterized by calcific nodule formation and stenosis of the valve. Klotho-deficient mice were used to study the molecular mechanisms of CAVD as they develop robust aortic valve (AoV) calcification. Through microarray analysis of AoV tissues from klotho-deficient and wild type mice, increased expression of the gene encoding cyclooxygenase 2/COX2 (Ptgs2) was found. COX2 activity contributes to bone differentiation and homeostasis, thus the contribution of COX2 activity to AoV calcification was assessed. Approach and Results In klotho-deficient mice, COX2 expression is increased throughout regions of valve calcification and is induced in the valvular interstitial cells (VICs) prior to calcification formation. Similarly, COX2 expression is increased in human diseased AoVs. Treatment of cultured porcine aortic VICs with osteogenic media induces bone marker gene expression and calcification in vitro, which is blocked by inhibition of COX2 activity. In vivo, genetic loss of function of COX2 cyclooxygenase activity partially rescues AoV calcification in klotho-deficient mice. Moreover, pharmacologic inhibition of COX2 activity in klotho-deficient mice via celecoxib-containing diet reduces AoV calcification and blocks osteogenic gene expression. Conclusions COX2 expression is upregulated in CAVD and its activity contributes to osteogenic gene induction and valve calcification in vitro and in vivo. PMID:25722432

The evolution of duplicate gene expression in mammalian organs

PubMed Central

Guschanski, Katerina; Warnefors, Maria; Kaessmann, Henrik

2017-01-01

Gene duplications generate genomic raw material that allows the emergence of novel functions, likely facilitating adaptive evolutionary innovations. However, global assessments of the functional and evolutionary relevance of duplicate genes in mammals were until recently limited by the lack of appropriate comparative data. Here, we report a large-scale study of the expression evolution of DNA-based functional gene duplicates in three major mammalian lineages (placental mammals, marsupials, egg-laying monotremes) and birds, on the basis of RNA sequencing (RNA-seq) data from nine species and eight organs. We observe dynamic changes in tissue expression preference of paralogs with different duplication ages, suggesting differential contribution of paralogs to specific organ functions during vertebrate evolution. Specifically, we show that paralogs that emerged in the common ancestor of bony vertebrates are enriched for genes with brain-specific expression and provide evidence for differential forces underlying the preferential emergence of young testis- and liver-specific expressed genes. Further analyses uncovered that the overall spatial expression profiles of gene families tend to be conserved, with several exceptions of pronounced tissue specificity shifts among lineage-specific gene family expansions. Finally, we trace new lineage-specific genes that may have contributed to the specific biology of mammalian organs, including the little-studied placenta. Overall, our study provides novel and taxonomically broad evidence for the differential contribution of duplicate genes to tissue-specific transcriptomes and for their importance for the phenotypic evolution of vertebrates. PMID:28743766

Altered Ca2+ Kinetics Associated with α-Actinin-3 Deficiency May Explain Positive Selection for ACTN3 Null Allele in Human Evolution

PubMed Central

Houweling, Peter J.; Quinlan, Kate G. R.; Murphy, Robyn; Wagner, Sören; Friedrich, Oliver; North, Kathryn N.

2015-01-01

Over 1.5 billion people lack the skeletal muscle fast-twitch fibre protein α-actinin-3 due to homozygosity for a common null polymorphism (R577X) in the ACTN3 gene. α-Actinin-3 deficiency is detrimental to sprint performance in elite athletes and beneficial to endurance activities. In the human genome, it is very difficult to find single-gene loss-of-function variants that bear signatures of positive selection, yet intriguingly, the ACTN3 null variant has undergone strong positive selection during recent evolution, appearing to provide a survival advantage where food resources are scarce and climate is cold. We have previously demonstrated that α-actinin-3 deficiency in the Actn3 KO mouse results in a shift in fast-twitch fibres towards oxidative metabolism, which would be more “energy efficient” in famine, and beneficial to endurance performance. Prolonged exposure to cold can also induce changes in skeletal muscle similar to those observed with endurance training, and changes in Ca2+ handling by the sarcoplasmic reticulum (SR) are a key factor underlying these adaptations. On this basis, we explored the effects of α-actinin-3 deficiency on Ca2+ kinetics in single flexor digitorum brevis muscle fibres from Actn3 KO mice, using the Ca2+-sensitive dye fura-2. Compared to wild-type, fibres of Actn3 KO mice showed: (i) an increased rate of decay of the twitch transient; (ii) a fourfold increase in the rate of SR Ca2+ leak; (iii) a threefold increase in the rate of SR Ca2+ pumping; and (iv) enhanced maintenance of tetanic Ca2+ during fatigue. The SR Ca2+ pump, SERCA1, and the Ca2+-binding proteins, calsequestrin and sarcalumenin, showed markedly increased expression in muscles of KO mice. Together, these changes in Ca2+ handling in the absence of α-actinin-3 are consistent with cold acclimatisation and thermogenesis, and offer an additional explanation for the positive selection of the ACTN3 577X null allele in populations living in cold environments during recent evolution. PMID:25590636

Imaging the secretory compartments involved in the intracellular traffic of CHS-4, a class IV chitin synthase, in Neurospora crassa.

PubMed

Rico-Ramírez, Adriana M; Roberson, Robert W; Riquelme, Meritxell

2018-03-27

In Neurospora crassa hyphae the localization of all seven chitin synthases (CHSs) at the Spitzenkörper (Spk) and at developing septa has been well analyzed. Hitherto, the mechanisms of CHSs traffic and sorting from synthesis to delivery sites remain largely unexplored. In Saccharomyces cerevisiae exit of Chs3p from the endoplasmic reticulum (ER) requires chaperone Chs7p. Here, we analyzed the role of CSE-7, N. crassa Chs7p orthologue in the biogenesis of CHS-4 (orthologue of Chs3p). In a N. crassa Δcse-7 mutant, CHS-4-GFP no longer accumulated at the Spk and septa. Instead, fluorescence was retained in hyphal subapical regions in an extensive network of elongated cisternae (NEC) referred to previously as tubular vacuoles. In a complemented strain expressing a copy of cse-7 the localization of CHS-4-GFP at the Spk and septa was restored, providing evidence that CSE-7 is necessary for the localization of CHS-4 at hyphal tips and septa. CSE-7 was revealed at delimited regions of the ER at the immediacies of nuclei, at the NEC, and remarkably also at septa and the Spk. The organization of the NEC was dependent on the cytoskeleton. SEC-63, an extensively used ER marker, and NCA-1, a SERCA-type ATPase previously localized at the nuclear envelope, were used as markers to discern the nature of the membranes containing CSE-7. Both SEC-63 and NCA-1 were found at the nuclear envelope, but also at regions of the NEC. However, at the NEC only NCA-1 co-localized extensively with CSE-7. Observations by transmission electron microscopy revealed abundant rough ER sheets and distinct electron translucent smooth flattened cisternae, which could correspond collectively to the NEC, thorough the subapical cytoplasm. This study identifies CSE-7 as the putative ER receptor for its cognate cargo, the polytopic membrane protein CHS-4, and elucidates the complexity of the ER system in filamentous fungi. Copyright © 2018. Published by Elsevier Inc.

Effect of TGFβ on calcium signaling in megakaryocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Jing; Schmid, Evi; Department of Pediatric Surgery and Pediatric Urology, University Children's Hospital Tübingen, Tübingen

2015-05-22

TGFβ is a powerful regulator of megakaryocyte maturation and platelet formation. As previously shown for other cell types, TGFβ may up-regulate the expression of the serum & glucocorticoid inducible kinase SGK1, an effect requiring p38 kinase. SGK1 has in turn recently been shown to participate in the regulation of cytosolic Ca{sup 2+} activity ([Ca{sup 2+}]{sub i}) in megakaryocytes and platelets. SGK1 phosphorylates the IκB kinase (IKKα/β), which in turn phosphorylates the inhibitor protein IκBα resulting in nuclear translocation of nuclear factor NFκB. Genes up-regulated by NFκB include Orai1, the pore forming ion channel subunit accomplishing store operated Ca{sup 2+} entrymore » (SOCE). The present study explored whether TGFβ influences Ca{sup 2+} signaling in megakaryocytes. [Ca{sup 2+}]{sub i} was determined by Fura-2 fluorescence and SOCE from the increase of [Ca{sup 2+}]{sub i} following re-addition of extracellular Ca{sup 2+} after store depletion by removal of extracellular Ca{sup 2+} and inhibition of the sarcoendoplasmatic Ca{sup 2+} ATPase (SERCA) with thapsigargin (1 μM). As a result, TGFβ (60 ng, 24 h) increased SOCE, an effect significantly blunted by p38 kinase inhibitor Skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) and NFκB inhibitor wogonin (100 μM). In conclusion, TGFβ is a powerful regulator of store operated Ca{sup 2+} entry into megakaryocytes, an effect mediated by a signaling cascade involving p38 kinase, SGK1 and NFκB. - Highlights: • TGFβ up-regulates store operated Ca{sup 2+} entry (SOCE) in megakaryocytes. • The effect of TGFβ on SOCE is blunted by p38 kinase inhibitor Skepinone-L. • The effect of TGFβ on SOCE is virtually abrogated by SGK1 inhibitor EMD638683. • The effect of TGFβ on SOCE is almost abolished by NFκB inhibitor wogonin. • The effect of TGFβ is expected to enhance sensitivity of platelets to activation.« less

Negative Regulation of NF-κB by the ING4 Tumor Suppressor in Breast Cancer

PubMed Central

Byron, Sara A.; Min, Elizabeth; Thal, Tanya S.; Hostetter, Galen; Watanabe, Aprill T.; Azorsa, David O.; Little, Tanya H.; Tapia, Coya; Kim, Suwon

2012-01-01

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer. PMID:23056468

Transcriptional over-expression of chloride intracellular channels 3 and 4 in malignant pleural mesothelioma.

PubMed

Tasiopoulou, Vasiliki; Magouliotis, Dimitrios; Solenov, Evgeniy I; Vavougios, Georgios; Molyvdas, Paschalis-Adam; Gourgoulianis, Konstantinos I; Hatzoglou, Chrissi; Zarogiannis, Sotirios G

2015-12-01

Chloride Intracellular Channels (CLICs) are contributing to the regulation of multiple cellular functions. CLICs have been found over-expressed in several malignancies, and therefore they are currently considered as potential drug targets. The goal of our study was to assess the gene expression levels of the CLIC's 1-6 in malignant pleural mesothelioma (MPM) as compared to controls. We used gene expression data from a publicly available microarray dataset comparing MPM versus healthy tissue in order to investigate the differential expression profile of CLIC 1-6. False discovery rates were calculated and the interactome of the significantly differentially expressed CLICs was constructed and Functional Enrichment Analysis for Gene Ontologies (FEAGO) was performed. In MPM, the gene expressions of CLIC3 and CLIC4 were significantly increased compared to controls (p=0.001 and p<0.001 respectively). A significant positive correlation between the gene expressions of CLIC3 and CLIC4 (p=0.0008 and Pearson's r=0.51) was found. Deming regression analysis provided an association equation between the CLIC3 and CLIC4 gene expressions: CLIC3=4.42CLIC4-10.07. Our results indicate that CLIC3 and CLIC4 are over-expressed in human MPM. Moreover, their expressions correlate suggesting that they either share common gene expression inducers or that their products act synergistically. FAEGO showed that CLIC interactome might contribute to TGF beta signaling and water transport. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of NMDA receptor-dependent LTP in the hippocampus: bridging the divide

PubMed Central

2013-01-01

A consensus has famously yet to emerge on the locus and mechanisms underlying the expression of the canonical NMDA receptor-dependent form of LTP. An objective assessment of the evidence leads us to conclude that both presynaptic and postsynaptic expression mechanisms contribute to this type of synaptic plasticity. PMID:23339575

Self-Injurious Behaviour: Limbic Dysregulation and Stress Effects in an Animal Model

ERIC Educational Resources Information Center

Muehlmann, A. M.; Kies, S. D.; Turner, C. A.; Wolfman, S.; Lewis, M. H.; Devine, D. P.

2012-01-01

Background: Self-injurious behaviour (SIB) is prevalent in neurodevelopmental disorders, but its expression is highly variable within, and between diagnostic categories. This raises questions about the factors that contribute to aetiology and expression of SIB. Expression of SIB is generally described in relation to social reinforcement. However,…

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu Ping; Jiang Xiaohong; Arcasoy, Murat O.

The role of erythropoietin receptor (EpoR) expression in tumor cells and the potential of EpoR-mediated signaling to contribute to cellular proliferation and invasiveness require further characterization. To determine whether EpoR expression and activation in tumor cells modulates intracellular signal transduction to promote cellular proliferation and migration, we employed a novel experimental model using human breast cancer cells engineered to stably express a constitutively active EpoR-R129C variant. EpoR-R129C expression resulted in increased cellular proliferation and migration of breast cancer cells and these effects were associated with significantly increased Epo-induced phosphorylation of ERK1/2, AKT and c-Jun-NH2-kinase (SAPK/JNK) proteins. Expression of the constitutivelymore » active EpoR-R129C receptor promoted the proliferation and migration of breast cancer cells via activation of ERK- and SAPK/JNK-dependent signaling pathways, respectively. These findings suggest that EpoR over-expression and activation in breast cancer cells has the potential to contribute to tumor progression by promoting the proliferation and invasiveness of the neoplastic cells.« less

Genome-Wide Survey on Genomic Variation, Expression Divergence, and Evolution in Two Contrasting Rice Genotypes under High Salinity Stress

PubMed Central

Jiang, Shu-Ye; Ma, Ali; Ramamoorthy, Rengasamy; Ramachandran, Srinivasan

2013-01-01

Expression profiling is one of the most important tools for dissecting biological functions of genes and the upregulation or downregulation of gene expression is sufficient for recreating phenotypic differences. Expression divergence of genes significantly contributes to phenotypic variations. However, little is known on the molecular basis of expression divergence and evolution among rice genotypes with contrasting phenotypes. In this study, we have implemented an integrative approach using bioinformatics and experimental analyses to provide insights into genomic variation, expression divergence, and evolution between salinity-sensitive rice variety Nipponbare and tolerant rice line Pokkali under normal and high salinity stress conditions. We have detected thousands of differentially expressed genes between these two genotypes and thousands of up- or downregulated genes under high salinity stress. Many genes were first detected with expression evidence using custom microarray analysis. Some gene families were preferentially regulated by high salinity stress and might play key roles in stress-responsive biological processes. Genomic variations in promoter regions resulted from single nucleotide polymorphisms, indels (1–10 bp of insertion/deletion), and structural variations significantly contributed to the expression divergence and regulation. Our data also showed that tandem and segmental duplication, CACTA and hAT elements played roles in the evolution of gene expression divergence and regulation between these two contrasting genotypes under normal or high salinity stress conditions. PMID:24121498

Spatially coordinated dynamic gene transcription in living pituitary tissue

PubMed Central

Featherstone, Karen; Hey, Kirsty; Momiji, Hiroshi; McNamara, Anne V; Patist, Amanda L; Woodburn, Joanna; Spiller, David G; Christian, Helen C; McNeilly, Alan S; Mullins, John J; Finkenstädt, Bärbel F; Rand, David A; White, Michael RH; Davis, Julian RE

2016-01-01

Transcription at individual genes in single cells is often pulsatile and stochastic. A key question emerges regarding how this behaviour contributes to tissue phenotype, but it has been a challenge to quantitatively analyse this in living cells over time, as opposed to studying snap-shots of gene expression state. We have used imaging of reporter gene expression to track transcription in living pituitary tissue. We integrated live-cell imaging data with statistical modelling for quantitative real-time estimation of the timing of switching between transcriptional states across a whole tissue. Multiple levels of transcription rate were identified, indicating that gene expression is not a simple binary ‘on-off’ process. Immature tissue displayed shorter durations of high-expressing states than the adult. In adult pituitary tissue, direct cell contacts involving gap junctions allowed local spatial coordination of prolactin gene expression. Our findings identify how heterogeneous transcriptional dynamics of single cells may contribute to overall tissue behaviour. DOI: http://dx.doi.org/10.7554/eLife.08494.001 PMID:26828110

Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

PubMed Central

Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

2013-01-01

Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

Calcium movements during pigment aggregation in freshwater shrimp chromatophores.

PubMed

Ribeiro, Márcia; McNamara, John Campbell

2007-02-01

Pigment granule migration within crustacean chromatophores provides an excellent model with which to investigate cytoplasmic movements, given the antagonistic, neurosecretory peptide regulation of granule translocation, and the absence of innervation in these large, brightly colored cells. Red pigment-concentrating hormone (RPCH) induces pigment aggregation in shrimp chromatophores via an increase in intracellular Ca2+; however, how this increase is brought about is not known. To examine the putative Ca2+ movements leading to pigment translocation in red, ovarian chromatophores of the freshwater shrimp, Macrobrachium olfersii, this study manipulates intra- and extracellular Ca2+ employing ER Ca2+-ATPase inhibitors, ryanodine-sensitive, ER Ca2+ channel blockers, and EDTA/EGTA-buffered A23187/Ca2+-containing salines. Our findings reveal that during pigment aggregation, cytosolic Ca2+ apparently increases from an intracellular source, the abundant SER, loaded by the SERCA and released through ryanodine-sensitive receptor/channels, triggered by capacitative calcium influx and/or calcium-induced calcium release mechanisms. Aggregation also depends on external calcium, which may modulate RPCH/receptor coupling. Such calcium-regulated pigment movements form the basis of a complex system of chromatic adaptation, which confers selective advantages like camouflage and protection against ultra-violet radiation to this palaemonid shrimp.

Signal transduction in red blood cells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae).

PubMed

Beraldo, F H; Sartorello, R; Lanari, R D; Garcia, C R

2001-06-01

The fluorescent calcium probe, Fluo-3, AM was used to measure the intracellular calcium concentration in red blood cells (RBCs) of the teiid lizards Ameiva ameiva and Tupinambis merianae. The cytosolic [Ca2+] is maintained around 20 nM and the cells contain membrane-bound Ca2+ pools. One pool appears to be identifiable with the endoplasmic reticulum (ER) inasmuch as addition of the sarco-endoplasmic reticulum Ca2+ ATPase, SERCA, inhibitor thapsigargin induces an increase in cytosolic [Ca2+ both in the presence and in the absence of extracellular Ca2+. In addition to the ER, an acidic compartment appears to be involved in Ca2+ storage, as collapse of intracellular pHgradients by monensin, a Na+ -H+ exchanger, and nigericin, a K+ -H+ exchanger, induce the release of Ca2+ from internal pools. A vacuolar H+ pump, sensitive to NBD-Cl and bafilomycin appears to be necessary to load the acidic Ca2+ pools. Finally, the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c in the cells from both lizard species, mostly by mobilization of the cation from internal stores. Copyright 2001 Harcourt Publishers Ltd.

Endocrine disrupting alkylphenols: structural requirements for their adverse effects on Ca2+ pumps, Ca2+ homeostasis & Sertoli TM4 cell viability.

PubMed

Michelangeli, Francesco; Ogunbayo, Oluseye A; Wootton, Laura L; Lai, Pei F; Al-Mousa, Fawaz; Harris, Robert M; Waring, Rosemary H; Kirk, Christopher J

2008-11-25

Alkylphenols such as nonylphenol are pollutants that are widely dispersed within our environment. They bio-accumulate within man, with levels in the muM concentration range reported in human tissues. These chemicals act as endocrine disruptors, having xenoestrogenic activity. More recently alkylphenols have also been shown to affect Ca2+ signalling pathways. Here we show that alkylphenols are potent inhibitors of sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) activity. For linear chain alkylphenols the potency of inhibition is related to chain length, with the IC50 values for inhibition ranging from 8 microM for 4-n-nonylphenol (C9) to 1.3 mM for 4-n-propylphenol (C3). Branched chain alkylphenols generally had lower potencies than their linear chain counterparts, however, good correlations for all alkylphenols were observed between their Ca2+ pump inhibition and hydrophobicity, molecular volume and flexibility, indicating that these parameters are all important factors. Alkylphenols cause abnormal elevations of intracellular [Ca2+] within TM4 Sertoli cells (cells involved in sperm maturation) depolarise their mitochondria and induce cell death in these cells, in an alkyl chain size-dependent manner.

