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Sample records for h9c2 rat myoblasts

  1. pPKCδ activates SC35 splicing factor during H9c2 myoblastic differentiation.

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    Zara, Susi; Falconi, Mirella; Rapino, Monica; Zago, Michela; Orsini, Giovanna; Mazzotti, Giovanni; Cataldi, Amelia; Teti, Gabriella

    2011-01-01

    Although Protein Kinase C (PKC) isoforms' role in the neonatal and adult cardiac tissue development and ageing has been widely described "in vivo", the interaction of such enzymes with specific nuclear substrates needs to be investigated. The aim of our research has been the study of the expression, localization and interaction with the splicing factor SC35 of PKC isoforms (α, δ, ε, ζ) and their potential role in modulating the transcription machinery. H9c2 cells induced to myoblast differentiation in the presence of 1% Horse Serum (HS) have represented our experimental model. The expression of PKC isoforms, their distribution and interaction with SC35 have been evaluated by western blotting, co-immunoprecipitation and double gold immunolabeling for transmission and scanning electron microscopy. Our results show PKCδ as the most expressed isoform in differentiated cells. Surprisingly, the distribution of PKCδ and SC35 does not show any significant modification between 10%FBS and 1%HS treated samples and no co-localization is observed. Moreover the interaction between the phosphorylated form of PKCδ (pPKCδ) and SC35 increases, is distributed and co-localizes within the nucleus of differentiated H9c2. These data represent reasonable evidence of pPKCδ mediated SC35 splicing factor activation, suggesting its direct effect on transcription via interaction with the transcription machinery. Furthermore, this co-localization represents a crucial event resulting in downstream changes in transcription of components which determine the morphological modifications related to cardiomyoblast differentiated phenotype.

  2. Endocytosis‒Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2).

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    Pooja, Sharma; Pushpanathan, Muthuirulan; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2015-01-01

    Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse‒ transcription PCR (RT‒PCR) and quantitative real time‒PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae‒infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose‒dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy‒dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction.

  3. Endocytosis‒Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2)

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    Pooja, Sharma; Pushpanathan, Muthuirulan; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2015-01-01

    Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae int...

  4. Norepinephrine-induced apoptotic and hypertrophic responses in H9c2 cardiac myoblasts are characterized by different repertoire of reactive oxygen species generation

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    Anita Thakur

    2015-08-01

    Full Text Available Despite recent advances, the role of ROS in mediating hypertrophic and apoptotic responses in cardiac myocytes elicited by norepinephrine (NE is rather poorly understood. We demonstrate through our experiments that H9c2 cardiac myoblasts treated with 2 µM NE (hypertrophic dose generate DCFH-DA positive ROS only for 2 h; while those treated with 100 µM NE (apoptotic dose sustains generation for 48 h, followed by apoptosis. Though the levels of DCFH fluorescence were comparable at early time points in the two treatment sets, its quenching by DPI, catalase and MnTmPyP suggested the existence of a different repertoire of ROS. Both doses of NE also induced moderate levels of H2O2 but with different kinetics. Sustained but intermittent generation of highly reactive species detectable by HPF was seen in both treatment sets but no peroxynitrite was generated in either conditions. Sustained generation of hydroxyl radicals with no appreciable differences were noticed in both treatment sets. Nevertheless, despite similar profile of ROS generation between the two conditions, extensive DNA damage as evident from the increase in 8-OH-dG content, formation of γ-H2AX and PARP cleavage was seen only in cells treated with the higher dose of NE. We therefore conclude that hypertrophic and apoptotic doses of NE generate distinct but comparable repertoire of ROS/RNS leading to two very distinct downstream responses.

  5. 4-Guanidino-n-butyl syringate (Leonurine, SCM 198) protects H9c2 rat ventricular cells from hypoxia-induced apoptosis.

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    Liu, Xin-hua; Chen, Pei-fang; Pan, Li-long; Silva, Ranil De; Zhu, Yi-zhun

    2009-11-01

    In the present study, we examined the ability of a chemically synthesized compound based on the structure of leonurine, a phytochemical component of Herba leonuri, to protect H9c2 rat ventricular cells from apoptosis induced by hypoxia and serum deprivation, as a model of ischemia. The results revealed a concentration-dependent increase in cell viability associated with leonurine treatment, accompanied by a consistent decline in lactate dehydrogenase leakage into the culture medium. The fraction of annexin V-fluorescein isothiocyanate-positive cells was increased by hypoxia but reduced by leonurine. These changes were associated with increased expression of the antiapoptotic gene, Bcl-2, and reduced expression of the proapoptotic gene, Bax. Leonurine also reduced the cytosolic Ca overload induced by hypoxia. These results suggest that leonurine elicits potent cardioprotective effects in H9c2 cells, and these effects may be mediated by inhibition of intracellular Ca overload and apoptosis during hypoxia.