Epimorphin expression in interstitial pneumonia

PubMed Central

Terasaki, Yasuhiro; Fukuda, Yuh; Suga, Moritaka; Ikeguchi, Naoki; Takeya, Motohiro

2005-01-01

Epimorphin modulates epithelial morphogenesis in embryonic mouse organs. We previously suggested that epimorphin contributes to repair of bleomycin-induced pulmonary fibrosis in mice via epithelium-mesenchyme interactions. To clarify the role of epimorphin in human lungs, we evaluated epimorphin expression and localization in normal lungs, lungs with nonspecific interstitial pneumonia (NSIP), and lungs with usual interstitial pneumonia (UIP); we also studied the effect of recombinant epimorphin on cultured human alveolar epithelial cells in vitro. Northern and Western blotting analyses revealed that epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP. Immunohistochemistry showed strong epimorphin expression in mesenchymal cells of early fibrotic lesions and localization of epimorphin protein on mesenchymal cells and extracellular matrix of early fibrotic lesions in the nonspecific interstitial pneumonia group. Double-labeled fluorescent images revealed expression of matrix metalloproteinase 2 in re-epithelialized cells overlying epimorphin-positive early fibrotic lesions. Immunohistochemistry and metalloproteinase activity assay demonstrated augmented expression of metalloproteinase induced by recombinant epimorphin in human alveolar epithelial cells. These findings suggest that epimorphin contributes to repair of pulmonary fibrosis in nonspecific interstitial pneumonia, perhaps partly by inducing expression of matrix metalloproteinase 2, which is an important proteolytic factor in lung remodeling. PMID:15651999

Deficiency in Aryl Hydrocarbon Receptor (AHR) Expression throughout Aging Alters Gene Expression Profiles in Murine Long-Term Hematopoietic Stem Cells

PubMed Central

Bennett, John A.; Singh, Kameshwar P.; Unnisa, Zeenath; Welle, Stephen L.; Gasiewicz, Thomas A.

2015-01-01

Dysregulation of hematopoietic stem cell (HSC) signaling can contribute to the development of diseases of the blood system. Lack of aryl hydrocarbon receptor (AhR) has been associated with alterations in gene expression related to HSC function and the subsequent development of a myeloproliferative disorder in aging female mice. We sorted the most primitive population of HSCs with the highest stem cell potential (Long-term, or LT-HSCs) from 18-month-old AhR-null-allele (AhR-KO) and WT mice and analyzed gene expression using microarray to determine alterations in gene expression and cell signaling networks in HSCs that could potentially contribute to the aging phenotype of AhR-KO mice. Comparisons with previous array data from 8-week old mice indicated that aging alone is sufficient to alter gene expression. In addition, a significant number of gene expression differences were observed in aged LT-HSCs that are dependent on both aging and lack of AhR. Pathway analysis of these genes revealed networks related to hematopoietic stem cell activity or function. qPCR was used to confirm the differential expression of a subset of these genes, focusing on genes that may represent novel AhR targets due to the presence of a putative AhR binding site in their upstream regulatory region. We verified differential expression of PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. Pathway analysis of this subset of genes revealed overlap between cellular functions of the novel AhR targets and AhR itself. Lentiviral-mediated knockdown of AhR in lineage-negative hematopoietic cells was sufficient to induce changes in all five of the candidate AhR targets identified. Taken together, these data suggest a role for AhR in HSC functional regulation, and identify novel HSC AhR target genes that may contribute to the phenotypes observed in AhR-KO mice. PMID:26208102

An alternative splicing program promotes adipose tissue thermogenesis

PubMed Central

Vernia, Santiago; Edwards, Yvonne JK; Han, Myoung Sook; Cavanagh-Kyros, Julie; Barrett, Tamera; Kim, Jason K; Davis, Roger J

2016-01-01

Alternative pre-mRNA splicing expands the complexity of the transcriptome and controls isoform-specific gene expression. Whether alternative splicing contributes to metabolic regulation is largely unknown. Here we investigated the contribution of alternative splicing to the development of diet-induced obesity. We found that obesity-induced changes in adipocyte gene expression include alternative pre-mRNA splicing. Bioinformatics analysis associated part of this alternative splicing program with sequence specific NOVA splicing factors. This conclusion was confirmed by studies of mice with NOVA deficiency in adipocytes. Phenotypic analysis of the NOVA-deficient mice demonstrated increased adipose tissue thermogenesis and improved glycemia. We show that NOVA proteins mediate a splicing program that suppresses adipose tissue thermogenesis. Together, these data provide quantitative analysis of gene expression at exon-level resolution in obesity and identify a novel mechanism that contributes to the regulation of adipose tissue function and the maintenance of normal glycemia. DOI: http://dx.doi.org/10.7554/eLife.17672.001 PMID:27635635

Distribution, expression and functional effects of small conductance Ca-activated potassium (SK) channels in rat myometrium.

PubMed

Noble, Karen; Floyd, Rachel; Shmygol, Andre; Shmygol, Anatoly; Mobasheri, A; Wray, Susan

2010-01-01

Calcium-activated potassium channels are important in a variety of smooth muscles, contributing to excitability and contractility. In the myometrium previous work has focussed on the large conductance channels (BK), and the role of small conductance channels (SK) has received scant attention, despite the finding that over-expression of an SK channel isoform (SK3) results in uterine dysfunction and delayed parturition. This study therefore characterises the expression of the three SK channel isoforms (SK1-3) in rat myometrium throughout pregnancy and investigates their effect on cytosolic [Ca] and force and compares this with that of BK channels. Consistent expression of all SK isoform transcripts and clear immunostaining of SK1-3 was found. Inhibition of SK1-3 channels (apamin, scyllatoxin) significantly inhibited outward current, caused membrane depolarisation and elicited action potentials in previously quiescent cells. Apamin or scyllatoxin increased the amplitude of [Ca] and force in spontaneously contracting myometrial strips throughout gestation. The functional effect of SK inhibition was larger than that of BK channel inhibition. Thus we show for the first time that SK1-3 channels are expressed and translated throughout pregnancy and contribute to outward current, regulate membrane potential and hence Ca signals in pregnant rat myometrium. They contribute more to quiescence that BK channels. 2009 Elsevier Ltd. All rights reserved.

Epigenetic regulation of RGS2 (Regulator of G-protein signaling 2) in chemoresistant ovarian cancer cells.

PubMed

Cacan, Ercan

2017-06-01

Regulator of G-protein signaling 2 (RGS2) is a GTPase-activating protein functioning as an inhibitor of G-protein coupled receptors (GPCRs). RGS2 dysregulation was implicated in solid tumour development and RGS2 downregulation has been reported in prostate and ovarian cancer progression. However, the molecular mechanism by which RGS2 expression is suppressed in ovarian cancer remains unknown. The expression and epigenetic regulation of RGS2 in chemosensitive and chemoresistant ovarian cancer cells were determined by qRT-PCR and chromatin immunoprecipitation assays, respectively. In the present study, the molecular mechanisms contributing to the loss of RGS2 expression were determined in ovarian cancer. The data indicated that suppression of RGS2 gene in chemoresistant ovarian cancer cells, in part, due to accumulation of histone deacetylases (HDACs) and DNA methyltransferase I (DNMT1) at the promoter region of RGS2. Inhibition of HDACs or DNMTs significantly increases RGS2 expression. These results suggest that epigenetic changes in histone modifications and DNA methylation may contribute to the loss of RGS2 expression in chemoresistant ovarian cancer cells. The results further suggest that class I HDACs and DNMT1 contribute to the suppression of RGS2 during acquired chemoresistance and support growing evidence that inhibition of HDACs/DNMTs represents novel therapeutic approaches to overcome ovarian cancer chemoresistance.

Transcriptional repression of epithelial cell adhesion molecule (EpCAM) contributes to p53 control of breast cancer invasion

PubMed Central

Sankpal, NV; Willman, MW; Fleming, TP; Mayfield, J; Gillanders, WE

2014-01-01

p53 is a tumor suppressor gene with well-characterized roles in cell cycle regulation, apoptosis and the maintenance of genome stability. Recent evidence suggests that p53 may also contribute to the regulation of migration and invasion. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is overexpressed in the majority of human epithelial carcinomas, including breast and colorectal carcinomas. We demonstrate by chromatin immunoprecipitation assays that p53 interacts with a candidate p53 binding site within the EpCAM gene. p53-mediated transcriptional repression of EpCAM was confirmed in gain-of-function, and loss-of-function experimental systems. Induction of wildtype p53 was associated with a significant dose-dependent decrease in EpCAM expression; conversely, specific ablation of p53 was associated with a significant increase in EpCAM expression. At the functional level, specific ablation of p53 expression is associated with increased breast cancer invasion, and this effect is abrogated by concomitant specific ablation of EpCAM expression. Taken together, these biochemical and functional data are the first demonstration that (1) wildtype p53 protein binds to a response element within the EpCAM gene and negatively regulates EpCAM expression, and (2) transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion. PMID:19141643

Dual Diathesis-Stressor Model of Emotional and Linguistic Contributions to Developmental Stuttering

ERIC Educational Resources Information Center

Walden, Tedra A.; Frankel, Carl B.; Buhr, Anthony P.; Johnson, Kia N.; Conture, Edward G.; Karrass, Jan M.

2012-01-01

This study assessed emotional and speech-language contributions to childhood stuttering. A dual diathesis-stressor framework guided this study, in which both linguistic requirements and skills, and emotion and its regulation, are hypothesized to contribute to stuttering. The language diathesis consists of expressive and receptive language skills.…

Human Mature Adipocytes Express Albumin and This Expression Is Not Regulated by Inflammation

PubMed Central

Sirico, Maria Luisa; Guida, Bruna; Procino, Alfredo; Pota, Andrea; Sodo, Maurizio; Grandaliano, Giuseppe; Simone, Simona; Pertosa, Giovanni; Riccio, Eleonora; Memoli, Bruno

2012-01-01

Aims. Our group investigated albumin gene expression in human adipocytes, its regulation by inflammation and the possible contribution of adipose tissue to albumin circulating levels. Methods. Both inflamed and healthy subjects provided adipose tissue samples. RT-PCR, Real-Time PCR, and Western Blot analysis on homogenates of adipocytes and pre-adipocytes were performed. In sixty-three healthy subjects and fifty-four micro-inflamed end stage renal disease (ESRD) patients circulating levels of albumin were measured by nephelometry; all subjects were also evaluated for body composition, calculated from bioelectrical measurements and an thropometric data. Results. A clear gene expression of albumin was showed in pre-adipocytes and, for the first time, in mature adipocytes. Albumin gene expression resulted significantly higher in pre-adipocytes than in adipocytes. No significant difference in albumin gene expression was showed between healthy controls and inflamed patients. A significant negative correlation was observed between albumin levels and fat mass in both healthy subjects and inflamed ESRD patients. Conclusions. In the present study we found first time evidence that human adipocytes express albumin. Our results also showed that systemic inflammation does not modulate albumin gene expression. The negative correlation between albumin and fat mass seems to exclude a significant contributing role of adipocyte in plasma albumin. PMID:22675238

An EG-VEGF-dependent decrease in homeobox gene NKX3.1 contributes to cytotrophoblast dysfunction: a possible mechanism in human fetal growth restriction.

PubMed

Murthi, P; Brouillet, S; Pratt, A; Borg, Aj; Kalionis, B; Goffin, F; Tsatsaris, V; Munaut, C; Feige, Jj; Benharouga, M; Fournier, T; Alfaidy, N

2015-07-21

Idiopathic fetal growth restriction (FGR) is frequently associated with placental insufficiency. Previous reports have provided evidence that EG-VEGF (endocrine gland derived-vascular endothelial growth factor), a placental secreted protein, is expressed during the first trimester of pregnancy, controls both trophoblast proliferation and invasion, and its increased expression is associated with human FGR. In this study, we hypothesise that EG-VEGF-dependent change in placental homeobox gene expressions contribute to trophoblast dysfunction in idiopathic FGR. The changes in EG-VEGF-dependent homeobox gene expressions were determined using a Homeobox gene cDNA array on placental explants of 8-12 weeks' gestation after stimulation with EG-VEGF in vitro for 24 hours. The Homeobox gene array identified a >5-fold increase in HOXA9, HOXC8, HOXC10, HOXD1, HOXD8, HOXD9 and HOXD11, while NKX 3.1 showed a >2 fold-decrease in mRNA expression compared to untreated controls. Homeobox gene NKX3.1 was selected as a candidate because it is a downstream target of EG-VEGF and its expression and functional role are largely unknown in control and idiopathic FGR-affected placentae. Real-time PCR and immunoblotting showed a significant decrease in NKX3.1 mRNA and protein levels, respectively, in placentae from FGR compared to control pregnancies. Gene inactivation in vitro using short-interference RNA specific for NKX3.1 demonstrated an increase in BeWo cell differentiation and a decrease in HTR8-SVneo proliferation. We conclude that the decreased expression of homeobox gene NKX3.1 down-stream of EG-VEGF may contribute to the trophoblast dysfunction associated with idiopathic FGR pregnancies.

Transcriptome study of differential expression in schizophrenia

PubMed Central

Sanders, Alan R.; Göring, Harald H. H.; Duan, Jubao; Drigalenko, Eugene I.; Moy, Winton; Freda, Jessica; He, Deli; Shi, Jianxin; Gejman, Pablo V.

2013-01-01

Schizophrenia genome-wide association studies (GWAS) have identified common SNPs, rare copy number variants (CNVs) and a large polygenic contribution to illness risk, but biological mechanisms remain unclear. Bioinformatic analyses of significantly associated genetic variants point to a large role for regulatory variants. To identify gene expression abnormalities in schizophrenia, we generated whole-genome gene expression profiles using microarrays on lymphoblastoid cell lines (LCLs) from 413 cases and 446 controls. Regression analysis identified 95 transcripts differentially expressed by affection status at a genome-wide false discovery rate (FDR) of 0.05, while simultaneously controlling for confounding effects. These transcripts represented 89 genes with functions such as neurotransmission, gene regulation, cell cycle progression, differentiation, apoptosis, microRNA (miRNA) processing and immunity. This functional diversity is consistent with schizophrenia's likely significant pathophysiological heterogeneity. The overall enrichment of immune-related genes among those differentially expressed by affection status is consistent with hypothesized immune contributions to schizophrenia risk. The observed differential expression of extended major histocompatibility complex (xMHC) region histones (HIST1H2BD, HIST1H2BC, HIST1H2BH, HIST1H2BG and HIST1H4K) converges with the genetic evidence from GWAS, which find the xMHC to be the most significant susceptibility locus. Among the differentially expressed immune-related genes, B3GNT2 is implicated in autoimmune disorders previously tied to schizophrenia risk (rheumatoid arthritis and Graves’ disease), and DICER1 is pivotal in miRNA processing potentially linking to miRNA alterations in schizophrenia (e.g. MIR137, the second strongest GWAS finding). Our analysis provides novel candidate genes for further study to assess their potential contribution to schizophrenia. PMID:23904455

hnRNP K plays a protective role in TNF-α-induced apoptosis in podocytes.

PubMed

Zhao, Shili; Feng, Junxia; Wang, Qi; Tian, Lu; Zhang, Yunfang; Li, Hongyan

2018-06-29

Apoptosis of podocytes contributes to proteinuria in many chronic kidney diseases. The cytokine, tumor necrosis factor-α (TNF-α) is thought to be involved in podocyte apoptosis, but the underlying mechanism is not understood. In our study, we established a model of TNF-α-induced apoptosis by isolating primary podocytes from mice. After exposing cells to TNF-α, we determined the expression levels of heterogeneous nuclear ribonucleoprotein K (hnRNP K) and cellular FLICE-inhibitory protein (c-FLIP) and the phosphorylation levels of glycogen synthase kinase β (GSK3β) and extracellular signal-regulated kinase (ERK). We then knocked down or overexpressed the levels of hnRNP K and observed its effects on the expressions of c-FLIP, caspase-8, caspase-3, and the phosphorylation of GSK3β and ERK. In addition, we examined the percentage of cells undergoing apoptosis and studied cell cycle distribution. We found that TNF-α induced apoptosis in podocytes and that the expressions of hnRNP K and c-FLIP were significantly decreased, whereas the phosphorylations of GSK3β and ERK were significantly increased. Both gene knockdown and overexpression of hnRPN K resulted in varied expressions/phosphorylations of c-FLIP, GSK3β, and ERK. Moreover, decreased hnRPN K expression contributed to increased levels of caspase-8 and capase-3, as well as an increase in cell apoptosis and G0/G1 arrest. In conclusion, down-regulated expression of hnRNP K by TNF-α resulted in a decrease in the expression of c-FLIP as well as increases in phosphorylated GSK3β, ERK, caspase-8, and caspase-3, and then critically contributed to the podocyte apoptosis. © 2018 The Author(s).

Hypothalamic neuropeptides, not leptin sensitivity, contributes to the hyperphagia in lactating Brandt's voles, Lasiopodomys brandtii.

PubMed

Cui, Jian-Guo; Tang, Gang-Bing; Wang, De-Hua

2011-07-01

Both pregnancy and lactation are associated with hyperphagia, and circulating leptin levels are elevated during pregnancy but decreased during lactation in Brandt's voles, Lasiopodomys brandtii. Previous findings suggest that impaired leptin sensitivity contributes to hyperphagia during pregnancy. The present study aimed to examine whether the decreased circulating leptin level and/or hypothalamic leptin sensitivity contributed to the hyperphagia during lactation in Brandt's voles. The serum leptin level and mRNA expression of the long form of the leptin receptor (Ob-Rb), suppressor-of-cytokine-signalling-3 (SOCS-3), neuropeptide Y (NPY), agouti-related protein (AgRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus were examined on dioestrous, day 5, day 17 of lactation and day 27 (1 week after weaning) in Brandt's voles. Compared with controls, hypothalamic Ob-Rb and SOCS-3 mRNA expression was not significantly changed during lactation. The serum leptin level was significantly lower in lactating females than in the non-reproductive group. Hypothalamic NPY and AgRP mRNA expression significantly increased whereas POMC mRNA expression was significantly decreased during lactation compared with controls. However, there were no significant changes in hypothalamic CART mRNA expression. Food intake was positively correlated with NPY and AgRP mRNA expression but negatively correlated with POMC mRNA expression during lactation. These data suggest that hyperphagia during lactation was associated with low leptin levels, but not impaired leptin sensitivity, and that the hypothalamic neuropeptides NPY, AgRP and POMC are involved in mediating the role of leptin in food intake regulation in lactating Brandt's voles.

Live imaging using a FRET glucose sensor reveals glucose delivery to all cell types in the Drosophila brain.

PubMed

Volkenhoff, Anne; Hirrlinger, Johannes; Kappel, Johannes M; Klämbt, Christian; Schirmeier, Stefanie

2018-04-01

All complex nervous systems are metabolically separated from circulation by a blood-brain barrier (BBB) that prevents uncontrolled leakage of solutes into the brain. Thus, all metabolites needed to sustain energy homeostasis must be transported across this BBB. In invertebrates, such as Drosophila, the major carbohydrate in circulation is the disaccharide trehalose and specific trehalose transporters are expressed by the glial BBB. Here we analyzed whether glucose is able to contribute to energy homeostasis in Drosophila. To study glucose influx into the brain we utilized a genetically encoded, FRET-based glucose sensor expressed in a cell type specific manner. When confronted with glucose all brain cells take up glucose within two minutes. In order to characterize the glucose transporter involved, we studied Drosophila Glut1, the homologue of which is primarily expressed by the BBB-forming endothelial cells and astrocytes in the mammalian nervous system. In Drosophila, however, Glut1 is expressed in neurons and is not found at the BBB. Thus, Glut1 cannot contribute to initial glucose uptake from the hemolymph. To test whether gap junctional coupling between the BBB forming cells and other neural cells contributes to glucose distribution we assayed these junctions using RNAi experiments and only found a minor contribution of gap junctions to glucose metabolism. Our results provide the entry point to further dissect the mechanisms underlying glucose distribution and offer new opportunities to understand brain metabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Factors contributing to the adaptation aftereffects of facial expression.

PubMed

Butler, Andrea; Oruc, Ipek; Fox, Christopher J; Barton, Jason J S

2008-01-29

Previous studies have demonstrated the existence of adaptation aftereffects for facial expressions. Here we investigated which aspects of facial stimuli contribute to these aftereffects. In Experiment 1, we examined the role of local adaptation to image elements such as curvature, shape and orientation, independent of expression, by using hybrid faces constructed from either the same or opposing expressions. While hybrid faces made with consistent expressions generated aftereffects as large as those with normal faces, there were no aftereffects from hybrid faces made from different expressions, despite the fact that these contained the same local image elements. In Experiment 2, we examined the role of facial features independent of the normal face configuration by contrasting adaptation with whole faces to adaptation with scrambled faces. We found that scrambled faces also generated significant aftereffects, indicating that expressive features without a normal facial configuration could generate expression aftereffects. In Experiment 3, we examined the role of facial configuration by using schematic faces made from line elements that in isolation do not carry expression-related information (e.g. curved segments and straight lines) but that convey an expression when arranged in a normal facial configuration. We obtained a significant aftereffect for facial configurations but not scrambled configurations of these line elements. We conclude that facial expression aftereffects are not due to local adaptation to image elements but due to high-level adaptation of neural representations that involve both facial features and facial configuration.