  6. The antioxidant edaravone prevents cardiac dysfunction by suppressing oxidative stress in type 1 diabetic rats and in high-glucose-induced injured H9c2 cardiomyoblasts.

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    Ji, Lei; Liu, Yingying; Zhang, Ying; Chang, Wenguang; Gong, Junli; Wei, Shengnan; Li, Xudong; Qin, Ling

    2016-09-01

    Edaravone, a radical scavenger, has been recognized as a potential protective agent for cardiovascular diseases. However, little is known about the effect of edaravone in cardiac complications associated with diabetes. Here, we have demonstrated that edaravone prevents cardiac dysfunction and apoptosis in the streptozotocin-induced type 1 diabetic rat heart. Mechanistic studies revealed that edaravone treatment improved cardiac function and restored superoxide dismutase levels. In addition, treatment of diabetic animals by edaravone increased protein expressions of sirtuin-1 (SIRT-1), peroxisome proliferator activated receptor γ coactivator α (PGC-1α), nuclear factor like-2 (NRF-2), and B cell lymphoma 2 (Bcl-2), and reduced protein expressions of Bax and Caspase-3 compared to the control group. High glucose incubation resulted in the production of reactive oxygen species (ROS) and cell death. Treatment of high-glucose-incubated H9c2 cells by edaravone reduced ROS production and cell death. In addition, the treatment of high-glucose-incubated H9c2 cells by edaravone increased the activity of antioxidative stress by increasing SIRT-1, PGC-1α, and NRF-2, and this treatment also reduced apoptosis by increasing Bcl-2 expression and reducing Bax and Caspase-3 expressions. Knockdown SIRT-1 with small interferer RNA abolished the effects of edaravone. Overall, our data demonstrated that edaravone may be an effective agent against the development of diabetic cardiomyopathy.

  7. Plant Natural Product Formononetin Protects Rat Cardiomyocyte H9c2 Cells against Oxygen Glucose Deprivation and Reoxygenation via Inhibiting ROS Formation and Promoting GSK-3β Phosphorylation

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    Yuanyuan Cheng

    2016-01-01

    Full Text Available The opening of mitochondrial permeability transition pore (mPTP is a major cause of cell death in ischemia reperfusion injury. Based on our pilot experiments, plant natural product formononetin enhanced the survival of rat cardiomyocyte H9c2 cells during oxygen glucose deprivation (OGD and reoxygenation. For mechanistic studies, we focused on two major cellular factors, namely, reactive oxygen species (ROS and glycogen synthase kinase 3β (GSK-3β, in the regulation of mPTP opening. We found that formononetin suppressed the formation of ROS and superoxide in a concentration-dependent manner. Formononetin also rescued OGD/reoxygenation-induced loss of mitochondrial membrane integrity. Further studies suggested that formononetin induced Akt activation and GSK-3β (Ser9 phosphorylation, thereby reducing GSK-3β activity towards mPTP opening. PI3K and PKC inhibitors abolished the effects of formononetin on mPTP opening and GSK-3β phosphorylation. Immunoprecipitation experiments further revealed that formononetin increased the binding of phosphor-GSK-3β to adenine nucleotide translocase (ANT while it disrupted the complex of ANT with cyclophilin D. Moreover, immunofluorescence revealed that phospho-GSK-3β (Ser9 was mainly deposited in the space between mitochondria and cell nucleus. Collectively, these results indicated that formononetin protected cardiomyocytes from OGD/reoxygenation injury via inhibiting ROS formation and promoting GSK-3β phosphorylation.

  8. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

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    Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der

    2013-01-01

    TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47 phox , p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF-α induces

  9. Feasibility of dual reporter gene in rat myoblast cell line using human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) gene

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    Lee, Yong Jin; Lee, You La; Ahn, Sohn Joo; Choi, Chang Ik; Lee, Sang Woo; Ahn, Byeong Cheol; Lee, Jae Tae [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)

    2007-07-01

    To develop a non-invasive combined imaging method of gamma camera and optical imaging to assess rat myoblast cell line, H9c2, we constructed retrovirus containing hNIS and EGFP gene, and transfected to rat myoblast cell and monitored hNIS and EGFP expression. Rat myoblast cell line, H9C2, was transfected with hNIS and EGFP gene using retrovirus (H9C2-NG). The expression of hNIS and EGFP gene was determined by RT-PCR and fluorescence microscopy, respectively. The uptake and efflux of I-125 were measured in the transfected and wild type cell lines. Each cell line was injected to 4 flank sites (H9c2: 1X107 or 2X107, H9C2-NG: 1X107 or 2X107) in nude mouse. Scintigraphic image was performed at 3h, 1 day after H9C2 and H9C2-NG cell inoculation. We performed gamma camera and animal PET imaging to evaluate NIS expression. Also, GFP image obtained using optical imaging system. The expression of hNIS and EGFP gene was confirmed by RT-PCR. In iodide uptake, H9C2-NG cells accumulated 274.52.2 pmol/ mg protein at 30 min. But wild type cell line did not uptake iodide. In fluorescent microscopy, H9C2-NG cells were highly fluorescent than that of H9C2 cells. In iodide efflux study, 50% of radioactivity flowed out during the first 10min. Scintigraphy showed increased uptake of Tc-99m in H9c2-NG than in H9C2 for 1 day. Also, H9C2-NG cells showed high signal-to-background fluorescent spots in animal body. In this study, NIS and EGFP reporter gene were successfully transfected by a retrovirus in myoblast cell line, and the transfected cell can be easily visualized in vivo. These results suggest that NIS and EGFP gene has an excellent feasibility as a reporter gene, and it can be used to monitor cell trafficking for monitoring.

  10. Microfluidic system for monitoring of cardiac (H9C2) cell proliferation

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    Kobuszewska, A.; Cwik, P.; Jastrzebska, E.; Brzozka, Z.; Chudy, M.; Renaud, P.; Dybko, A.

    2017-05-01

    The paper presents the application of electrical impedance spectroscopy (EIS) analysis for investigation of cardiac cell (H9C2 - rat cardiomyoblast) proliferation after verapamil hydrochloride exposure. For this purpose, two different PDMS/glass microsystems with circular microchamber and longitudinal microchannel integrated with Pt/Al electrodes were used. The microchambers were fabricated in PDMS using photolithography and replica moulding techniques. Pt/Al electrodes were fabricated on a 4-inch glass substrate using Physical Vapor Deposition (PVD). Solution of verapamil hydrochloride was continuously introduced into the microsystems with H9C2 cell culture (a flow rate of 1 μl/min) for 72 h. The impedance spectra were recorded from 100 Hz to 1 MHz. We confirmed that impedance spectroscopy can be used for non-invasive, label-free and real-time analysis of cardiac cells proliferation based on cells dielectric properties and biological structure.

  11. Overexpression of MIP2, a novel WD-repeat protein, promotes proliferation of H9c2 cells

    International Nuclear Information System (INIS)

    Wei, Xing; Song, Lan; Jiang, Lei; Wang, Guiliang; Luo, Xinjing; Zhang, Bin; Xiao, Xianzhong

    2010-01-01

    WD40 repeat proteins have a wide range of diverse biological functions including signal transduction, cell cycle regulation, RNA splicing, and transcription. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a novel member of the WD40 repeat proteins superfamily that contains five WD40 repeats. Little is known about its biological role, and the purpose of this study was to determine the role of MIP2 in regulating cellular proliferation. Transfection and constitutive expression of MIP2 in the rat cardiomyoblast cell line H9c2 results in enhanced growth of those cells as measured by cell number and is proportional to the amount of MIP2 expressed. Overexpression of MIP2 results in a shorter cell cycle, as measured by flow cytometry. Collectively, these data suggest that MIP2 may participate in the progression of cell proliferation in H9c2 cells.

  12. Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

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    Mali, Vishal R; Deshpande, Mandar; Pan, Guodong; Thandavarayan, Rajarajan A; Palaniyandi, Suresh S