Methamphetamine-induced stereotypy correlates negatively with patch-enhanced prodynorphin and arc mRNA expression in the rat caudate putamen: the role of mu opioid receptor activation.

PubMed

Horner, Kristen A; Noble, Erika S; Gilbert, Yamiece E

2010-06-01

Amphetamines induce stereotypy, which correlates with patch-enhanced c-Fos expression the patch compartment of caudate putamen (CPu). Methamphetamine (METH) treatment also induces patch-enhanced expression of prodynorphin (PD), arc and zif/268 in the CPu. Whether patch-enhanced activation of any of these genes correlates with METH-induced stereotypy is unknown, and the factors that contribute to this pattern of expression are poorly understood. Activation of mu opioid receptors, which are expressed by the neurons of the patch compartment, may underlie METH-induced patch-enhanced gene expression and stereotypy. The current study examined whether striatal mu opioid receptor blockade altered METH-induced stereotypy and patch-enhanced gene expression, and if there was a correlation between the two responses. Animals were intrastriatally infused with the mu antagonist CTAP (10 microg/microl), treated with METH (7.5 mg/kg, s.c.), placed in activity chambers for 3h, and then sacrificed. CTAP pretreatment attenuated METH-induced increases in PD, arc and zif/268 mRNA expression and significantly reduced METH-induced stereotypy. Patch-enhanced PD and arc mRNA expression in the dorsolateral CPu correlated negatively with METH-induced stereotypy. These data indicate that mu opioid receptor activation contributes to METH-induced gene expression in the CPu and stereotypy, and that patch-enhanced PD and arc expression may be a homeostatic response to METH treatment. Copyright 2010 Elsevier Inc. All rights reserved.

Methamphetamine-Induced Stereotypy Correlates Negatively with Patch-Enhanced Prodynorphin and ARC mRNA Expression in the Rat Caudate Putamen: The Role of Mu Opioid Receptor Activation

PubMed Central

Horner, Kristen A.; Noble, Erika S.; Gilbert, Yamiece E.

2010-01-01

Amphetamines induce stereotypy, which correlates with patch-enhanced c-Fos expression the patch compartment of caudate putamen (CPu). Methamphetamine (METH) treatment also induces patch-enhanced expression of prodynorphin (PD), arc and zif/268 in the CPu. Whether patch-enhanced activation of any of these genes correlates with METH-induced stereotypy is unknown, and the factors that contribute to this pattern of expression are poorly understood. Activation of mu opioid receptors, which are expressed by the neurons of the patch compartment, may underlie METH-induced patch-enhanced gene expression and stereotypy. The current study examined whether striatal mu opioid receptor blockade altered METH-induced stereotypy and patch-enhanced gene expression, and if there was a correlation between the two responses. Animals were intrastriatally infused with the mu antagonist CTAP (10 μg/μl), treated with METH (7.5 mg/kg, s.c.), placed in activity chambers for 3h, and then sacrificed. CTAP pretreatment attenuated METH-induced increases in PD, arc and zif/268 mRNA expression and significantly reduced METH-induced stereotypy. Patch-enhanced PD and arc mRNA expression in the dorsolateral CPu correlated negatively with METH-induced stereotypy. These data indicate that mu opioid receptor activation contributes to METH-induced gene expression in the CPu and stereotypy, and that patch-enhanced PD and arc expression may be a homeostatic response to METH treatment. PMID:20298714

Macrophage activation by factors released from acetaminophen-injured hepatocytes: Potential role of HMGB1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dragomir, Ana-Cristina; Laskin, Jeffrey D.; Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu

2011-06-15

Toxic doses of acetaminophen (AA) cause hepatocellular necrosis. Evidence suggests that activated macrophages contribute to the pathogenic process; however, the factors that activate these cells are unknown. In these studies, we assessed the role of mediators released from AA-injured hepatocytes in macrophage activation. Treatment of macrophages with conditioned medium (CM) collected 24 hr after treatment of mouse hepatocytes with 5 mM AA (CM-AA) resulted in increased production of reactive oxygen species (ROS). Macrophage expression of heme oxygenase-1 (HO-1) and catalase mRNA was also upregulated by CM-AA, as well as cyclooxygenase (COX)-2 and 12/15-lipoxygenase (LOX). CM-AA also upregulated expression of themore » proinflammatory chemokines, MIP-1{alpha} and MIP-2. The effects of CM-AA on expression of COX-2, MIP-1{alpha} and MIP-2 were inhibited by blockade of p44/42 MAP kinase, suggesting a biochemical mechanism mediating macrophage activation. Hepatocytes injured by AA were found to release HMGB1, a potent macrophage activator. This was inhibited by pretreatment of hepatocytes with ethyl pyruvate (EP), which blocks HMGB1 release. EP also blocked CM-AA induced ROS production and antioxidant expression, and reduced expression of COX-2, but not MIP-1{alpha} or MIP-2. These findings suggest that HMGB1 released by AA-injured hepatocytes contributes to macrophage activation. This is supported by our observation that expression of the HMGB1 receptor RAGE is upregulated in macrophages in response to CM-AA. These data indicate that AA-injured hepatocytes contribute to the inflammatory environment in the liver through the release of mediators such as HMGB1. Blocking HMGB1/RAGE may be a useful approach to limiting classical macrophage activation and AA-induced hepatotoxicity. - Research Highlights: > These studies analyze macrophage activation by mediators released from acetaminophen-damaged hepatocytes. > Factors released from acetaminophen-injured hepatocytes induce macrophage ROS production and expression of COX-2, chemokines, and RAGE. > Hepatocyte-mediated macrophage activation involves p44/42 MAP kinase signaling. > HMGB1 is released from acetaminophen-injured hepatocytes and contributes to macrophage activation.« less

Contributions of Phonological Memory, Language Comprehension and Hearing to the Expressive Language of Adolescents and Young Adults with Down Syndrome

ERIC Educational Resources Information Center

Laws, Glynis

2004-01-01

Background: Expressive language constitutes a major challenge to the development of individuals with Down syndrome. This paper investigates the relationships between expressive language abilities, language comprehension and the deficits in verbal short-term memory and hearing which are also associated with the syndrome. Methods: Tests of nonverbal…

Las Expresiones Pragmaticas en la Conversacion Espontanea (Pragmatic Expressions in Spontaneous Conversation).

ERIC Educational Resources Information Center

Carranza, Isolda

The starting point of this thesis is the hypothesis that in Spanish there are conventionalized expressions that signal both the articulation of text parts and the speaker's attitude towards the utterance. "Pragmatic Expressions" (PEs) are fixed lexical forms that contribute to text creation in relation to the context of enunciation, are oriented…

Astrocytic expression of HIV-1 Nef impairs spatial and recognition memory

PubMed Central

Chompre, Gladys; Cruz, Emmanuel; Maldonado, Lucianette; Rivera-Amill, Vanessa; Porter, James T.; Noel, Richard J.

2012-01-01

Despite the widespread use of antiretroviral therapy that effectively limits viral replication, memory impairment remains a dilemma for HIV infected people. In the CNS, HIV infection of astrocytes leads to the production of the HIV-1 Nef protein without viral replication. Post mortem studies have found Nef expression in hippocampal astrocytes of people with HIV associated dementia suggesting that astrocytic Nef may contribute to HIV associated cognitive impairment even when viral replication is suppressed. To test whether astrocytic expression of Nef is sufficient to induce cognitive deficits, we examined the effect of implanting primary rat astrocytes expressing Nef into the hippocampus on spatial and recognition memory. Rats implanted unilaterally with astrocytes expressing Nef showed impaired novel location and novel object recognition in comparison with controls implanted with astrocytes expressing green fluorescent protein (GFP). This impairment was correlated with an increase in chemokine ligand 2 (CCL2) expression and the infiltration of peripheral macrophages into the hippocampus at the site of injection. Furthermore, the Nef exposed rats exhibited a bilateral loss of CA3 neurons. These results suggest that Nef protein expressed by the implanted astrocytes activates the immune system leading to neuronal damage and spatial and recognition memory deficits. Therefore, the continued expression of Nef by astrocytes in the absence of viral replication has the potential to contribute to HIV associated cognitive impairment. PMID:22926191

PI3K/Akt contributes to increased expression of Toll-like receptor 4 in macrophages exposed to hypoxic stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, So Young; Jeong, Eunshil; Joung, Sun Myung

2012-03-16

Highlights: Black-Right-Pointing-Pointer Hypoxic stress-induced TLR4 expression is mediated by PI3K/Akt in macrophages. Black-Right-Pointing-Pointer PI3K/Akt regulated HIF-1 activation leading to TLR4 expression. Black-Right-Pointing-Pointer p38 mitogen-activated protein kinase was not involved in TLR4 expression by hypoxic stress. Black-Right-Pointing-Pointer Sulforaphane suppressed hypoxia-mediated TLR4 expression by inhibiting PI3K/Akt. -- Abstract: Toll-like receptors (TLRs) play critical roles in triggering immune and inflammatory responses by detecting invading microbial pathogens and endogenous danger signals. Increased expression of TLR4 is implicated in aggravated inflammatory symptoms in ischemic tissue injury and chronic diseases. Results from our previous study showed that TLR4 expression was upregulated by hypoxic stress mediated bymore » hypoxia-inducible factor-1 (HIF-1) at a transcriptional level in macrophages. In this study, we further investigated the upstream signaling pathway that contributed to the increase of TLR4 expression by hypoxic stress. Either treatment with pharmacological inhibitors of PI3K and Akt or knockdown of Akt expression by siRNA blocked the increase of TLR4 mRNA and protein levels in macrophages exposed to hypoxia and CoCl{sub 2}. Phosphorylation of Akt by hypoxic stress preceded nuclear accumulation of HIF-1{alpha}. A PI3K inhibitor (LY294002) attenuated CoCl{sub 2}-induced nuclear accumulation and transcriptional activation of HIF-1{alpha}. In addition, HIF-1{alpha}-mediated upregulation of TLR4 expression was blocked by LY294002. Furthermore, sulforaphane suppressed hypoxia- and CoCl{sub 2}-induced upregulation of TLR4 mRNA and protein by inhibiting PI3K/Akt activation and the subsequent nuclear accumulation and transcriptional activation of HIF-1{alpha}. However, p38 was not involved in HIF-1{alpha} activation and TLR4 expression induced by hypoxic stress in macrophages. Collectively, our results demonstrate that PI3K/Akt contributes to hypoxic stress-induced TLR4 expression at least partly through the regulation of HIF-1 activation. These reveal a novel mechanism for regulation of TLR4 expression upon hypoxic stress and provide a therapeutic target for chronic diseases related to hypoxic stress.« less

Inappropriate expression of hepcidin by liver congestion contributes to anemia and relative iron deficiency.

PubMed

Suzuki, Tomoyasu; Hanawa, Haruo; Jiao, Shuang; Ohno, Yukako; Hayashi, Yuka; Yoshida, Kaori; Kashimura, Takeshi; Obata, Hiroaki; Minamino, Tohru

2014-04-01

Anemia and relative iron deficiency (RID) are prevalent in patients with heart failure (HF). The etiology of anemia and RID in HF patients is unclear. Hepcidin expression may be closely related to anemia and RID in HF patients. Although hepcidin is produced mainly by the liver, and the most frequent histologic appearance of liver in HF patients is congestion, the influence of liver congestion (LC) on hepcidin production has not yet been investigated. We investigated whether hepcidin contributed to anemia and RID in rats with LC. LC was induced in rats by ligating the inferior vena cava and compared with bleeding anemia (BA) model induced by phlebotomy and hemolytic anemia (HA) model induced by injection of phenylhydrazine. BA and HA strongly suppressed expression of hepcidin in liver and so did not cause decrease in serum iron and transferrin saturation. However, hepcidin expression did not decrease in LC rats, which resulted in anemia and lower transferrin saturation. In addition, many cells with hemosiderin deposits were observed in the liver and spleen and not in the bone marrow, and this appeared to be related to suppression of hepcidin expression. Iron accumulated in hepatocytes, and bone morphogenetic protein 6, which induces hepcidin, increased. Inflammation was observed in the congestive liver, and there was an increase in interleukin-6, which also induced hepcidin and was induced by free heme and hemoglobin via Toll-like receptor 4. We conclude that LC contributes to RID and anemia, and it does so via inappropriate expression of hepcidin. Copyright © 2014 Elsevier Inc. All rights reserved.

Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and differential roles in pathogenicity

PubMed Central

Recinos, David A.; Sekedat, Matthew D.; Hernandez, Adriana; Cohen, Taylor Sitarik; Sakhtah, Hassan; Prince, Alice S.; Price-Whelan, Alexa; Dietrich, Lars E. P.

2012-01-01

Evolutionary biologists have postulated that several fitness advantages may be conferred by the maintenance of duplicate genes, including environmental adaptation resulting from differential regulation. We examined the expression and physiological contributions of two redundant operons in the adaptable bacterium Pseudomonas aeruginosa PA14. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode nearly identical sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid, the precursor for several phenazine derivatives. Phenazines perform diverse roles in P. aeruginosa physiology and act as virulence factors during opportunistic infections of plant and animal hosts. Although reports have indicated that phz1 is regulated by the Pseudomonas quinolone signal, factors controlling phz2 expression have not been identified, and the relative contributions of these redundant operons to phenazine biosynthesis have not been evaluated. We found that in liquid cultures, phz1 was expressed at higher levels than phz2, although phz2 showed a greater contribution to phenazine production. In colony biofilms, phz2 was expressed at high levels, whereas phz1 expression was not detectable, and phz2 was responsible for virtually all phenazine production. Analysis of mutants defective in quinolone signal synthesis revealed a critical role for 4-hydroxy-2-heptylquinoline in phz2 induction. Finally, deletion of phz2, but not of phz1, decreased lung colonization in a murine model of infection. These results suggest that differential regulation of the redundant phz operons allows P. aeruginosa to adapt to diverse environments. PMID:23129634

Differentiation of EL4 lymphoma cells by tumoral environment is associated with inappropriate expression of the large chondroitin sulfate proteoglycan PG-M and the tumor-associated antigen HTgp-175.

PubMed

Rottiers, P; Verfaillie, T; Contreras, R; Revets, H; Desmedt, M; Dooms, H; Fiers, W; Grooten, J

1998-11-09

Progression to malignancy of transformed cells involves complex genetic alterations and aberrant gene expression patterns. While aberrant gene expression is often caused by alterations in individual genes, the contribution of the tumoral environment to the triggering of this gene expression is less well established. The stable but heterogeneous expression in cultured EL4/13 cells of a novel tumor-associated antigen, designated as HTgp-175, was chosen for the investigation of gene expression during tumor formation. Homogeneously HTgp-175-negative EL4/13 cells, isolated by cell sorting or obtained by subcloning, acquired HTgp-175 expression as a result of tumor formation. The tumorigenicity of HTgp-175-negative vs. HTgp-175-positive EL4 variants was identical, indicating that induction but not selection accounted for the phenotypic switch from HTgp-175-negative to HTgp-175-positive. Although mutagenesis experiments showed that the protein was not essential for tumor establishment, tumor-derived cells showed increased malignancy, linking HTgp-175 expression with genetic changes accompanying tumor progression. This novel gene expression was not an isolated event, since it was accompanied by ectopic expression of the large chondroitin sulfate proteoglycan PG-M and of normal differentiation antigens. We conclude that signals derived from the tumoral microenvironment contribute significantly to the aberrant gene expression pattern of malignant cells, apparently by fortuitous activation of differentiation processes and cause expression of novel differentiation antigens as well as of inappropriate tumor-associated and ectopic antigens.

Comparative ecological transcriptomics and the contribution of gene expression to the evolutionary potential of a threatened fish.

PubMed

Brauer, Chris J; Unmack, Peter J; Beheregaray, Luciano B

2017-12-01

Understanding whether small populations with low genetic diversity can respond to rapid environmental change via phenotypic plasticity is an outstanding research question in biology. RNA sequencing (RNA-seq) has recently provided the opportunity to examine variation in gene expression, a surrogate for phenotypic variation, in nonmodel species. We used a comparative RNA-seq approach to assess expression variation within and among adaptively divergent populations of a threatened freshwater fish, Nannoperca australis, found across a steep hydroclimatic gradient in the Murray-Darling Basin, Australia. These populations evolved under contrasting selective environments (e.g., dry/hot lowland; wet/cold upland) and represent opposite ends of the species' spectrum of genetic diversity and population size. We tested the hypothesis that environmental variation among isolated populations has driven the evolution of divergent expression at ecologically important genes using differential expression (DE) analysis and an anova-based comparative phylogenetic expression variance and evolution model framework based on 27,425 de novo assembled transcripts. Additionally, we tested whether gene expression variance within populations was correlated with levels of standing genetic diversity. We identified 290 DE candidate transcripts, 33 transcripts with evidence for high expression plasticity, and 50 candidates for divergent selection on gene expression after accounting for phylogenetic structure. Variance in gene expression appeared unrelated to levels of genetic diversity. Functional annotation of the candidate transcripts revealed that variation in water quality is an important factor influencing expression variation for N. australis. Our findings suggest that gene expression variation can contribute to the evolutionary potential of small populations. © 2017 John Wiley & Sons Ltd.

Ocean to Outback: Leonie Rennie's Contribution to Science Education in Australia

ERIC Educational Resources Information Center

Venville, Grady

2009-01-01

In this article I initially borrow a metaphor from an art exhibition, "Ocean to Outback," as a way to express my perspective on the contribution that Leonie Rennie has made to science education in Australia. I then consider Leonie's contributions as overlapping themes. In particular, Leonie's well-known research on gender and issues of…

Contributions of Print Exposure to First and Second Grade Oral Language and Reading in Chile

ERIC Educational Resources Information Center

Strasser, Katherine; Vergara, Daniela; del Río, M. Francisca

2017-01-01

This study examines the contribution of print exposure to oral language (expressive vocabulary and listening comprehension) and reading (word reading and reading comprehension) in first and second grade in Chile, and tests whether the contribution of print exposure to reading comprehension is mediated by language and word reading skills.…

The potential role of Brachyury in inducing epithelial-to-mesenchymal transition (EMT) and HIF-1α expression in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Chao; Zhang, Jingjing, E-mail: jingjingzhangzs@163.com; Fu, Jianhua

One of transcription factors of the T-box family, Brachyury has been implicated in tumorigenesis of many types of cancers, regulating cancer cell proliferation, metastasis, invasion and epithelial-to-mesenchymal transition (EMT). However, the role of Brachyury in breast cancer cells has been scarcely reported. The present study aimed to investigate the expression and role of Brachyury in breast cancer. Brachyury expression was analyzed by qRT-PCR and Western blot. The correlations between Brachyury expression and clinicopathological factors of breast cancer were determined. Involvement of EMT stimulation and hypoxia-inducible factor-1α (HIF-1α) expression induction by Brachyury was also evaluated. Moreover, the effect of Brachyury onmore » tumor growth and metastasis in vivo was examined in a breast tumor xenograft model. Brachyury expression was enhanced in primary breast cancer tissues and Brachyury expression was correlated with tumor stage and lymph node metastasis. Hypoxia enhanced Brachyury expression, the silencing of which blocked the modulation effect of hypoxia on E-cadherin and vimentin expression. Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling as well as accelerated cell proliferation and migration in vitro. Additionally, Brachyury accelerated breast tumor xenograft growth and increased lung metastasis in nude mice. In summary, our data confirmed that Brachyury might contribute to hypoxia-induced EMT of breast cancer and trigger HIF-1alpha expression via PTEN/Akt signaling. - Highlights: • Brachyury expression was correlated with tumor stage and lymph node metastasis. • Hypoxia enhanced Brachyury expression, which contributes to hypoxia-induced EMT. • Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling. • Brachyury accelerated tumor xenograft growth and increased lung metastasis.« less

MYC and the Control of DNA Replication

PubMed Central

Dominguez-Sola, David; Gautier, Jean

2014-01-01

The MYC oncogene is a multifunctional protein that is aberrantly expressed in a significant fraction of tumors from diverse tissue origins. Because of its multifunctional nature, it has been difficult to delineate the exact contributions of MYC’s diverse roles to tumorigenesis. Here, we review the normal role of MYC in regulating DNA replication as well as its ability to generate DNA replication stress when overexpressed. Finally, we discuss the possible mechanisms by which replication stress induced by aberrant MYC expression could contribute to genomic instability and cancer. PMID:24890833

Cultural modes of expressing emotions influence how emotions are experienced.

PubMed

Immordino-Yang, Mary Helen; Yang, Xiao-Fei; Damasio, Hanna

2016-10-01

The brain's mapping of bodily responses during emotion contributes to emotional experiences, or feelings. Culture influences emotional expressiveness, that is, the magnitude of individuals' bodily responses during emotion. So, are cultural influences on behavioral expressiveness associated with differences in how individuals experience emotion? Chinese and American young adults reported how strongly admiration- and compassion-inducing stories made them feel, first in a private interview and then during functional magnetic resonance imaging (fMRI). As expected, Americans were more expressive in the interview. Although expressiveness did not predict stronger reported feelings or neural responses during fMRI, in both cultural groups more-expressive people showed tighter trial-by-trial correlations between their experienced strength of emotion and activations in visceral-somatosensory cortex, even after controlling for individuals' overall strength of reactions (neural and felt). Moreover, expressiveness mediated a previously described cultural effect in which activations in visceral-somatosensory cortex correlated with feeling strength among Americans but not among Chinese. Post hoc supplementary analyses revealed that more-expressive individuals reached peak activation of visceral-somatosensory cortex later in the emotion process and took longer to decide how strongly they felt. The results together suggest that differences in expressiveness correspond to differences in how somatosensory mechanisms contribute to constructing conscious feelings. By influencing expressiveness, culture may therefore influence how individuals know how strongly they feel, what conscious feelings are based on, or possibly what strong versus weak emotions "feel like." (PsycINFO Database Record (c) 2016 APA, all rights reserved).

A deep auto-encoder model for gene expression prediction.

PubMed

Xie, Rui; Wen, Jia; Quitadamo, Andrew; Cheng, Jianlin; Shi, Xinghua

2017-11-17

Gene expression is a key intermediate level that genotypes lead to a particular trait. Gene expression is affected by various factors including genotypes of genetic variants. With an aim of delineating the genetic impact on gene expression, we build a deep auto-encoder model to assess how good genetic variants will contribute to gene expression changes. This new deep learning model is a regression-based predictive model based on the MultiLayer Perceptron and Stacked Denoising Auto-encoder (MLP-SAE). The model is trained using a stacked denoising auto-encoder for feature selection and a multilayer perceptron framework for backpropagation. We further improve the model by introducing dropout to prevent overfitting and improve performance. To demonstrate the usage of this model, we apply MLP-SAE to a real genomic datasets with genotypes and gene expression profiles measured in yeast. Our results show that the MLP-SAE model with dropout outperforms other models including Lasso, Random Forests and the MLP-SAE model without dropout. Using the MLP-SAE model with dropout, we show that gene expression quantifications predicted by the model solely based on genotypes, align well with true gene expression patterns. We provide a deep auto-encoder model for predicting gene expression from SNP genotypes. This study demonstrates that deep learning is appropriate for tackling another genomic problem, i.e., building predictive models to understand genotypes' contribution to gene expression. With the emerging availability of richer genomic data, we anticipate that deep learning models play a bigger role in modeling and interpreting genomics.

Cultural Modes of Expressing Emotions Influence How Emotions Are Experienced

PubMed Central

Immordino-Yang, Mary Helen; Yang, Xiao-Fei; Damasio, Hanna

2016-01-01

The brain’s mapping of bodily responses during emotion contributes to emotional experiences, or feelings. Culture influences emotional expressiveness, i.e. the magnitude of individuals’ bodily responses during emotion. So, are cultural influences on behavioral expressiveness associated with differences in how individuals experience emotion? Chinese and American young adults reported how strongly admiration and compassion-inducing stories made them feel, first in a private interview and then during fMRI. As expected, Americans were more expressive in the interview. While expressiveness did not predict stronger reported feelings or neural responses during fMRI, in both cultural groups more expressive people showed tighter trial-by-trial correlations between their experienced strength of emotion and activations in visceral-somatosensory cortex, even after controlling for individuals’ overall strength of reactions (neural and felt). Moreover, expressiveness mediated a previously described cultural effect in which activations in visceral-somatosensory cortex correlated with feeling strength among Americans but not among Chinese. Post-hoc supplementary analyses revealed that more expressive individuals reached peak activation of visceral-somatosensory cortex later in the emotion process and took longer to decide how strongly they felt. The results together suggest that differences in expressiveness correspond to differences in how somatosensory mechanisms contribute to constructing conscious feelings. By influencing expressiveness, culture may therefore influence how individuals know how strongly they feel, what conscious feelings are based on, or possibly what strong versus weak emotions “feel like.” PMID:27270077

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME).

PubMed

Petty, Robert D; McCarthy, Neil E; Le Dieu, Rifca; Kerr, Jonathan R

2016-01-01

Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function.

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME)

PubMed Central

Petty, Robert D.; McCarthy, Neil E.; Le Dieu, Rifca; Kerr, Jonathan R.

2016-01-01

Background Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. Methods miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. Results Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. Conclusion This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function. PMID:26967895

Detection of the human endogenous retrovirus ERV3-encoded Env-protein in human tissues using antibody-based proteomics.

PubMed

Fei, Chen; Atterby, Christina; Edqvist, Per-Henrik; Pontén, Fredrik; Zhang, Wei Wei; Larsson, Erik; Ryan, Frank P

2014-01-01

There is growing evidence to suggest that human endogenous retroviruses (HERVs) have contributed to human evolution, being expressed in development, normal physiology and disease. A key difficulty in the scientific evaluation of this potential viral contribution is the accurate demonstration of virally expressed protein in specific human cells and tissues. In this study, we have adopted the endogenous retrovirus, ERV3, as our test model in developing a reliable high-capacity methodology for the expression of such endogenous retrovirus-coded protein. Two affinity-purified polyclonal antibodies to ERV3 Env-encoded protein were generated to detect the corresponding protein expression pattern in specific human cells, tissues and organs. Sampling included normal tissues from 144 individuals ranging from childhood to old age. This included more than forty different tissues and organs and some 216 different cancer tissues representing the twenty commonest forms of human cancer. The Rudbeck Laboratory, Uppsala University and Uppsala University Hospital, Uppsala, Sweden. The potential expression at likely physiological level of the ERV3Env encoded protein in a wide range of human cells, tissues and organs. We found that ERV3 encoded Env protein is expressed at substantive levels in placenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes, bronchial epithelium and the ducts of the salivary glands. Substantive expression was also seen in a variety of epithelial cells as well as cells known to undergo fusion in inflammation and in normal physiology, including fused macrophages, myocardium and striated muscle. This contrasted strongly with the low levels expressed in other tissues types. These findings suggest that this virus plays a significant role in human physiology and may also play a possible role in disease. This technique can now be extended to the study of other HERV genomes within the human chromosomes that may have contributed to human evolution, physiology and disease.

Forager bees (Apis mellifera) highly express immune and detoxification genes in tissues associated with nectar processing.

PubMed

Vannette, Rachel L; Mohamed, Abbas; Johnson, Brian R

2015-11-09

Pollinators, including honey bees, routinely encounter potentially harmful microorganisms and phytochemicals during foraging. However, the mechanisms by which honey bees manage these potential threats are poorly understood. In this study, we examine the expression of antimicrobial, immune and detoxification genes in Apis mellifera and compare between forager and nurse bees using tissue-specific RNA-seq and qPCR. Our analysis revealed extensive tissue-specific expression of antimicrobial, immune signaling, and detoxification genes. Variation in gene expression between worker stages was pronounced in the mandibular and hypopharyngeal gland (HPG), where foragers were enriched in transcripts that encode antimicrobial peptides (AMPs) and immune response. Additionally, forager HPGs and mandibular glands were enriched in transcripts encoding detoxification enzymes, including some associated with xenobiotic metabolism. Using qPCR on an independent dataset, we verified differential expression of three AMP and three P450 genes between foragers and nurses. High expression of AMP genes in nectar-processing tissues suggests that these peptides may contribute to antimicrobial properties of honey or to honey bee defense against environmentally-acquired microorganisms. Together, these results suggest that worker role and tissue-specific expression of AMPs, and immune and detoxification enzymes may contribute to defense against microorganisms and xenobiotic compounds acquired while foraging.

Mapping of Human FOXP2 Enhancers Reveals Complex Regulation.

PubMed

Becker, Martin; Devanna, Paolo; Fisher, Simon E; Vernes, Sonja C

2018-01-01

Mutations of the FOXP2 gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with FOXP2 mutations have structural and functional alterations affecting brain circuits that overlap with sites of FOXP2 expression, including regions of the cortex, striatum, and cerebellum. FOXP2 displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of FOXP2 or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human FOXP2 locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the FOXP2 promoter and enhancer regions by candidate regulators - FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human FOXP2 . Understanding the upstream regulatory pathways controlling FOXP2 expression will bring new insight into the molecular networks contributing to human language and related disorders.

Mapping of Human FOXP2 Enhancers Reveals Complex Regulation

PubMed Central

Becker, Martin; Devanna, Paolo; Fisher, Simon E.; Vernes, Sonja C.

2018-01-01

Mutations of the FOXP2 gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with FOXP2 mutations have structural and functional alterations affecting brain circuits that overlap with sites of FOXP2 expression, including regions of the cortex, striatum, and cerebellum. FOXP2 displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of FOXP2 or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human FOXP2 locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the FOXP2 promoter and enhancer regions by candidate regulators – FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human FOXP2. Understanding the upstream regulatory pathways controlling FOXP2 expression will bring new insight into the molecular networks contributing to human language and related disorders. PMID:29515369

Forager bees (Apis mellifera) highly express immune and detoxification genes in tissues associated with nectar processing

PubMed Central

Vannette, Rachel L.; Mohamed, Abbas; Johnson, Brian R.

2015-01-01

Pollinators, including honey bees, routinely encounter potentially harmful microorganisms and phytochemicals during foraging. However, the mechanisms by which honey bees manage these potential threats are poorly understood. In this study, we examine the expression of antimicrobial, immune and detoxification genes in Apis mellifera and compare between forager and nurse bees using tissue-specific RNA-seq and qPCR. Our analysis revealed extensive tissue-specific expression of antimicrobial, immune signaling, and detoxification genes. Variation in gene expression between worker stages was pronounced in the mandibular and hypopharyngeal gland (HPG), where foragers were enriched in transcripts that encode antimicrobial peptides (AMPs) and immune response. Additionally, forager HPGs and mandibular glands were enriched in transcripts encoding detoxification enzymes, including some associated with xenobiotic metabolism. Using qPCR on an independent dataset, we verified differential expression of three AMP and three P450 genes between foragers and nurses. High expression of AMP genes in nectar-processing tissues suggests that these peptides may contribute to antimicrobial properties of honey or to honey bee defense against environmentally-acquired microorganisms. Together, these results suggest that worker role and tissue-specific expression of AMPs, and immune and detoxification enzymes may contribute to defense against microorganisms and xenobiotic compounds acquired while foraging. PMID:26549293

A Role for the Nucleosome Assembly Proteins TAF-Iβ and NAP1 in the Activation of BZLF1 Expression and Epstein-Barr Virus Reactivation

PubMed Central

Frappier, Lori

2013-01-01

The reactivation of Epstein-Barr virus (EBV) from latent to lytic infection begins with the expression of the viral BZLF1 gene, leading to a subsequent cascade of viral gene expression and amplification of the EBV genome. Using RNA interference, we show that nucleosome assembly proteins NAP1 and TAF-I positively contribute to EBV reactivation in epithelial cells through the induction of BZLF1 expression. In addition, overexpression of NAP1 or the β isoform of TAF-I (TAF-Iβ) in AGS cells latently infected with EBV was sufficient to induce BZLF1 expression. Chromatin immunoprecipitation experiments performed in AGS-EBV cells showed that TAF-I associated with the BZLF1 promoter upon lytic induction and affected local histone modifications by increasing H3K4 dimethylation and H4K8 acetylation. MLL1, the host protein known to dimethylate H3K4, was found to associate with the BZLF1 promoter upon lytic induction in a TAF-I-dependent manner, and MLL1 depletion decreased BZLF1 expression, confirming its contribution to lytic reactivation. The results indicate that TAF-Iβ promotes BZLF1 expression and subsequent lytic infection by affecting chromatin at the BZLF1 promoter. PMID:23691099

A role for the nucleosome assembly proteins TAF-Iβ and NAP1 in the activation of BZLF1 expression and Epstein-Barr virus reactivation.

PubMed

Mansouri, Sheila; Wang, Shan; Frappier, Lori

2013-01-01

The reactivation of Epstein-Barr virus (EBV) from latent to lytic infection begins with the expression of the viral BZLF1 gene, leading to a subsequent cascade of viral gene expression and amplification of the EBV genome. Using RNA interference, we show that nucleosome assembly proteins NAP1 and TAF-I positively contribute to EBV reactivation in epithelial cells through the induction of BZLF1 expression. In addition, overexpression of NAP1 or the β isoform of TAF-I (TAF-Iβ) in AGS cells latently infected with EBV was sufficient to induce BZLF1 expression. Chromatin immunoprecipitation experiments performed in AGS-EBV cells showed that TAF-I associated with the BZLF1 promoter upon lytic induction and affected local histone modifications by increasing H3K4 dimethylation and H4K8 acetylation. MLL1, the host protein known to dimethylate H3K4, was found to associate with the BZLF1 promoter upon lytic induction in a TAF-I-dependent manner, and MLL1 depletion decreased BZLF1 expression, confirming its contribution to lytic reactivation. The results indicate that TAF-Iβ promotes BZLF1 expression and subsequent lytic infection by affecting chromatin at the BZLF1 promoter.

Streptococcal Adhesin P (SadP) contributes to Streptococcus suis adhesion to the human intestinal epithelium.

PubMed

Ferrando, Maria Laura; Willemse, Niels; Zaccaria, Edoardo; Pannekoek, Yvonne; van der Ende, Arie; Schultsz, Constance

2017-01-01

Streptococcus suis is a zoonotic pathogen, causing meningitis and septicemia. We previously demonstrated that the gastrointestinal tract (GIT) is an entry site for zoonotic S. suis infection. Here we studied the contribution of Streptococcal adhesin Protein (SadP) to host-pathogen interaction at GIT level. SadP expression in presence of Intestinal Epithelial Cells (IEC) was compared with expression of other virulence factors by measuring transcript levels using quantitative Real Time PCR (qRT-PCR). SadP variants were identified by phylogenetic analysis of complete DNA sequences. The interaction of SadP knockout and complementation mutants with IEC was tested in vitro. Expression of sadP was significantly increased in presence of IEC. Sequence analysis of 116 invasive strains revealed five SadP sequence variants, correlating with genotype. SadP1, present in zoonotic isolates of clonal complex 1, contributed to binding to both human and porcine IEC and translocation across human IEC. Antibodies against the globotriaosylceramide Gb3/CD77 receptor significantly inhibited adhesion to human IEC. SadP is involved in the host-pathogen interaction in the GIT. Differences between SadP variants may determine different affinities to the Gb3/CD77 host-receptor, contributing to variation in adhesion capacity to host IEC and thus to S. suis zoonotic potential.

Dopaminergic dysregulation in mice selectively bred for excessive exercise or obesity.

PubMed

Mathes, Wendy Foulds; Nehrenberg, Derrick L; Gordon, Ryan; Hua, Kunjie; Garland, Theodore; Pomp, Daniel

2010-07-11

Dysregulation of the dopamine system is linked to various aberrant behaviors, including addiction, compulsive exercise, and hyperphagia leading to obesity. The goal of the present experiments was to determine how dopamine contributes to the expression of opposing phenotypes, excessive exercise and obesity. We hypothesized that similar alterations in dopamine and dopamine-related gene expression may underly obesity and excessive exercise, as competing traits for central reward pathways. Moreover, we hypothesized that selective breeding for high levels of exercise or obesity may have influenced genetic variation controlling these pathways, manifesting as opposing complex traits. Dopamine, dopamine-related peptide concentrations, and gene expression were evaluated in dorsal striatum (DS) and nucleus accumbens (NA) of mice from lines selectively bred for high rates of wheel running (HR) or obesity (M16), and the non-selected ICR strain from which these lines were derived. HPLC analysis showed significantly greater neurotransmitter concentrations in DS and NA of HR mice compared to M16 and ICR. Microarray analysis showed significant gene expression differences between HR and M16 compared to ICR in both brain areas, with changes revealed throughout the dopamine pathway including D1 and D2 receptors, associated G-proteins (e.g., Golf), and adenylate cyclase (e.g., Adcy5). The results suggest that similar modifications within the dopamine system may contribute to the expression of opposite phenotypes in mice, demonstrating that alterations within central reward pathways can contribute to both obesity and excessive exercise. Copyright 2010 Elsevier B.V. All rights reserved.

Dopaminergic Dysregulation in Mice Selectively Bred for Excessive Exercise or Obesity

PubMed Central

Nehrenberg, Derrick L.; Gordon, Ryan; Hua, Kunjie; Garland, Theodore; Pomp, Daniel

2010-01-01

Dysregulation of the dopamine system is linked to various aberrant behaviors, including addiction, compulsive exercise, and hyperphagia leading to obesity. The goal of the present experiments was to determine how dopamine contributes to the expression of opposing phenotypes, excessive exercise and obesity. We hypothesized that similar alterations in dopamine and dopamine-related gene expression may underly obesity and excessive exercise, as competing traits for central reward pathways. Moreover, we hypothesized that selective breeding for high levels of exercise or obesity may have influenced genetic variation controlling these pathways, manifesting as opposing complex traits. Dopamine, dopamine-related peptide concentrations, and gene expression were evaluated in dorsal striatum (DS) and nucleus accumbens (NA) of mice from lines selectively bred for high rates of wheel running (HR) or obesity (M16), and the non-selected ICR strain from which these lines were derived. HPLC analysis showed significantly greater neurotransmitter concentrations in DS and NA of HR mice compared to M16 and ICR. Microarray analysis showed significant gene expression differences between HR and M16 compared to ICR in both brain areas, with changes revealed throughout the dopamine pathway including D1 and D2 receptors, associated G-proteins (eg. Golf), and adenylate cyclase (eg. Adcy5). The results suggest similar modifications within the dopamine system may contribute to the expression of opposite phenotypes in mice, demonstrating that alterations within central reward pathways can contribute to both obesity and excessive exercise. PMID:20156488

Effects of castration on expression of lipid metabolism genes in the liver of korean cattle.

PubMed

Baik, Myunggi; Nguyen, Trang Hoa; Jeong, Jin Young; Piao, Min Yu; Kang, Hyeok Joong

2015-01-01

Castration induces the accumulation of body fat and deposition of intramuscular fat in Korean cattle, resulting in improved beef quality. However, little is known about the metabolic adaptations in the liver following castration. To understand changes in lipid metabolism following castration, hepatic expression levels of lipid metabolism genes were compared between Korean bulls and steers. Steers had higher (p<0.001) hepatic lipids contents and higher (p<0.01) mRNA levels of lipogenic acetyl-CoA carboxylase. This differential gene expression may, in part, contribute to increased hepatic lipid content following the castration of bulls. However, we found no differences in the hepatic expression levels of genes related to triglyceride synthesis (mitochondrial glycerol-3-phosphate acyltransferase, diacylglycerol O-acyltransferase 1 and 2) and fatty acid (FA) oxidation (carnitine palmitoyltransferase 1A, C-4 to C-12 straight chain acyl-CoA dehydrogenase, very long chain acyl-CoA dehydrogenase) between bulls and steers. No differences in gene expression for very-low-density lipoprotein (VLDL) secretion, including apolipoprotein B mRNA and microsomal triglyceride transfer protein (MTTP) protein, were observed in the liver although MTTP mRNA levels were higher in steers compared to bulls. In conclusion, FA synthesis may contribute to increased hepatic lipid deposition in steers following castration. However, hepatic lipid metabolism, including triglyceride synthesis, FA oxidation, and VLDL secretion, was not significantly altered by castration. Our results suggest that hepatic lipid metabolism does not significantly contribute to increased body fat deposition in steers following castration.

H63D mutation in hemochromatosis alters cholesterol metabolism and induces memory impairment.