    2016-02-01

    Reactive oxygen species (ROS)-mediated reactive aldehydes induce cellular stress. In cardiovascular diseases such as ischemia-reperfusion injury, lipid-peroxidation derived reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are known to contribute to the pathogenesis. 4HNE is involved in ROS formation, abnormal calcium handling and more importantly defective mitochondrial respiration. Aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)(+)-dependent isozymes which can detoxify endogenous and exogenous aldehydes into non-toxic carboxylic acids. Therefore we hypothesize that 4HNE afflicts mitochondrial respiration and leads to cell death by impairing ALDH2 activity in cultured H9C2 cardiomyocyte cell lines. H9C2 cardiomyocytes were treated with 25, 50 and 75 μM 4HNE and its vehicle, ethanol as well as 25, 50 and 75 μM disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4 h. 4HNE significantly decreased ALDH2 activity, ALDH2 protein levels, mitochondrial respiration and mitochondrial respiratory reserve capacity, and increased 4HNE adduct formation and cell death in cultured H9C2 cardiomyocytes. ALDH2 inhibition by DSF and ALDH2 siRNA attenuated ALDH2 activity besides reducing ALDH2 levels, mitochondrial respiration and mitochondrial respiratory reserve capacity and increased cell death. Our results indicate that ALDH2 impairment can lead to poor mitochondrial respiration and increased cell death in cultured H9C2 cardiomyocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Fluoride induces apoptosis in H9c2 cardiomyocytes via the mitochondrial pathway.

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    Yan, Xiaoyan; Wang, Lu; Yang, Xia; Qiu, Yulan; Tian, Xiaolin; Lv, Yi; Tian, Fengjie; Song, Guohua; Wang, Tong

    2017-09-01

    Numerous studies have shown that chronic excessive fluoride intake can adversely affect different organ systems. In particular, the cardiovascular system is susceptible to disruption by a high concentration of fluoride. The objectives of this study were to explore the mechanism of apoptosis by detecting the toxic effects of different concentrations of sodium fluoride (NaF) in H9c2 cells exposed for up to 96 h. NaF not only inhibited H9c2 cell proliferation but also induced apoptosis and morphological damage. With increasing NaF concentrations, early apoptosis of H9c2 cells was increased while the mitochondrial membrane potential was decreased. Compared with the control group, the mRNA levels of caspase-3, caspase-9, and cytochrome c all increased with increasing concentrations of NaF. In summary, these data suggest that apoptosis is involved in NaF-induced H9c2 cell toxicity and that activation of the mitochondrial pathway may occur. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Proliferation and skeletal myotube formation capability of C2C12 and H9c2 cells on isotropic and anisotropic electrospun nanofibrous PHB scaffolds

    International Nuclear Information System (INIS)

    Ricotti, Leonardo; Genchi, Giada G; Menciassi, Arianna; Polini, Alessandro; Iandolo, Donata; Pisignano, Dario; Ciofani, Gianni; Mattoli, Virgilio; Vazão, Helena; Ferreira, Lino

    2012-01-01

    This study aims at investigating the behavior in terms of the proliferation and skeletal muscle differentiation capability of two myoblastic cell lines, C2C12 and H9c2, on both isotropic and anisotropic electrospun nanofibrous poly(hydroxybutyrate) (PHB) scaffolds, as well as on PHB films and polystyrene controls. After a careful characterization of the matrices in terms of surface morphology, surface roughness and mechanical properties, the proliferation rate and the capability of the two cell lines to form skeletal myotubes were evaluated. Genetic analyses were also performed in order to assess the differentiation level of the cells on the different substrates. We demonstrated that the aligned nanofibrous mesh decreases the proliferation activity and provides a higher differentiative stimulus. We also clarified how the nanofibrous substrate influences myotube formation, and quantified a series of myotube-related parameters for both C2C12 and H9c2 cells. (paper)

  15. The Metabolic Effects of Traditional Chinese Medication Qiliqiangxin on H9C2 Cardiomyocytes

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    Shenghui Lin

    2015-11-01

    Full Text Available Background/Aims: A traditional Chinese medicine, Qiliqiangxin (QLQX has been identified to perform protective effects on myocardium energy metabolism in mice with acute myocardial infarction, though the effects of QLQX on myocardial mitochondrial biogenesis under physiological condition is still largely elusive. Methods: H9C2 cells were treated with different concentrations of QLQX (0.25, 0.5, and 1.0 µg/mL from 6 to 48 hours. Oxidative metabolism and glycolysis were measured by oxygen consumption and extracellular acidification with XF96 analyzer (SeaHorse. Mitochondrial content and ultrastructure were assessed by Mitotracker staining, confocal microscopy, flow cytometry, and transmission electron microscopy. Mitochondrial biogenesis-related genes were measured by qRT-PCR and Western blot. Results: H9C2 cells treated with QLQX exhibited increased glycolysis at earlier time points (6, 12, and 24 hours, while QLQX could enhance oxidative metabolism and mitochondrial uncoupling in H9C2 cells with longer duration of treatment (48 hours. QLQX also increased mitochondrial content and mitochondrial biogenesis-related gene expression levels, including 16sRNA, SSBP1, TWINKLE, TOP1MT and PLOG, with an activation of peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α and its downstream effectors. Silencing PGC-1α could abolish the increased mitochondrial content in H9C2 cells treated with QLQX. Conclusion: Our study is the first to document enhanced metabolism in cardiomyocytes treated with QLQX, which is linked to increased mitochondrial content and mitochondrial biogenesis via activation of PGC-1α.