PubMed

Ali-Rahmani, Fatima; Grigson, Patricia S; Lee, Sang; Neely, Elizabeth; Connor, James R; Schengrund, Cara-Lynne

2014-06-01

The H63D variant of the hemochromatosis (HFE) gene, when expressed in carriers of the apolipoprotein E4 allele, is implicated as a risk factor for earlier onset of Alzheimer's disease (AD). We tested the hypothesis that like expression of apolipoprotein E4, expression of H63D-HFE disrupts cholesterol metabolism contributing to an increase in neurodegeneration and memory deficits. Analysis of SH-SY5Y human neuroblastoma cells transfected to stably express either wild type- (WT) or H63D-HFE indicated about a 50% reduction in cholesterol content in cells expressing H63D-HFE. This was accompanied by a significant decrease in expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase, and a significant increase in expression of cholesterol 24-hydroxylase. Consistent with these studies, H67D-HFE (orthologous to human H63D-HFE) knock-in mice, showed a greater age dependent decline in brain cholesterol than WT-HFE animals and changes in expression of proteins regulating cholesterol metabolism. Brains of aged H67D-HFE mice also exhibited a significant decrease in expression of synapse proteins and a significant increase in caspase-3 expression relative to WT-HFE controls. H67D-HFE mice also had a greater reduction in brain volume and poorer recognition and spatial memory than WT-HFE mice, symptoms associated with AD. These results indicate that the alterations in cholesterol metabolism associated with expression of H63D-HFE may contribute to the development of AD. Copyright © 2014 Elsevier Inc. All rights reserved.

KSHV LANA and EBV LMP1 induce the expression of UCH-L1 following viral transformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bentz, Gretchen L.; Bheda-Malge, Anjali; Wang, Ling

Ubiquitin C-terminal Hydrolase L1 (UCH-L1) has oncogenic properties and is highly expressed during malignancies. We recently documented that Epstein-Barr virus (EBV) infection induces uch-l1 expression. Here we show that Kaposi's Sarcoma-associated herpesvirus (KSHV) infection induced UCH-L1 expression, via cooperation of KSHV Latency-Associated Nuclear Antigen (LANA) and RBP-Jκ and activation of the uch-l1 promoter. UCH-L1 expression was also increased in Primary Effusion Lymphoma (PEL) cells co-infected with KSHV and EBV compared with PEL cells infected only with KSHV, suggesting EBV augments the effect of LANA on uch-l1. EBV latent membrane protein 1 (LMP1) is one of the few EBV products expressedmore » in PEL cells. Results showed that LMP1 was sufficient to induce uch-l1 expression, and co-expression of LMP1 and LANA had an additive effect on uch-l1 expression. These results indicate that viral latency products of both human γ-herpesviruses contribute to uch-l1 expression, which may contribute to the progression of lymphoid malignancies. - Highlights: • Infection of endothelial cells with KSHV induced UCH-L1 expression. • KSHV LANA is sufficient for the induction of uch-l1. • Co-infection with KSHV and EBV (observed in some PELs) results in the additive induction of uch-l1. • EBV LMP1 also induced UCH-L1 expression. • LANA- and LMP1-mediated activation of the uch-l1 promoter is in part through RBP-Jκ.« less

Nuclear Multidrug-Resistance Related Protein 1 Contributes to Multidrug-Resistance of Mucoepidermoid Carcinoma Mainly via Regulating Multidrug-Resistance Protein 1: A Human Mucoepidermoid Carcinoma Cells Model and Spearman's Rank Correlation Analysis

PubMed Central

Liu, Yuan; Xu, Xiaofang; Guan, Sumin; Wu, Junzheng; Liu, Yanpu

2013-01-01

Background Multidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of mucoepidermoid carcinoma (MEC). The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells. Methods Western blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1) in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi). Immunohistochemistry (IHC) staining of 127 cases of MEC tissues was scored with the expression index (EI). The EI of MDR1 and MRP1 (or nuclear MRP1) was analyzed with Spearman's rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter. Results MRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues. Conclusions Our findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors. PMID:24013781

Mechanisms of impaired nephrogenesis with fetal growth restriction: altered renal transcription and growth factor expression

PubMed Central

Abdel-Hakeem, Ahmed K; Henry, Tasmia Q; Magee, Thomas R; Desai, Mina; Ross, Michael; Mansano, Roy; Torday, John; Nast, Cynthia C.

2010-01-01

Objective Maternal food restriction during pregnancy results in growth restricted newborns and reduced glomerular number, contributing to programmed offspring hypertension. We investigated whether reduced nephrogenesis may be programmed by dysregulation of factors controlling ureteric bud branching and mesenchyme to epithelial transformation. Study Design 10 to 20 days gestation, Sprague Dawley pregnant rats (n=6/group) received ad libitum food; FR rats were 50% food restricted. At embryonic day 20, mRNA and protein expression of WT1, Pax2, FGF2, GDNF, cRET, WNT4, WNT11, BMP4, BMP7, and FGF7 were determined by real-time PCR and Western blotting. Results Maternal FR resulted in up-regulated mRNA expression for WT1, FGF2, and BMP7 whereas Pax2, GDNF, FGF7, BMP4, WNT4, and WNT11 mRNAs were down-regulated. Protein expression was concordant for WT1, GDNF, Pax2, FGF7, BMP4 and WNT4. Conclusion Maternal FR altered gene expression of fetal renal transcription and growth factors, and likely contributes to development of offspring hypertension. PMID:18639218

Writing Abilities Longitudinally Predict Academic Outcomes of Adolescents with ADHD

PubMed Central

Molitor, Stephen J.; Langberg, Joshuah M.; Bourchtein, Elizaveta; Eddy, Laura D.; Dvorsky, Melissa R.; Evans, Steven W.

2016-01-01

Students with ADHD often experience a host of negative academic outcomes and deficits in reading and mathematics abilities contribute to these academic impairments. Students with ADHD may also have difficulties with written expression but there has been minimal research in this area and it is not clear whether written expression abilities uniquely contribute to the academic functioning of students with ADHD. The current study included a sample of 104 middle school students diagnosed with ADHD (grades 6–8). Participants were followed longitudinally to evaluate whether written expression abilities at baseline predicted student GPA and parent ratings of academic impairment 18 months later, after controlling for reading ability and additional relevant covariates. Written expression abilities longitudinally predicted both academic outcomes above and beyond ADHD and ODD symptoms, medication use, reading ability, and baseline values of GPA and parent-rated academic impairment. Follow-up analyses revealed that no single aspect of written expression was demonstrably more impactful on academic outcomes than the others, suggesting that writing as an entire process should be the focus of intervention. PMID:26783650

MicroRNAs shape circadian hepatic gene expression on a transcriptome-wide scale

PubMed Central

Du, Ngoc-Hien; Arpat, Alaaddin Bulak; De Matos, Mara; Gatfield, David

2014-01-01

A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation. DOI: http://dx.doi.org/10.7554/eLife.02510.001 PMID:24867642

Endometrial Expression of Steroidogenic Factor 1 Promotes Cystic Glandular Morphogenesis

PubMed Central

Vasquez, Yasmin M.; Wu, San-Pin; Anderson, Matthew L.; Hawkins, Shannon M.; Creighton, Chad J.; Ray, Madhumita; Tsai, Sophia Y.; Tsai, Ming-Jer; Lydon, John P.

2016-01-01

Epigenetic silencing of steroidogenic factor 1 (SF1) is lost in endometriosis, potentially contributing to de novo local steroidogenesis favoring inflammation and growth of ectopic endometrial tissue. In this study, we examine the impact of SF1 expression in the eutopic uterus by a novel mouse model that conditionally expresses SF1 in endometrium. In vivo SF1 expression promoted the development of enlarged endometrial glands and attenuated estrogen and progesterone responsiveness. Endometriosis induction by autotransplantation of uterine tissue to the mesenteric membrane resulted in the increase in size of ectopic lesions from SF1-expressing mice. By integrating the SF1-dependent transcriptome with the whole genome binding profile of SF1, we identified uterine-specific SF1-regulated genes involved in Wingless and Progesterone receptor-Hedgehog-Chicken ovalbumin upstream promoter transcription factor II signaling for gland development and epithelium-stroma interaction, respectively. The present results indicate that SF1 directly contributes to the abnormal uterine gland morphogenesis, an inhibition of steroid hormone signaling and activation of an immune response, in addition to previously postulated estrogen production. PMID:27018534

In vivo gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials.

PubMed Central

Heithoff, D M; Sinsheimer, R L; Low, D A; Mahan, M J

2000-01-01

Microbial pathogens possess a repertoire of virulence determinants that each make unique contributions to fitness during infection. Analysis of these in vivo-expressed functions reveals the biology of the infection process, encompassing the bacterial infection strategies and the host ecological and environmental retaliatory strategies designed to combat them (e.g. thermal, osmotic, oxygen, nutrient and acid stress). Many of the bacterial virulence functions that contribute to a successful infection are normally only expressed during infection. A genetic approach was used to isolate mutants that ectopically expressed many of these functions in a laboratory setting. Lack of DNA adenine methylase (Dam) in Salmonella typhimurium abolishes the preferential expression of many bacterial virulence genes in host tissues. Dam- Salmonella were proficient in colonization of mucosal sites but were defective in colonization of deeper tissue sites. Additionally, Dam- mutants were totally avirulent and effective as live vaccines against murine typhoid fever. Since dam is highly conserved in many pathogenic bacteria that cause significant morbidity and mortality worldwide, Dams are potentially excellent targets for both vaccines and antimicrobials. PMID:10874736

Site-Specific Expression of Polycomb-Group Genes Encoding the HPC-HPH/PRC1 Complex in Clinically Defined Primary Nodal and Cutaneous Large B-Cell Lymphomas

PubMed Central

Raaphorst, Frank M.; Vermeer, Maarten; Fieret, Elly; Blokzijl, Tjasso; Dukers, Danny; Sewalt, Richard G.A.B.; Otte, Arie P.; Willemze, Rein; Meijer, Chris J.L.M.

2004-01-01

Polycomb-group (PcG) genes preserve cell identity by gene silencing, and contribute to regulation of lymphopoiesis and malignant transformation. We show that primary nodal large B-cell lymphomas (LBCLs), and secondary cutaneous deposits from such lymphomas, abnormally express the BMI-1, RING1, and HPH1 PcG genes in cycling neoplastic cells. By contrast, tumor cells in primary cutaneous LBCLs lacked BMI-1 expression, whereas RING1 was variably detected. Lack of BMI-1 expression was characteristic for primary cutaneous LBCLs, because other primary extranodal LBCLs originating from brain, testes, and stomach were BMI-1-positive. Expression of HPH1 was rarely detected in primary cutaneous LBCLs of the head or trunk and abundant in primary cutaneous LBCLs of the legs, which fits well with its earlier recognition as a distinct clinical pathological entity with different clinical behavior. We conclude that clinically defined subclasses of primary LBCLs display site-specific abnormal expression patterns of PcG genes of the HPC-HPH/PRC1 PcG complex. Some of these patterns (such as the expression profile of BMI-1) may be diagnostically relevant. We propose that distinct expression profiles of PcG genes results in abnormal formation of HPC-HPH/PRC1 PcG complexes, and that this contributes to lymphomagenesis and different clinical behavior of clinically defined LBCLs. PMID:14742259

Determination of the Genetic Architecture Underlying Short Wavelength Sensitivity in Lake Malawi Cichlids.

PubMed

Nandamuri, Sri Pratima; Dalton, Brian E; Carleton, Karen L

2017-06-01

African cichlids are an exemplary system to study organismal diversity and rapid speciation. Species differ in external morphology including jaw shape and body coloration, but also differ in sensory systems including vision. All cichlids have 7 cone opsin genes with species differing broadly in which opsins are expressed. The differential opsin expression results in closely related species with substantial differences in spectral sensitivity of their photoreceptors. In this work, we take a first step in determining the genetic basis of opsin expression in cichlids. Using a second generation cross between 2 species with different opsin expression patterns, we make a conservative estimate that short wavelength opsin expression is regulated by a few loci. Genetic mapping in 96 F2 hybrids provides clear evidence of a cis-regulatory region for SWS1 opsin that explains 34% of the variation in expression between the 2 species. Additionally, in situ hybridization has shown that SWS1 and SWS2B opsins are coexpressed in individual single cones in the retinas of F2 progeny. Results from this work will contribute to a better understanding of the genetic architecture underlying opsin expression. This knowledge will help answer long-standing questions about the evolutionary processes fundamental to opsin expression variation and how this contributes to adaptive cichlid divergence. © The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Central Role for Heme Oxygenase-1 in the Control of Intestinal Epithelial Chemokine Expression.

PubMed

Onyiah, Joseph C; Schaefer, Rachel E M; Colgan, Sean P

2018-05-23

In mucosal inflammatory disorders, the protective influence of heme oxygenase-1 (HO-1) and its metabolic byproducts, carbon monoxide (CO) and biliverdin, is a topic of significant interest. Mechanisms under investigation include the regulation of macrophage function and mucosal cytokine expression. While there is an increasing recognition of the importance of epithelial-derived factors in the maintenance of intestinal mucosal homeostasis, the contribution of intestinal epithelial cell (IEC) HO-1 on inflammatory responses has not previously been investigated. We examined the influence of modulating HO-1 expression on the inflammatory response of human IECs. Engineered deficiency of HO-1 in Caco-2 and T84 IECs led to increased proinflammatory chemokine expression in response to pathogenic bacteria and inflammatory cytokine stimulation. Crosstalk with activated leukocytes also led to increased chemokine expression in HO-1-deficient cells in an IL-1β dependent manner. Treatment of Caco-2 cells with a pharmacological inducer of HO-1 led to the inhibition of chemokine expression. Mechanistic studies suggest that HO-1 and HO-1-related transcription factors, but not HO-1 metabolic products, are partly responsible for the influence of HO-1 on chemokine expression. In conclusion, our data identify HO-1 as a central regulator of IEC chemokine expression that may contribute to homeo-stasis in the intestinal mucosa. © 2018 S. Karger AG, Basel.

Estimating the contribution of genetic variants to difference in incidence of disease between population groups.

PubMed

Moonesinghe, Ramal; Ioannidis, John P A; Flanders, W Dana; Yang, Quanhe; Truman, Benedict I; Khoury, Muin J

2012-08-01

Genome-wide association studies have identified multiple genetic susceptibility variants to several complex human diseases. However, risk-genotype frequency at loci showing robust associations might differ substantially among different populations. In this paper, we present methods to assess the contribution of genetic variants to the difference in the incidence of disease between different population groups for different scenarios. We derive expressions for the contribution of a single genetic variant, multiple genetic variants, and the contribution of the joint effect of a genetic variant and an environmental factor to the difference in the incidence of disease. The contribution of genetic variants to the difference in incidence increases with increasing difference in risk-genotype frequency, but declines with increasing difference in incidence between the two populations. The contribution of genetic variants also increases with increasing relative risk and the contribution of joint effect of genetic and environmental factors increases with increasing relative risk of the gene-environmental interaction. The contribution of genetic variants to the difference in incidence between two populations can be expressed as a function of the population attributable risks of the genetic variants in the two populations. The contribution of a group of genetic variants to the disparity in incidence of disease could change considerably by adding one more genetic variant to the group. Any estimate of genetic contribution to the disparity in incidence of disease between two populations at this stage seems to be an elusive goal.

Estimating the contribution of genetic variants to difference in incidence of disease between population groups

PubMed Central

Moonesinghe, Ramal; Ioannidis, John PA; Flanders, W Dana; Yang, Quanhe; Truman, Benedict I; Khoury, Muin J

2012-01-01

Genome-wide association studies have identified multiple genetic susceptibility variants to several complex human diseases. However, risk-genotype frequency at loci showing robust associations might differ substantially among different populations. In this paper, we present methods to assess the contribution of genetic variants to the difference in the incidence of disease between different population groups for different scenarios. We derive expressions for the contribution of a single genetic variant, multiple genetic variants, and the contribution of the joint effect of a genetic variant and an environmental factor to the difference in the incidence of disease. The contribution of genetic variants to the difference in incidence increases with increasing difference in risk-genotype frequency, but declines with increasing difference in incidence between the two populations. The contribution of genetic variants also increases with increasing relative risk and the contribution of joint effect of genetic and environmental factors increases with increasing relative risk of the gene–environmental interaction. The contribution of genetic variants to the difference in incidence between two populations can be expressed as a function of the population attributable risks of the genetic variants in the two populations. The contribution of a group of genetic variants to the disparity in incidence of disease could change considerably by adding one more genetic variant to the group. Any estimate of genetic contribution to the disparity in incidence of disease between two populations at this stage seems to be an elusive goal. PMID:22333905

Response to the commentaries of Melissa S Anderson and Murray J Dyck.

PubMed

Kovacs, Jozsef

2013-08-01

Anderson and Dyck claim that the current trend of almost exclusively using citation-based evaluative metrics to assess the research output of scholars is unsound. I agree with them in this, but I feel that, for practical reasons, this system will not disappear in the near future, so we must concentrate on making it fairer. Both commentators doubt whether numerically expressing each contributor's relative contribution is feasible. I admit that an important precondition for this task is the possibility of an informed, democratic debate among equals about the relative contribution of each contributor to the article. Mechanisms should be established to protect vulnerable researchers in the academic field in the same way as safeguards exist today to protect vulnerable research participants. Theoretically, however, I think that the fair allocation of authorship credit is possible, and much of this task is already being performed routinely when contributors determine the order of their names in the byline, being well aware of the widespread assumption that this order mostly mirrors the order of their relative contributions. All they would have to do as an additional task is to express this order in numbers. If they cannot reach a consensus, they could always choose not to express their relative contribution in numbers, in which case the presumption would be that they contributed equally. My proposal could, at best, make the system fairer and, at worst, not reduce the options that evaluators already have.

Short communication: expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos.

PubMed

Laporta, J; Driver, A; Khatib, H

2011-08-01

Early embryo loss is a major contributing factor to cow infertility and that 70 to 80% of this loss occurs between d 8 and 16 postfertilization. However, little is known about the molecular mechanisms and the nature of genes involved in normal and abnormal embryonic development. Moreover, information is limited on the contributions of the genomes of dams and of embryos to the development and survival of preimplantation embryos. We hypothesized that proper gene expression level in the developing embryo is essential for embryo survival and pregnancy success. As such, the characterization of expression profiles in early embryos could lead to a better understanding of the mechanisms involved in normal and abnormal embryo development. To test this hypothesis, 2 d-8 embryo populations (degenerate embryos and blastocysts) that differed in morphology and developmental status were investigated. Expression levels of POU1F1 pathway genes were estimated in 4 sets of biological replicate pools of degenerate embryos and blastocysts. The OPN and STAT5A genes were found to be upregulated in degenerate embryos compared with blastocysts, whereas STAT5B showed similar expression levels in both embryo groups. Analysis of splice variants of OPN and STAT5A revealed expression patterns different from the total expression values of these genes. As such, measuring expression of individual transcripts should be considered in gene expression studies. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

ACTIVATION OF MU OPIOID RECEPTORS IN THE STRIATUM DIFFERENTIALLY AUGMENTS METHAMPHETAMINE-INDUCED GENE EXPRESSION AND ENHANCES STEREOTYPIC BEHAVIOR

PubMed Central

Horner, Kristen A.; Hebbard, John C.; Logan, Anna S.; Vanchipurakel, Golda A.; Gilbert, Yamiece E.

2013-01-01

Mu opioid receptors are densely expressed in the patch compartment of striatum and contribute to methamphetamine-induced patch-enhanced gene expression and stereotypy. In order to further elucidate the role of mu opioid receptor activation in these phenomena, we examined whether activation of mu opioid receptors would enhance methamphetamine-induced stereotypy and prodynorphin, c-fos, arc and zif/268 expression in the patch and/or matrix compartments of striatum, as well as the impact of mu opioid receptor activation on the relationship between patch-enhanced gene expression and stereotypy. Male Sprague-Dawley rats were intrastriatally infused with D-Ala(2)-N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO; 1 μg/μl), treated with methamphetamine (0.5 mg/kg) and sacrificed at 45 minutes or 2 hours later. DAMGO augmented methamphetamine-induced zif/268 mRNA expression in the patch and matrix compartments, while prodynorphin expression was increased in the dorsolateral patch compartment. DAMGO pretreatment did not affect methamphetamine-induced arc and c-fos expression. DAMGO enhanced methamphetamine-induced stereotypy and resulted in greater patch versus matrix expression of prodynorphin in the dorsolateral striatum, leading to a negative correlation between the two. These findings indicate that mu opioid receptors contribute to methamphetamine-induced stereotypy, but can differentially influence the genomic responses to methamphetamine. These data also suggest that prodynorphin may offset the overstimulation of striatal neurons by methamphetamine. PMID:22150526

Unsteady lift and thrust of a 2D flapping thin airfoil in the presence of additional leading edge vortices

NASA Astrophysics Data System (ADS)

Alaminos-Quesada, Javier; Fernandez-Feria, Ramon

2017-11-01

The effect of leading-edge vortices (LEVs) on the lift, thrust and moment of a two-dimensional heaving and pitching foil is analyzed from the unsteady, linear potential theory. General expressions taking into account the effect of unsteady point vortices interacting with the oscillatory trailing wake are first derived. Then, simplified expressions for the initial stages of the growing LEV on each half-stroke are used to obtain analytical closed expressions for the main contribution of these vortices to the lift, thrust and moment. It is found that, within the linear potential framework and the Brown-Michael model, the LEV contributes to the aerodynamic forces and moment only for combined pitching and heaving motions of the foil, being a relevant contribution for sufficiently large values of the product of the reduced frequency and the amplitude of the heaving and/or pitching motions. The results are compared with available experimental data and numerical simulations. Supported by the Ministerio de Economia y Competitividad of Spain Grants No. DPI2013-40479-P and DPI2016-76151-C2-1-R.

IL-13Rα1 expression in dopaminergic neurons contributes to their oxidative stress-mediated loss following chronic peripheral treatment with LPS

PubMed Central

Morrison, Brad E.; Marcondes, Maria Cecilia Garibaldi; Nomura, Daniel K.; Sanchez-Alavez, Manuel; Sanchez-Gonzalez, Alejandro; Saar, Indrek; Kim, Kwang-Soo; Bartfai, Tamas; Maher, Pamela; Sugama, Shuei; Conti, Bruno

2012-01-01

Inflammation and its mediators, including cytokines and reactive oxigen species, are believed to contribute to neurodegeneration. In the mouse brain, we found that the interleukin 13 receptor alpha 1 chain (IL-13Rα1) was expressed in the dopaminergic (DA) neurons of the substantia nigra pars compacta which are preferentially lost in human Parkinson’s disease (PD). Mice deficient for Il13ra1 exhibited resistance to loss of DA neurons in a model of chronic peripheral inflammation using bacterial lipopolysaccharide. Interleukin-13, as well as interleukin-4, potentiated the cytotoxic effects of t-butyl hydroperoxide and hydrogen peroxide on mouse dopaminergic MN9D cells. Collectively, our data indicate that expression of IL-13Rα1 on DA neurons can increase their susceptibility to oxidative stress-mediated damage thereby contributing to their preferential loss. In humans, Il13ra1 lies on the X chromosome within the PARK12 locus of susceptibility to PD suggesting that IL-13Rα1 may have a role in the pathogenesis of this neurodegenerative disease. PMID:23169588

Activation of the hexosamine pathway causes oxidative stress and abnormal embryo gene expression: involvement in diabetic teratogenesis.

PubMed

Horal, Melissa; Zhang, Zhiquan; Stanton, Robert; Virkamäki, Antti; Loeken, Mary R

2004-08-01

Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific biochemical pathways responsible for oxidative stress have not been fully elucidated. The hexosamine pathway is activated in many tissues during diabetes and could contribute to oxidative stress by inhibiting the pentose shunt pathway, thereby diminishing production of the cellular antioxidant, reduced glutathione (GSH). To test the hypothesis that activation of the hexosamine pathway might contribute to the teratogenic effects of diabetic pregnancy, pregnant mice were injected with glucose, to induce hyperglycemia, or glucosamine, to directly activate the hexosamine pathway. Embryo tissue fragments were also cultured in physiological glucose, high glucose, or physiological glucose plus glucosamine, to test effects on oxidative stress and embryo gene expression. Glucosamine increased hexosamine synthesis and inhibited pentose shunt activity. There was a trend for transient hyperglycemia to have the same effects, but they did not reach statistical significance. However, both glucose and glucosamine significantly decreased GSH, and increased oxidative stress, as indicated by 2',7'-dichloro-dihydrofluorescein fluorescence. Glucose and glucosamine inhibited expression of Pax-3, a gene required for neural tube closure both in vivo and in vitro, and increased neural tube defects (NTDs) in vivo; these effects were prevented by GSH ethyl ester. High glucose and glucosamine inhibited Pax-3 expression by embryo culture, but culture in glutamine-free media to block the hexosamine pathway prevented the inhibition of Pax-3 expression by high glucose. Activation of the hexosamine pathway causes oxidative stress through depletion of GSH and consequent disruption of embryo gene expression. Activation of this pathway may contribute to diabetic teratogenesis.

Collagen XIX Is Expressed by Interneurons and Contributes to the Formation of Hippocampal Synapses

PubMed Central

Su, Jianmin; Gorse, Karen; Ramirez, Francesco; Fox, Michael A.

2010-01-01

Extracellular matrix (ECM) molecules contribute to the formation and maintenance of synapses in the mammalian nervous system. We previously discovered a family of nonfibrillar collagens that organize synaptic differentiation at the neuromuscular junction (NMJ). Although many NMJ-organizing cues contribute to central nervous system (CNS) synaptogenesis, whether similar roles for collagens exist at central synapses remained unclear. In the present study we discovered that col19a1, the gene encoding nonfibrillar collagen XIX, is expressed by subsets of hippocampal neurons. Colocalization with the interneuron-specific enzyme glutamate decarboxylase 67 (Gad67), but not other cell-type-specific markers, suggests that hippocampal expression of col19a1 is restricted to interneurons. However, not all hippocampal interneurons express col19a1 mRNA; subsets of neuropeptide Y (NPY)-, somatostatin (Som)-, and calbindin (Calb)-immunoreactive interneurons express col19a1, but those containing parvalbumin (Parv) or calretinin (Calr) do not. To assess whether collagen XIX is required for the normal formation of hippocampal synapses, we examined synaptic morphology and composition in targeted mouse mutants lacking collagen XIX. We show here that subsets of synaptotagmin 2 (Syt2)-containing hippocampal nerve terminals appear malformed in the absence of collagen XIX. The presence of Syt2 in inhibitory hippocampal synapses, the altered distribution of Gad67 in collagen XIX-deficient subiculum, and abnormal levels of gephyrin in collagen XIX-deficient hippocampal extracts all suggest inhibitory synapses are affected by the loss of collagen XIX. Together, these data not only reveal that collagen XIX is expressed by central neurons, but show for the first time that a nonfibrillar collagen is necessary for the formation of hippocampal synapses. PMID:19937713

Heterogeneity in Kv2 Channel Expression Shapes Action Potential Characteristics and Firing Patterns in CA1 versus CA2 Hippocampal Pyramidal Neurons

PubMed Central

Chevaleyre, Vivien; Murray, Karl D.; Piskorowski, Rebecca A.

2017-01-01

Abstract The CA1 region of the hippocampus plays a critical role in spatial and contextual memory, and has well-established circuitry, function and plasticity. In contrast, the properties of the flanking CA2 pyramidal neurons (PNs), important for social memory, and lacking CA1-like plasticity, remain relatively understudied. In particular, little is known regarding the expression of voltage-gated K+ (Kv) channels and the contribution of these channels to the distinct properties of intrinsic excitability, action potential (AP) waveform, firing patterns and neurotransmission between CA1 and CA2 PNs. In the present study, we used multiplex fluorescence immunolabeling of mouse brain sections, and whole-cell recordings in acute mouse brain slices, to define the role of heterogeneous expression of Kv2 family Kv channels in CA1 versus CA2 pyramidal cell excitability. Our results show that the somatodendritic delayed rectifier Kv channel subunits Kv2.1, Kv2.2, and their auxiliary subunit AMIGO-1 have region-specific differences in expression in PNs, with the highest expression levels in CA1, a sharp decrease at the CA1-CA2 boundary, and significantly reduced levels in CA2 neurons. PNs in CA1 exhibit a robust contribution of Guangxitoxin-1E-sensitive Kv2-based delayed rectifier current to AP shape and after-hyperpolarization potential (AHP) relative to that seen in CA2 PNs. Our results indicate that robust Kv2 channel expression confers a distinct pattern of intrinsic excitability to CA1 PNs, potentially contributing to their different roles in hippocampal network function. PMID:28856240

Epigenetic Silencing of TAP1 in Aldefluor+ Breast Cancer Stem Cells Contributes to Their Enhanced Immune Evasion.

PubMed

Sultan, Mohammad; Vidovic, Dejan; Paine, Arianne S; Huynh, Thomas T; Coyle, Krysta M; Thomas, Margaret L; Cruickshank, Brianne M; Dean, Cheryl A; Clements, Derek R; Kim, Youra; Lee, Kristen; Gujar, Shashi A; Weaver, Ian C G; Marcato, Paola

2018-05-01

Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor + population was expanded, suggesting inherent resistance mechanisms. Gene expression analysis of the sorted tumor cells revealed that the Aldefluor + tumor cells has decreased expression of transporter associated with antigen processing (TAP) genes and co-stimulatory molecule CD80, which would decrease susceptibility to T cells. Similarly, the Aldefluor + population of patient tumors and 4T1 murine mammary cells had decreased expression of TAP and co-stimulatory molecule genes. In contrast, breast CSCs identified by CD44 + CD24 - do not have decreased expression of these genes, but do have increased expression of C-X-C chemokine receptor type 4. Decitabine treatment and bisulfite pyrosequencing suggests that DNA hypermethylation contributes to decreased TAP gene expression in Aldefluor + CSCs. TAP1 knockdown resulted in increased tumor growth of 4T1 cells in immunocompetent mice. Together, this suggests immune evasion mechanisms in breast CSCs are marker specific and epigenetic silencing of TAP1 in Aldefluor + breast CSCs contributes to their enhanced survival under immune pressure. Stem Cells 2018;36:641-654. © AlphaMed Press 2018.

Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

PubMed Central

Taubitz, Anela; Schwarz, Martin; Eltrich, Nuru; Lindenmeyer, Maja T.; Vielhauer, Volker

2013-01-01

TNF is an important mediator of glomerulonephritis. The two TNF-receptors TNFR1 and TNFR2 contribute differently to glomerular inflammation in vivo, but specific mechanisms of TNFR-mediated inflammatory responses in glomeruli are unknown. We investigated their expression and function in murine kidneys, isolated glomeruli ex vivo, and glomerular cells in vitro. In normal kidney TNFR1 and TNFR2 were preferentially expressed in glomeruli. Expression of both TNFRs and TNF-induced upregulation of TNFR2 mRNA was confirmed in murine glomerular endothelial and mesangial cell lines. In vivo, TNF exposure rapidly induced glomerular accumulation of leukocytes. To examine TNFR-specific inflammatory responses in intrinsic glomerular cells but not infiltrating leukocytes we performed microarray gene expression profiling on intact glomeruli isolated from wildtype and Tnfr-deficient mice following exposure to soluble TNF ex vivo. Most TNF-induced effects were exclusively mediated by TNFR1, including induced glomerular expression of adhesion molecules, chemokines, complement factors and pro-apoptotic molecules. However, TNFR2 contributed to TNFR1-dependent mRNA expression of inflammatory mediators in glomeruli when exposed to low TNF concentrations. Chemokine secretion was absent in TNF-stimulated Tnfr1-deficient glomeruli, but also significantly decreased in glomeruli lacking TNFR2. In vivo, TNF-induced glomerular leukocyte infiltration was abrogated in Tnfr1-deficient mice, whereas Tnfr2-deficiency decreased mononuclear phagocytes infiltrates, but not neutrophils. These data demonstrate that activation of intrinsic glomerular cells by soluble TNF requires TNFR1, whereas TNFR2 is not essential, but augments TNFR1-dependent effects. Previously described TNFR2-dependent glomerular inflammation may therefore require TNFR2 activation by membrane-bound, but not soluble TNF. PMID:23869211

Lower expression of glutamic acid decarboxylase 67 in the prefrontal cortex in schizophrenia: contribution of altered regulation by Zif268.

PubMed

Kimoto, Sohei; Bazmi, H Holly; Lewis, David A

2014-09-01

Cognitive deficits of schizophrenia may be due at least in part to lower expression of the 67-kDa isoform of glutamic acid decarboxylase (GAD67), a key enzyme for GABA synthesis, in the dorsolateral prefrontal cortex of individuals with schizophrenia. However, little is known about the molecular regulation of lower cortical GAD67 levels in schizophrenia. The GAD67 promoter region contains a conserved Zif268 binding site, and Zif268 activation is accompanied by increased GAD67 expression. Thus, altered expression of the immediate early gene Zif268 may contribute to lower levels of GAD67 mRNA in the dorsolateral prefrontal cortex in schizophrenia. The authors used polymerase chain reaction to quantify GAD67 and Zif268 mRNA levels in dorsolateral prefrontal cortex area 9 from 62 matched pairs of schizophrenia and healthy comparison subjects, and in situ hybridization to assess Zif268 expression at laminar and cellular levels of resolution. The effects of potentially confounding variables were assessed in human subjects, and the effects of antipsychotic treatments were tested in antipsychotic-exposed monkeys. The specificity of the Zif268 findings was assessed by quantifying mRNA levels for other immediate early genes. GAD67 and Zif268 mRNA levels were significantly lower and were positively correlated in the schizophrenia subjects. Both Zif268 mRNA-positive neuron density and Zif268 mRNA levels per neuron were significantly lower in the schizophrenia subjects. These findings were robust to the effects of the confounding variables examined and differed from other immediate early genes. Deficient Zif268 mRNA expression may contribute to lower cortical GAD67 levels in schizophrenia, suggesting a potential mechanistic basis for altered cortical GABA synthesis and impaired cognition in schizophrenia.

A Distinctive Cytoplasmic Tail Contributes to Low Surface Expression and Intracellular Retention of the Patr-AL MHC class I molecule1

PubMed Central

Goyos, Ana; Guethlein, Lisbeth A.; Horowitz, Amir; Hilton, Hugo G.; Gleimer, Michael; Brodsky, Frances M.; Parham, Peter

2015-01-01

Chimpanzees have orthologs of the six, fixed, functional human MHC class I genes. But in addition, the chimpanzee has a seventh functional gene, Patr-AL, which is not polymorphic but contributes substantially to population diversity by its presence on only 50% of MHC haplotypes. The ancestral AL gene emerged long before the separation of human and chimpanzee ancestors and then subsequently and specifically lost function during human evolution, but was maintained in chimpanzees. Patr-AL is an alloantigen that participates in negative and positive selection of the T-cell repertoire. The three-dimensional structure and the peptide-binding repertoire of Patr-AL and HLA-A*02 are surprisingly similar. In contrast, the expression of these two molecules is very different as shown using specific monoclonal and polyclonal antibodies made against Patr-AL. Peripheral blood cells and B cell lines express low levels of Patr-AL at the cell surface. Higher levels are seen for 221-cell transfectants expressing Patr-AL, but in these cells a large majority of Patr-AL molecules are retained in the early compartments of the secretory pathway: mainly the endoplasmic reticulum but also cis-Golgi. Replacing the cytoplasmic tail of Patr-AL with that of HLA-A*02 increased the cell-surface expression of Patr-AL substantially. Four substitutions distinguish the Patr-AL and HLA-A*02 cytoplasmic tails. Systematic mutagenesis showed that each substitution contributes changes in cell-surface expression. The combination of residues present in Patr-AL appears unique, but each individual residue is present in other primate MHC class I molecules, notably MHC-E, the most ancient of the functional human MHC class I molecules. PMID:26371256

Expression of DISC1-interactome members correlates with cognitive phenotypes related to schizophrenia.

PubMed

Rampino, Antonio; Walker, Rosie May; Torrance, Helen Scott; Anderson, Susan Maguire; Fazio, Leonardo; Di Giorgio, Annabella; Taurisano, Paolo; Gelao, Barbara; Romano, Raffaella; Masellis, Rita; Ursini, Gianluca; Caforio, Grazia; Blasi, Giuseppe; Millar, J Kirsty; Porteous, David John; Thomson, Pippa Ann; Bertolino, Alessandro; Evans, Kathryn Louise

2014-01-01

Cognitive dysfunction is central to the schizophrenia phenotype. Genetic and functional studies have implicated Disrupted-in-Schizophrenia 1 (DISC1), a leading candidate gene for schizophrenia and related psychiatric conditions, in cognitive function. Altered expression of DISC1 and DISC1-interactors has been identified in schizophrenia. Dysregulated expression of DISC1-interactome genes might, therefore, contribute to schizophrenia susceptibility via disruption of molecular systems required for normal cognitive function. Here, the blood RNA expression levels of DISC1 and DISC1-interacting proteins were measured in 63 control subjects. Cognitive function was assessed using neuropsychiatric tests and functional magnetic resonance imaging was used to assess the activity of prefrontal cortical regions during the N-back working memory task, which is abnormal in schizophrenia. Pairwise correlations between gene expression levels and the relationship between gene expression levels and cognitive function and N-back-elicited brain activity were assessed. Finally, the expression levels of DISC1, AKAP9, FEZ1, NDEL1 and PCM1 were compared between 63 controls and 69 schizophrenic subjects. We found that DISC1-interactome genes showed correlated expression in the blood of healthy individuals. The expression levels of several interactome members were correlated with cognitive performance and N-back-elicited activity in the prefrontal cortex. In addition, DISC1 and NDEL1 showed decreased expression in schizophrenic subjects compared to healthy controls. Our findings highlight the importance of the coordinated expression of DISC1-interactome genes for normal cognitive function and suggest that dysregulated DISC1 and NDEL1 expression might, in part, contribute to susceptibility for schizophrenia via disruption of prefrontal cortex-dependent cognitive functions.

Expression of DISC1-Interactome Members Correlates with Cognitive Phenotypes Related to Schizophrenia

PubMed Central

Rampino, Antonio; Walker, Rosie May; Torrance, Helen Scott; Anderson, Susan Maguire; Fazio, Leonardo; Di Giorgio, Annabella; Taurisano, Paolo; Gelao, Barbara; Romano, Raffaella; Masellis, Rita; Ursini, Gianluca; Caforio, Grazia; Blasi, Giuseppe; Millar, J. Kirsty; Porteous, David John; Thomson, Pippa Ann; Bertolino, Alessandro; Evans, Kathryn Louise

2014-01-01

Cognitive dysfunction is central to the schizophrenia phenotype. Genetic and functional studies have implicated Disrupted-in-Schizophrenia 1 (DISC1), a leading candidate gene for schizophrenia and related psychiatric conditions, in cognitive function. Altered expression of DISC1 and DISC1-interactors has been identified in schizophrenia. Dysregulated expression of DISC1-interactome genes might, therefore, contribute to schizophrenia susceptibility via disruption of molecular systems required for normal cognitive function. Here, the blood RNA expression levels of DISC1 and DISC1-interacting proteins were measured in 63 control subjects. Cognitive function was assessed using neuropsychiatric tests and functional magnetic resonance imaging was used to assess the activity of prefrontal cortical regions during the N-back working memory task, which is abnormal in schizophrenia. Pairwise correlations between gene expression levels and the relationship between gene expression levels and cognitive function and N-back-elicited brain activity were assessed. Finally, the expression levels of DISC1, AKAP9, FEZ1, NDEL1 and PCM1 were compared between 63 controls and 69 schizophrenic subjects. We found that DISC1-interactome genes showed correlated expression in the blood of healthy individuals. The expression levels of several interactome members were correlated with cognitive performance and N-back-elicited activity in the prefrontal cortex. In addition, DISC1 and NDEL1 showed decreased expression in schizophrenic subjects compared to healthy controls. Our findings highlight the importance of the coordinated expression of DISC1-interactome genes for normal cognitive function and suggest that dysregulated DISC1 and NDEL1 expression might, in part, contribute to susceptibility for schizophrenia via disruption of prefrontal cortex-dependent cognitive functions. PMID:24940743

Expression for time travel based on diffusive wave theory: applicability and considerations

NASA Astrophysics Data System (ADS)

Aguilera, J. C.; Escauriaza, C. R.; Passalacqua, P.; Gironas, J. A.

2017-12-01

Prediction of hydrological response is of utmost importance when dealing with urban planning, risk assessment, or water resources management issues. With the advent of climate change, special care must be taken with respect to variations in rainfall and runoff due to rising temperature averages. Nowadays, while typical workstations have adequate power to run distributed routing hydrological models, it is still not enough for modeling on-the-fly, a crucial ability in a natural disaster context, where rapid decisions must be made. Semi-distributed time travel models, which compute a watershed's hydrograph without explicitly solving the full shallow water equations, appear as an attractive approach to rainfall-runoff modeling since, like fully distributed models, also superimpose a grid on the watershed, and compute runoff based on cell parameter values. These models are heavily dependent on the travel time expression for an individual cell. Many models make use of expressions based on kinematic wave theory, which is not applicable in cases where watershed storage is important, such as mild slopes. This work presents a new expression for concentration times in overland flow, based on diffusive wave theory, which considers not only the effects of storage but also the effects on upstream contribution. Setting upstream contribution equal to zero gives an expression consistent with previous work on diffusive wave theory; on the other hand, neglecting storage effects (i.e.: diffusion,) is shown to be equivalent to kinematic wave theory, currently used in many spatially distributed time travel models. The newly found expression is shown to be dependent on plane discretization, particularly when dealing with very non-kinematic cases. This is shown to be the result of upstream contribution, which gets larger downstream, versus plane length. This result also provides some light on the limits on applicability of the expression: when a certain kinematic threshold is reached, the expression is no longer valid, and one must fall back to kinematic wave theory, for lack of a better option. This expression could be used for improving currently published spatially distributed time travel models, since they would become applicable in many new cases.

CHARACTERIZATION OF INFLAMMATORY GENE EXPRESSION AND GALECTIN-3 FUNCTION AFTER SPINAL CORD INJURY IN MICE

PubMed Central

Pajoohesh-Ganji, Ahdeah; Knoblach, Susan M.; Faden, Alan I.; Byrnes, Kimberly R.

2012-01-01

Inflammation has long been implicated in secondary tissue damage after spinal cord injury (SCI). Our previous studies of inflammatory gene expression in rats after SCI revealed two temporally correlated clusters: the first was expressed early after injury and the second was up-regulated later, with peak expression at 1–2 weeks and persistent up-regulation through 6 months. To further address the role of inflammation after SCI, we examined inflammatory genes in a second species, mice, through 28 days after SCI. Using anchor gene clustering analysis, we found similar expression patterns for both the acute and chronic gene clusters previously identified after rat SCI. The acute group returned to normal expression levels by 7 days post-injury. The chronic group, which included C1qB, p22phox and galectin-3, showed peak expression at 7 days and remained up-regulated through 28 days. Immunohistochemistry and western blot analysis showed that the protein expression of these genes was consistent with the mRNA expression. Further exploration of the role of one of these genes, galectin-3, suggests that galectin-3 may contribute to secondary injury. In summary, our findings extend our prior gene profiling data by demonstrating the chronic expression of a cluster of microglial associated inflammatory genes after SCI in mice. Moreover, by demonstrating that inhibition of one such factor improves recovery, the findings suggest that such chronic up-regulation of inflammatory processes may contribute to secondary tissue damage after SCI, and that there may be a broader therapeutic window for neuroprotection than generally accepted. PMID:22884909

Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

PubMed Central

Harden, Mallory E.; Prasad, Nripesh; Griffiths, Anthony

2017-01-01

ABSTRACT The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation. PMID:28049151

Diverse Cis-Regulatory Mechanisms Contribute to Expression Evolution of Tandem Gene Duplicates

PubMed Central

Baudouin-Gonzalez, Luís; Santos, Marília A; Tempesta, Camille; Sucena, Élio; Roch, Fernando; Tanaka, Kohtaro

2017-01-01

Abstract Pairs of duplicated genes generally display a combination of conserved expression patterns inherited from their unduplicated ancestor and newly acquired domains. However, how the cis-regulatory architecture of duplicated loci evolves to produce these expression patterns is poorly understood. We have directly examined the gene-regulatory evolution of two tandem duplicates, the Drosophila Ly6 genes CG9336 and CG9338, which arose at the base of the drosophilids between 40 and 60 Ma. Comparing the expression patterns of the two paralogs in four Drosophila species with that of the unduplicated ortholog in the tephritid Ceratitis capitata, we show that they diverged from each other as well as from the unduplicated ortholog. Moreover, the expression divergence appears to have occurred close to the duplication event and also more recently in a lineage-specific manner. The comparison of the tissue-specific cis-regulatory modules (CRMs) controlling the paralog expression in the four Drosophila species indicates that diverse cis-regulatory mechanisms, including the novel tissue-specific enhancers, differential inactivation, and enhancer sharing, contributed to the expression evolution. Our analysis also reveals a surprisingly variable cis-regulatory architecture, in which the CRMs driving conserved expression domains change in number, location, and specificity. Altogether, this study provides a detailed historical account that uncovers a highly dynamic picture of how the paralog expression patterns and their underlying cis-regulatory landscape evolve. We argue that our findings will encourage studying cis-regulatory evolution at the whole-locus level to understand how interactions between enhancers and other regulatory levels shape the evolution of gene expression. PMID:28961967

Hypoxic regulation of the expression of cell proliferation related genes in U87 glioma cells upon inhibition of ire1 signaling enzyme

PubMed

Minchenko, O H; Tsymbal, D O; Minchenko, D O; Riabovol, O O; Ratushna, O O; Karbovskyi, L L

2016-01-01

We have studied the effect of inhibition of IRE1 (inositol requiring enzyme 1), which is a central mediator of endoplasmic reticulum stress and a controller of cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in U87 glioma cells. It was shown that hypoxia leads to up-regulation of the expression of IL13RA2, CD24, ING1, ING2, ENDOG, and POLG genes and to down-regulation – of KRT18, TRAPPC3, TSFM, and MTIF2 genes at the mRNA level in control glioma cells. Changes for ING1 and CD24 genes were more significant. At the same time, inhibition of IRE1 modifies the effect of hypoxia on the expression of all studied genes. In particular, it increases sensitivity to hypoxia of the expression of IL13RA2, TRAPPC3, ENDOG, and PLOG genes and suppresses the effect of hypoxia on the expression of ING1 gene. Additionally, it eliminates hypoxic regulation of KRT18, CD24, ING2, TSFM, and MTIF2 genes expressions and introduces sensitivity to hypoxia of the expression of BET1 gene in glioma cells. The present study demonstrates that hypoxia, which often contributes to tumor growth, affects the expression of almost all studied genes. Additionally, inhibition of IRE1 can both enhance and suppress the hypoxic regulation of these gene expressions in a gene specific manner and thus possibly contributes to slower glioma growth, but several aspects of this regulation must be further clarified.

Interleukin-like EMT inducer regulates partial phenotype switching in MITF-low melanoma cell lines

PubMed Central

Noguchi, Ken; Dalton, Annamarie C.; Howley, Breege V.; McCall, Buckley J.; Yoshida, Akihiro; Diehl, J. Alan

2017-01-01

ILEI (FAM3C) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell biological process that confers metastatic properties to a tumor cell. Initially, we found that ILEI mRNA is highly expressed in melanoma metastases but not in primary tumors, suggesting that ILEI contributes to the malignant properties of melanoma. While melanoma is not an epithelial cell-derived tumor and does not undergo a traditional EMT, melanoma undergoes a similar process known as phenotype switching in which high (micropthalmia-related transcription factor) MITF expressing (MITF-high) proliferative cells switch to a low expressing (MITF-low) invasive state. We observed that MITF-high proliferative cells express low levels of ILEI (ILEI-low) and MITF-low invasive cells express high levels of ILEI (ILEI-high). We found that inducing phenotype switching towards the MITF-low invasive state increases ILEI mRNA expression, whereas phenotype switching towards the MITF-high proliferative state decreases ILEI mRNA expression. Next, we used in vitro assays to show that knockdown of ILEI attenuates invasive potential but not MITF expression or chemoresistance. Finally, we used gene expression analysis to show that ILEI regulates several genes involved in the MITF-low invasive phenotype including JARID1B, HIF-2α, and BDNF. Gene set enrichment analysis suggested that ILEI-regulated genes are enriched for JUN signaling, a known regulator of the MITF-low invasive phenotype. In conclusion, we demonstrate that phenotype switching regulates ILEI expression, and that ILEI regulates partial phenotype switching in MITF-low melanoma cell lines. PMID:28545079

NORMAL NASAL GENE EXPRESSION LEVELS USING CDNA ARRAY TECHNOLOGY

EPA Science Inventory

Normal Nasal Gene Expression Levels Using cDNA Array Technology.

The nasal epithelium is a target site for chemically-induced toxicity and carcinogenicity. To detect and analyze genetic events which contribute to nasal tumor development, we first defined the gene expressi...

Direct testing for allele-specific expression differences between conditions

USDA-ARS?s Scientific Manuscript database

Genetic differences in cis regulatory regions contribute to the phenotypic variation observed in natural and human populations, including beneficial, potentially adaptive, traits as well as disease states. The two alleles in a diploid cell can differ in their allele-specific expression leading to al...

Dance and Essence: Reflections on Morality and Education.

ERIC Educational Resources Information Center

Hodes, Stuart

1995-01-01

Asserts that the self-actualizing and expressive nature of dance contributes to an intuitive sense of morality. Criticizes the body versus mind dualism and antibody bias implicit in the Western intellectual tradition. Contrasts the aggressive nature of sports with the expressive character of dance. (MJP)

Intramuscular injection of AAV8 in mice and macaques is associated with substantial hepatic targeting and transgene expression.

PubMed

Greig, Jenny A; Peng, Hui; Ohlstein, Jason; Medina-Jaszek, C Angelica; Ahonkhai, Omua; Mentzinger, Anne; Grant, Rebecca L; Roy, Soumitra; Chen, Shu-Jen; Bell, Peter; Tretiakova, Anna P; Wilson, James M

2014-01-01

Intramuscular (IM) administration of adeno-associated viral (AAV) vectors has entered the early stages of clinical development with some success, including the first approved gene therapy product in the West called Glybera. In preparation for broader clinical development of IM AAV vector gene therapy, we conducted detailed pre-clinical studies in mice and macaques evaluating aspects of delivery that could affect performance. We found that following IM administration of AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly to total expression of secreted transgenes. The contribution from liver could be controlled by altering injection volume and by the use of traditional (promoter) and non-traditional (tissue-specific microRNA target sites) expression control elements. Hepatic distribution of vector following IM injection was also noted in rhesus macaques. These pre-clinical data on AAV delivery should inform safe and efficient development of future AAV products.

Reduced expression of G protein-coupled receptor kinases in schizophrenia but not in schizoaffective disorder

PubMed Central

Bychkov, ER; Ahmed, MR; Gurevich, VV; Benovic, JL; Gurevich, EV

2011-01-01

Alterations of multiple G protein-mediated signaling pathways are detected in schizophrenia. G protein-coupled receptor kinases (GRKs) and arrestins terminate signaling by G protein-coupled receptors exerting powerful influence on receptor functions. Modifications of arrestin and/or GRKs expression may contribute to schizophrenia pathology. Cortical expression of arrestins and GRKs was measured postmortem in control and subjects with schizophrenia or schizoaffective disorder. Additionally, arrestin/GRK expression was determined in elderly patients with schizophrenia and age-matched control. Patients with schizophrenia, but not schizoaffective disorder, displayed reduced concentration of arrestin and GRK mRNAs and GRK3 protein. Arrestins and GRK significantly decreased with age. In elderly patients, GRK6 was reduced, with other GRKs and arrestins unchanged. Reduced cortical concentration of GRKs in schizophrenia (resembling that in aging) may result in altered G protein-dependent signaling, thus contributing to prefrontal deficits in schizophrenia. The data suggest distinct molecular mechanisms underlying schizophrenia and schizoaffective disorder. PMID:21784156

Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans

PubMed Central

Hulver, Matthew W.; Berggren, Jason R.; Carper, Michael J.; Miyazaki, Makoto; Ntambi, James M.; Hoffman, Eric P.; Thyfault, John P.; Stevens, Robert; Dohm, G. Lynis; Houmard, Joseph A.; Muoio, Deborah M.

2014-01-01

Summary Obesity and type 2 diabetes are strongly associated with abnormal lipid metabolism and accumulation of intramyocellular triacylglycerol, but the underlying cause of these perturbations are yet unknown. Herein, we show that the lipogenic gene, stearoyl-CoA desaturase 1 (SCD1), is robustly up-regulated in skeletal muscle from extremely obese humans. High expression and activity of SCD1, an enzyme that catalyzes the synthesis of monounsaturated fatty acids, corresponded with low rates of fatty acid oxidation, increased triacylglycerol synthesis and increased monounsaturation of muscle lipids. Elevated SCD1 expression and abnormal lipid partitioning were retained in primary skeletal myocytes derived from obese compared to lean donors, implying that these traits might be driven by epigenetic and/or heritable mechanisms. Overexpression of human SCD1 in myotubes from lean subjects was sufficient to mimic the obese phenotype. These results suggest that elevated expression of SCD1 in skeletal muscle contributes to abnormal lipid metabolism and progression of obesity. PMID:16213227

Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans.

PubMed

Hulver, Matthew W; Berggren, Jason R; Carper, Michael J; Miyazaki, Makoto; Ntambi, James M; Hoffman, Eric P; Thyfault, John P; Stevens, Robert; Dohm, G Lynis; Houmard, Joseph A; Muoio, Deborah M

2005-10-01

Obesity and type 2 diabetes are strongly associated with abnormal lipid metabolism and accumulation of intramyocellular triacylglycerol, but the underlying cause of these perturbations are yet unknown. Herein, we show that the lipogenic gene, stearoyl-CoA desaturase 1 (SCD1), is robustly up-regulated in skeletal muscle from extremely obese humans. High expression and activity of SCD1, an enzyme that catalyzes the synthesis of monounsaturated fatty acids, corresponded with low rates of fatty acid oxidation, increased triacylglycerol synthesis and increased monounsaturation of muscle lipids. Elevated SCD1 expression and abnormal lipid partitioning were retained in primary skeletal myocytes derived from obese compared to lean donors, implying that these traits might be driven by epigenetic and/or heritable mechanisms. Overexpression of human SCD1 in myotubes from lean subjects was sufficient to mimic the obese phenotype. These results suggest that elevated expression of SCD1 in skeletal muscle contributes to abnormal lipid metabolism and progression of obesity.

Associations between transcriptional changes and protein phenotypes provide insights into immune regulation in corals.

PubMed

Fuess, Lauren E; Pinzόn C, Jorge H; Weil, Ernesto; Mydlarz, Laura D

2016-09-01

Disease outbreaks in marine ecosystems have driven worldwide declines of numerous taxa, including corals. Some corals, such as Orbicella faveolata, are particularly susceptible to disease. To explore the mechanisms contributing to susceptibility, colonies of O. faveolata were exposed to immune challenge with lipopolysaccharides. RNA sequencing and protein activity assays were used to characterize the response of corals to immune challenge. Differential expression analyses identified 17 immune-related transcripts that varied in expression post-immune challenge. Network analyses revealed several groups of transcripts correlated to immune protein activity. Several transcripts, which were annotated as positive regulators of immunity were included in these groups, and some were downregulated following immune challenge. Correlations between expression of these transcripts and protein activity results further supported the role of these transcripts in positive regulation of immunity. The observed pattern of gene expression and protein activity may elucidate the processes contributing to the disease susceptibility of species like O. faveolata. Copyright © 2016 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharpe, Laura J.; Brown, Andrew J.

As a key regulator of cholesterol homeostasis, sterol-regulatory element binding protein-2 (SREBP-2) up-regulates expression of genes involved in cholesterol synthesis (e.g., 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) Reductase) and uptake (the low density lipoprotein (LDL)-receptor). Previously, we showed that Akt, a critical kinase in cell growth and proliferation, contributes to SREBP-2 activation. However, the specific Akt target involved is unknown. A potential candidate is the mammalian target of rapamycin, mTOR. Rapamycin can cause hyperlipidaemia clinically, and we hypothesised that this may be mediated via an effect of mTOR on SREBP-2. Herein, we found that SREBP-2 activation and HMG-CoA Reductase gene expression were unaffectedmore » by rapamycin treatment. However, LDL-receptor gene expression was decreased by rapamycin, suggesting that this may contribute to the hyperlipidaemia observed in rapamycin-treated patients. Rapamycin did not affect mRNA stability, so the decrease in LDL-receptor gene expression is likely to be occurring at the transcriptional level, although independently of SREBP-2.« less

Lorentz-covariant coordinate-space representation of the leading hadronic contribution to the anomalous magnetic moment of the muon

NASA Astrophysics Data System (ADS)

Meyer, Harvey B.

2017-09-01

We present a Lorentz-covariant, Euclidean coordinate-space expression for the hadronic vacuum polarisation, the Adler function and the leading hadronic contribution to the anomalous magnetic moment of the muon. The representation offers a high degree of flexibility for an implementation in lattice QCD. We expect it to be particularly helpful for the quark-line disconnected contributions.

Parabiosis and single-cell RNA sequencing reveal a limited contribution of monocytes to myofibroblasts in kidney fibrosis.

PubMed

Kramann, Rafael; Machado, Flavia; Wu, Haojia; Kusaba, Tetsuro; Hoeft, Konrad; Schneider, Rebekka K; Humphreys, Benjamin D

2018-05-03

Fibrosis is the common final pathway of virtually all chronic injury to the kidney. While it is well accepted that myofibroblasts are the scar-producing cells in the kidney, their cellular origin is still hotly debated. The relative contribution of proximal tubular epithelium and circulating cells, including mesenchymal stem cells, macrophages, and fibrocytes, to the myofibroblast pool remains highly controversial. Using inducible genetic fate tracing of proximal tubular epithelium, we confirm that the proximal tubule does not contribute to the myofibroblast pool. However, in parabiosis models in which one parabiont is genetically labeled and the other is unlabeled and undergoes kidney fibrosis, we demonstrate that a small fraction of genetically labeled renal myofibroblasts derive from the circulation. Single-cell RNA sequencing confirms this finding but indicates that these cells are circulating monocytes, express few extracellular matrix or other myofibroblast genes, and express many proinflammatory cytokines. We conclude that this small circulating myofibroblast progenitor population contributes to renal fibrosis by paracrine rather than direct mechanisms.

Epigenetic modification of DRG neuronal gene expression subsequent to nerve injury: etiological contribution to complex regional pain syndromes (Part II).

PubMed

Wang, Fuzhou; Stefano, George B; Kream, Richard M

2014-07-12

Cumulating evidence indicated that nerve injury-associated cellular and molecular changes play an essential role in contributing to the development of pathological pain, and more recent findings implicated the critical role of epigenetic mechanisms in pain-related sensitization in the DRG subsequent to nerve injury. In this part of the dyad review (Part II), we reviewed and paid special attention on the etiological contribution of DGR gene expression modulated by epigenetic mechanisms of CRPS. As essential effectors to different molecular activation, we first discussed the activation of various signaling pathways that subsequently from nerve injury, and in further illustrated the fundamental and functional underpinnings of nerve injury-induced pain, in which we argued for the potential epigenetic mechanisms in response to sensitizing stimuli or injury. Therefore, understanding the specific mediating factors that influence individual epigenetic differences contributing to pain sensitivity and responsiveness to analgesics possesses crucial clinical implications.

Expression of Neuropeptides and Cytokines in a Rabbit Model of Diabetic Neuroischemic Wound-Healing

PubMed Central

Nabzdyk, Leena Pradhan; Kuchibhotla, Sarada; Guthrie, Patrick; Chun, Maggie; Auster, Michael E; Nabzdyk, Christoph; Deso, Steven; Andersen, Nicholas; LoGerfo, Frank W.; Veves, Aristidis

2013-01-01

Objective The present study is designed to understand the contribution of peripheral vascular disease and peripheral neuropathy to the wound-healing impairment associated with diabetes. Using a rabbit model of diabetic neuroischemic wound-healing we investigated rate of healing, leukocyte infiltration and expression of cytokines, Interleukin (IL)-8 and IL-6, and, neuropeptides, Substance P (SP) and Neuropeptide Y (NPY). Design of study Diabetes was induced in White New Zealand rabbits by administering alloxan while control rabbits received saline. Ten days later animals in both groups underwent surgery. One ear served as a sham and the other was made ischemic (ligation of central+rostral arteries), or neuroischemic (ischemia+ resection of central+rostral nerves). Four, 6mm punch biopsy wounds were created in both ears and wound-healing was followed for ten days using computerized planimetry. Results Non-diabetic sham and ischemic wounds healed significantly more rapidly than diabetic sham and ischemic wounds. Healing was slowest in neuroischemic wounds, irrespective of diabetic status. A high M1/M2 macrophage ratio and a high pro-inflammatory cytokine expression, both indicators of chronic-proinflammatory state, and low neuropeptide expression were seen in pre-injury diabetic skin. Post-injury, in diabetic wounds M1/M2 ratio remained high, the reactive increase in cytokine expression was low and neuropeptide expression was further decreased in neuroischemic wounds. Conclusion This rabbit model illustrates how a combination of a high M1/M2 ratio, a failure to mount post-injury cytokine response as well as a diminished neuropeptide expression contribute to wound-healing impairment in diabetes. The addition of neuropathy to ischemia leads to equivalently severe impaired wound-healing irrespective of diabetes status, suggesting that in the presence of ischemia, loss of neuropeptide function contributes to the impaired healing associated with diabetes. PMID:23755976

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis

PubMed Central

Clarke, Cassie J.; Berg, Tracy J.; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L.; Vermeulen, Peter B.; Foo, Shane; Kostaras, Eleftherios; Jones, J. Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R.; Norman, Jim C.

2016-01-01

Summary Expression of the initiator methionine tRNA (tRNAiMet) is deregulated in cancer. Despite this fact, it is not currently known how tRNAiMet expression levels influence tumor progression. We have found that tRNAiMet expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAiMet in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAiMet contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAiMet gene (2+tRNAiMet mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAiMet mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAiMet mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAiMet significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAiMet-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAiMet-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAiMet mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAiMet levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. PMID:26948875

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis.

PubMed

Clarke, Cassie J; Berg, Tracy J; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L; Vermeulen, Peter B; Foo, Shane; Kostaras, Eleftherios; Jones, J Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R; Norman, Jim C

2016-03-21

Expression of the initiator methionine tRNA (tRNAi(Met)) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi(Met) expression levels influence tumor progression. We have found that tRNAi(Met) expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi(Met) in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi(Met) contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi(Met) gene (2+tRNAi(Met) mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi(Met) mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi(Met) mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi(Met) significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi(Met)-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi(Met)-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi(Met) mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi(Met) levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

MiR-34a regulates the invasive capacity of canine osteosarcoma cell lines

PubMed Central

Lopez, Cecilia M.; Yu, Peter Y.; Zhang, Xiaoli; Yilmaz, Ayse Selen; London, Cheryl A.

2018-01-01

Background Osteosarcoma (OSA) is the most common bone tumor in children and dogs; however, no substantial improvement in clinical outcome has occurred in either species over the past 30 years. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play a fundamental role in cancer. The purpose of this study was to investigate the potential contribution of miR-34a loss to the biology of canine OSA, a well-established spontaneous model of the human disease. Methodology and principal findings RT-qPCR demonstrated that miR-34a expression levels were significantly reduced in primary canine OSA tumors and canine OSA cell lines as compared to normal canine osteoblasts. In canine OSA cell lines stably transduced with empty vector or pre-miR-34a lentiviral constructs, overexpression of miR-34a inhibited cellular invasion and migration but had no effect on cell proliferation or cell cycle distribution. Transcriptional profiling of canine OSA8 cells possessing enforced miR-34a expression demonstrated dysregulation of numerous genes, including significant down-regulation of multiple putative targets of miR-34a. Moreover, gene ontology analysis of down-regulated miR-34a target genes showed enrichment of several biological processes related to cell invasion and motility. Lastly, we validated changes in miR-34a putative target gene expression, including decreased expression of KLF4, SEM3A, and VEGFA transcripts in canine OSA cells overexpressing miR-34a and identified KLF4 and VEGFA as direct target genes of miR-34a. Concordant with these data, primary canine OSA tumor tissues demonstrated increased expression levels of putative miR-34a target genes. Conclusions These data demonstrate that miR-34a contributes to invasion and migration in canine OSA cells and suggest that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OSA. PMID:29293555

MiR-34a regulates the invasive capacity of canine osteosarcoma cell lines.

PubMed

Lopez, Cecilia M; Yu, Peter Y; Zhang, Xiaoli; Yilmaz, Ayse Selen; London, Cheryl A; Fenger, Joelle M

2018-01-01

Osteosarcoma (OSA) is the most common bone tumor in children and dogs; however, no substantial improvement in clinical outcome has occurred in either species over the past 30 years. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play a fundamental role in cancer. The purpose of this study was to investigate the potential contribution of miR-34a loss to the biology of canine OSA, a well-established spontaneous model of the human disease. RT-qPCR demonstrated that miR-34a expression levels were significantly reduced in primary canine OSA tumors and canine OSA cell lines as compared to normal canine osteoblasts. In canine OSA cell lines stably transduced with empty vector or pre-miR-34a lentiviral constructs, overexpression of miR-34a inhibited cellular invasion and migration but had no effect on cell proliferation or cell cycle distribution. Transcriptional profiling of canine OSA8 cells possessing enforced miR-34a expression demonstrated dysregulation of numerous genes, including significant down-regulation of multiple putative targets of miR-34a. Moreover, gene ontology analysis of down-regulated miR-34a target genes showed enrichment of several biological processes related to cell invasion and motility. Lastly, we validated changes in miR-34a putative target gene expression, including decreased expression of KLF4, SEM3A, and VEGFA transcripts in canine OSA cells overexpressing miR-34a and identified KLF4 and VEGFA as direct target genes of miR-34a. Concordant with these data, primary canine OSA tumor tissues demonstrated increased expression levels of putative miR-34a target genes. These data demonstrate that miR-34a contributes to invasion and migration in canine OSA cells and suggest that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OSA.

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion

PubMed Central

Valle-Leija, Pablo; Cancino-Rodezno, Angeles; Sánchez-Tafolla, Berardo M.; Arias, Erwin; Elinos, Diana; Feria, Jessica; Zetina, María E.; Morales, Miguel A.; Cifuentes, Fredy

2017-01-01

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity. PMID:28744222

Estrogen Receptor-Related Receptor α Mediates Up-Regulation of Aromatase Expression by Prostaglandin E2 in Prostate Stromal Cells

PubMed Central

Miao, Lin; Shi, Jiandang; Wang, Chun-Yu; Zhu, Yan; Du, Xiaoling; Jiao, Hongli; Mo, Zengnan; Klocker, Helmut; Lee, Chung; Zhang, Ju

2010-01-01

Estrogen receptor-related receptor α (ERRα) is an orphan member of the nuclear receptor superfamily of transcription factors. ERRα is highly expressed in the prostate, especially in prostate stromal cells. However, little is known about the regulation and function of ERRα, which may contribute to the progression of prostatic diseases. We previously found that prostaglandin E2 (PGE2) up-regulated the expression of aromatase in prostate stromal cells. Here we show that PGE2 also up-regulates the expression of ERRα, which, as a transcription factor, further mediates the regulatory effects of PGE2 on the expression of aromatase. ERRα expression was up-regulated by PGE2 in prostate stromal cell line WPMY-1, which was mediated mainly through the protein kinase A signaling pathway by PGE2 receptor EP2. Suppression of ERRα activity by chlordane (an antagonist of ERRα) or small interfering RNA knockdown of ERRα blocked the increase of expression and promoter activity of aromatase induced by PGE2. Overexpression of ERRα significantly increased aromatase expression and promoter activity, which were further augmented by PGE2. Chromatin immunoprecipitation assay demonstrated that ERRα directly bound to the aromatase promoter in vivo, and PGE2 enhanced the recruitment of ERRα and promoted transcriptional regulatory effects on aromatase expression in WPMY-1. 17β-Estradiol concentration in WPMY-1 medium was up-regulated by ERRα expression, and that was further increased by PGE2. Our results provided evidence that ERRα contributed to local estrogen production by up-regulating aromatase expression in response to PGE2 and provided further insights into the potential role of ERRα in estrogen-related prostatic diseases. PMID:20351196

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion.

PubMed

Valle-Leija, Pablo; Cancino-Rodezno, Angeles; Sánchez-Tafolla, Berardo M; Arias, Erwin; Elinos, Diana; Feria, Jessica; Zetina, María E; Morales, Miguel A; Cifuentes, Fredy

2017-01-01

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.

Mapping the emotional face. How individual face parts contribute to successful emotion recognition.

PubMed

Wegrzyn, Martin; Vogt, Maria; Kireclioglu, Berna; Schneider, Julia; Kissler, Johanna

2017-01-01

Which facial features allow human observers to successfully recognize expressions of emotion? While the eyes and mouth have been frequently shown to be of high importance, research on facial action units has made more precise predictions about the areas involved in displaying each emotion. The present research investigated on a fine-grained level, which physical features are most relied on when decoding facial expressions. In the experiment, individual faces expressing the basic emotions according to Ekman were hidden behind a mask of 48 tiles, which was sequentially uncovered. Participants were instructed to stop the sequence as soon as they recognized the facial expression and assign it the correct label. For each part of the face, its contribution to successful recognition was computed, allowing to visualize the importance of different face areas for each expression. Overall, observers were mostly relying on the eye and mouth regions when successfully recognizing an emotion. Furthermore, the difference in the importance of eyes and mouth allowed to group the expressions in a continuous space, ranging from sadness and fear (reliance on the eyes) to disgust and happiness (mouth). The face parts with highest diagnostic value for expression identification were typically located in areas corresponding to action units from the facial action coding system. A similarity analysis of the usefulness of different face parts for expression recognition demonstrated that faces cluster according to the emotion they express, rather than by low-level physical features. Also, expressions relying more on the eyes or mouth region were in close proximity in the constructed similarity space. These analyses help to better understand how human observers process expressions of emotion, by delineating the mapping from facial features to psychological representation.

Mapping the emotional face. How individual face parts contribute to successful emotion recognition

PubMed Central

Wegrzyn, Martin; Vogt, Maria; Kireclioglu, Berna; Schneider, Julia; Kissler, Johanna

2017-01-01

Which facial features allow human observers to successfully recognize expressions of emotion? While the eyes and mouth have been frequently shown to be of high importance, research on facial action units has made more precise predictions about the areas involved in displaying each emotion. The present research investigated on a fine-grained level, which physical features are most relied on when decoding facial expressions. In the experiment, individual faces expressing the basic emotions according to Ekman were hidden behind a mask of 48 tiles, which was sequentially uncovered. Participants were instructed to stop the sequence as soon as they recognized the facial expression and assign it the correct label. For each part of the face, its contribution to successful recognition was computed, allowing to visualize the importance of different face areas for each expression. Overall, observers were mostly relying on the eye and mouth regions when successfully recognizing an emotion. Furthermore, the difference in the importance of eyes and mouth allowed to group the expressions in a continuous space, ranging from sadness and fear (reliance on the eyes) to disgust and happiness (mouth). The face parts with highest diagnostic value for expression identification were typically located in areas corresponding to action units from the facial action coding system. A similarity analysis of the usefulness of different face parts for expression recognition demonstrated that faces cluster according to the emotion they express, rather than by low-level physical features. Also, expressions relying more on the eyes or mouth region were in close proximity in the constructed similarity space. These analyses help to better understand how human observers process expressions of emotion, by delineating the mapping from facial features to psychological representation. PMID:28493921

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression

PubMed Central

Tavares, Catarina; Eloy, Catarina; Melo, Miguel; Gaspar da Rocha, Adriana; Pestana, Ana; Batista, Rui; Rios, Elisabete; Sobrinho Simões, Manuel

2018-01-01

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression. PMID:29757257

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression.

PubMed

Tavares, Catarina; Eloy, Catarina; Melo, Miguel; Gaspar da Rocha, Adriana; Pestana, Ana; Batista, Rui; Bueno Ferreira, Luciana; Rios, Elisabete; Sobrinho Simões, Manuel; Soares, Paula

2018-05-13

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression.

Gestalt Imagery: A Critical Factor in Language Comprehension.

ERIC Educational Resources Information Center

Bell, Nanci

1991-01-01

Lack of gestalt imagery (the ability to create imaged wholes) can contribute to language comprehension disorder characterized by weak reading comprehension, weak oral language comprehension, weak oral language expression, weak written language expression, difficulty following directions, and a weak sense of humor. Sequential stimulation using an…

Gene Expression Profiles in Developing Corn Kernels in Response to Drought Stress

USDA-ARS?s Scientific Manuscript database

Preharvest aflatoxin contamination of corn is aggravated by abiotic stress and the hot and humid weather patterns along with suboptimal summer rainfall favor the development. Drought stress is a major factor known to contribute to preharvest aflatoxin contamination. The gene expression studies were ...

Deficiency of the protein-tyrosine phosphatase DEP-1/PTPRJ promotes matrix metalloproteinase-9 expression in meningioma cells.

PubMed

Petermann, Astrid; Stampnik, Yvonn; Cui, Yan; Morrison, Helen; Pachow, Doreen; Kliese, Nadine; Mawrin, Christian; Böhmer, Frank-D

2015-05-01

Brain-invasive growth of a subset of meningiomas is associated with less favorable prognosis. The molecular mechanisms causing invasiveness are only partially understood, however, the expression of matrix metalloproteinases (MMPs) has been identified as a contributing factor. We have previously found that loss of density enhanced phosphatase-1 (DEP-1, also designated PTPRJ), a transmembrane protein-tyrosine phosphatase, promotes meningioma cell motility and invasive growth in an orthotopic xenotransplantation model. We have now analyzed potential alterations of the expression of genes involved in motility control, caused by DEP-1 loss in meningioma cell lines. DEP-1 depleted cells exhibited increased expression of mRNA encoding MMP-9, and the growth factors EGF and FGF-2. The increase of MMP-9 expression in DEP-1 depleted cells was also readily detectable at the protein level by zymography. MMP-9 upregulation was sensitive to chemical inhibitors of growth factor signal transduction. Conversely, MMP-9 mRNA levels could be stimulated with growth factors (e.g. EGF) and inflammatory cytokines (e.g. TNFα). Increase of MMP-9 expression by DEP-1 depletion, or growth factor/cytokine stimulation qualitatively correlated with increased invasiveness in vitro scored as transmigration through matrigel-coated membranes. The studies suggest induction of MMP-9 expression promoted by DEP-1 deficiency, or potentially by growth factors and inflammatory cytokines, as a mechanism contributing to meningioma brain invasiveness.

Cytokine-induced CEACAM1 expression on keratinocytes is characteristic for psoriatic skin and contributes to a prolonged lifespan of neutrophils.

PubMed

Rahmoun, Massilva; Molès, Jean-Pierre; Pedretti, Nathalie; Mathieu, Marc; Fremaux, Isabelle; Raison-Peyron, Nadia; Lecron, Jean-Claude; Yssel, Hans; Pène, Jérôme

2009-03-01

Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a cell-surface glycoprotein, belonging to the carcinoembryonic antigen family, expressed by human neutrophils, epithelial cells, activated T and NK cells. CEACAM1 is expressed as a cell-surface molecule with different isoforms or can be secreted as a soluble protein. Here, we show that keratinocytes in the outer epidermal layer of psoriatic skin express CEACAM1, unlike those in healthy skin or in cutaneous lesions of patients with atopic or nummular dermatitis. Stimulation of primary human keratinocytes or in vitro reconstituted epidermis with culture supernatants of activated psoriatic lesion-infiltrating T cells, IFN-gamma or oncostatin M, but not IL-17, induced the expression of transcripts for the CEACAM1-long and -short isoforms and cell-surface CEACAM1, whereas soluble CEACAM1 was not produced. The uppermost layers of the epidermis in psoriatic lesions also contain neutrophils, a cell type with inflammatory and antimicrobial properties. Coculture of CEACAM1-expressing keratinocytes or CHO transfectants with neutrophils delayed spontaneous apoptosis of the latter cells. These results show that cytokine-induced cell-surface expression of CEACAM1 by keratinocytes in the context of a psoriatic environment might contribute to the persistence of neutrophils and thus to ongoing inflammation and the decreased propensity for skin infection, typical for patients with psoriasis.

HIV-1-negative female sex workers sustain high cervical IFNɛ, low immune activation, and low expression of HIV-1-required host genes.

PubMed

Abdulhaqq, S A; Zorrilla, C; Kang, G; Yin, X; Tamayo, V; Seaton, K E; Joseph, J; Garced, S; Tomaras, G D; Linn, K A; Foulkes, A S; Azzoni, L; VerMilyea, M; Coutifaris, C; Kossenkov, A V; Showe, L; Kraiselburd, E N; Li, Q; Montaner, L J

2016-07-01

Sex workers practicing in high HIV endemic areas have been extensively targeted to test anti-HIV prophylactic strategies. We hypothesize that in women with high levels of genital exposure to semen changes in cervico-vaginal mucosal and/or systemic immune activation will contribute to a decreased susceptibility to HIV-1 infection. To address this question, we assessed sexual activity and immune activation status (in peripheral blood), as well as cellular infiltrates and gene expression in ectocervical mucosa biopsies in female sex workers (FSWs; n=50), as compared with control women (CG; n=32). FSWs had low-to-absent HIV-1-specific immune responses with significantly lower CD38 expression on circulating CD4(+) or CD8(+) T-cells (both: P<0.001) together with lower cervical gene expression of genes associated with leukocyte homing and chemotaxis. FSWs also had increased levels of interferon-ɛ (IFNɛ) gene and protein expression in the cervical epithelium together with reduced expression of genes associated with HIV-1 integration and replication. A correlative relationship between semen exposure and elevated type-1 IFN expression in FSWs was also established. Overall, our data suggest that long-term condomless sex work can result in multiple changes within the cervico-vaginal compartment that would contribute to sustaining a lower susceptibility for HIV-1 infection in the absence of HIV-specific responses.

The impact of rare variation on gene expression across tissues.

PubMed

Li, Xin; Kim, Yungil; Tsang, Emily K; Davis, Joe R; Damani, Farhan N; Chiang, Colby; Hess, Gaelen T; Zappala, Zachary; Strober, Benjamin J; Scott, Alexandra J; Li, Amy; Ganna, Andrea; Bassik, Michael C; Merker, Jason D; Hall, Ira M; Battle, Alexis; Montgomery, Stephen B

2017-10-11

Rare genetic variants are abundant in humans and are expected to contribute to individual disease risk. While genetic association studies have successfully identified common genetic variants associated with susceptibility, these studies are not practical for identifying rare variants. Efforts to distinguish pathogenic variants from benign rare variants have leveraged the genetic code to identify deleterious protein-coding alleles, but no analogous code exists for non-coding variants. Therefore, ascertaining which rare variants have phenotypic effects remains a major challenge. Rare non-coding variants have been associated with extreme gene expression in studies using single tissues, but their effects across tissues are unknown. Here we identify gene expression outliers, or individuals showing extreme expression levels for a particular gene, across 44 human tissues by using combined analyses of whole genomes and multi-tissue RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project v6p release. We find that 58% of underexpression and 28% of overexpression outliers have nearby conserved rare variants compared to 8% of non-outliers. Additionally, we developed RIVER (RNA-informed variant effect on regulation), a Bayesian statistical model that incorporates expression data to predict a regulatory effect for rare variants with higher accuracy than models using genomic annotations alone. Overall, we demonstrate that rare variants contribute to large gene expression changes across tissues and provide an integrative method for interpretation of rare variants in individual genomes.

Morphometric analysis of the cerebral expression of ATP-binding cassette transporter protein ABCB1 in chronic schizophrenia: Circumscribed deficits in the habenula.

PubMed

Bernstein, Hans-Gert; Hildebrandt, Jens; Dobrowolny, Henrik; Steiner, Johann; Bogerts, Bernhard; Pahnke, Jens

2016-11-01

There is increasing evidence that microvascular abnormalities and malfunction of the blood-brain barrier (BBB) significantly contribute to schizophrenia pathophysiology. The ATP-binding cassette transporter ABCB1 is an important molecular component of the intact BBB, which has been implicated in a number of neurodegenerative and psychiatric disorders, including schizophrenia. However, the regional and cellular expression of ABCB1 in schizophrenia is yet unexplored. Therefore, we studied ABCB1 protein expression immunohistochemically in twelve human post-mortem brain regions known to play a role in schizophrenia, in 13 patients with schizophrenia and nine controls. In ten out of twelve brain regions under study, no significant differences were found with regard to the numerical density of ABCB1-expressing capillaries between all patients with schizophrenia and control cases. The left and right habenular complex, however, showed significantly reduced capillary densities in schizophrenia patients. In addition, we found a significantly reduced density of ABCB1-expressing neurons in the left habenula. Reduced ABCB1 expression in habenular capillaries might contribute to increased brain levels of proinflammatory cytokines in patients with schizophrenia, while decreased expression of this protein in a subpopulation of medial habenular neurons (which are probably purinergic) might be related to abnormalities of purines and their receptors found in this disease. Copyright © 2015 Elsevier B.V. All rights reserved.