  16. Cardiomyocyte H9c2 cells present a valuable alternative to fish lethal testing for azoxystrobin

    International Nuclear Information System (INIS)

    Rodrigues, Elsa T.; Pardal, Miguel Â.; Laizé, Vincent; Cancela, M. Leonor; Oliveira, Paulo J.; Serafim, Teresa L.

    2015-01-01

    The present study aims at identifying, among six mammalian and fish cell lines, a sensitive cell line whose in vitro median inhibitory concentration (IC_5_0) better matches the in vivo short-term Sparus aurata median lethal concentration (LC_5_0). IC_5_0_s and LC_5_0 were assessed after exposure to the widely used fungicide azoxystrobin (AZX). Statistical results were relevant for most cell lines after 48 h of AZX exposure, being H9c2 the most sensitive cells, as well as the ones which provided the best prediction of fish toxicity, with a LC_5_0_,_9_6_h/IC_5_0_,_4_8_h = 0.581. H9c2 cell proliferation upon 72 h of AZX exposure revealed a LC_5_0_,_9_6_h/IC_5_0_,_7_2_h = 0.998. Therefore, identical absolute sensitivities were attained for both in vitro and in vivo assays. To conclude, the H9c2 cell-based assay is reliable and represents a suitable ethical alternative to conventional fish assays for AZX, and could be used to get valuable insights into the toxic effects of other pesticides. - Highlights: • Fish toxicity data are still considered standard information in ecotoxicology. • Alternatives to animal testing have become an important topic of research. • Cell-based assays are currently a promising in vitro alternative. • Comparative studies to accelerate the validation of cell-based methods are required. • H9c2 cell line proved to produce in vitro reliable toxicity results for azoxystrobin. - The application of cell-based assays for environmental toxicity studies would greatly reduce the number of fish needed for toxicity testing without any loss of reliability.

  17. Sodium Ferulate Prevents Daunorubicin - Induced Apoptosis in H9c2 Cells via Inhibition of the ERKs Pathway

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    Zhi-Juan Wu

    2015-07-01

    Full Text Available Background: Daunorubicin (DNR-induced cardiotoxicity, which is closely associated with cardiomyocyte apoptosis, limits the drug's clinical application. The activation of the extracellular regulated protein kinases (ERKs pathway is responsible for the pro-apoptosis effect of DNR Sodium ferulate (SF has recently been found to attenuate both DNR-induced cardiotoxicity and mitochondrial apoptosis in juvenile rats. Nonetheless, the precise mechanism underlying SF-induced cardio-protection remains unclear. Methods: The DNR-injured H9c2 cell model was prepared by incubating the cells in 1 µM DNR for 24 h. Amounts of 15.6, 31.3 or 62.5 µM SF were simultaneously added to the cells. The effect of SF on the cytotoxic and apoptotic parameters of the cells was studied by monitoring apoptosis regulation via the ERKs pathway. Results: SF attenuated DNR-induced cell death (particularly apoptotic death, cTnI and β-tubulin degradation, and cellular morphological changes. SF reduced mitochondrial membrane potential depolarization, cytochrome c leakage, and caspase-9 and caspase-3 activation. SF also decreased ERK1/2, phospho-ERK1/2, p53 and Bax expression and increased Bcl-2 expression. These effects were similar to the results observed when using the pharmacological ERKs phosphorylation inhibitor, AZD6244. Conclusion: We determined that SF protects H9c2 cells from DNR-induced apoptosis through a mechanism that involves the interruption of the ERKs signaling pathway.

  18. Antiapoptotic effect of novel compound from Herba leonuri - leonurine (SCM-198): a mechanism through inhibition of mitochondria dysfunction in H9c2 cells.

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    Liu, Xin Hua; Pan, Li Long; Gong, Qi Hai; Zhu, Yi Zhun

    2010-12-01

    Apoptosis of cardiomyocytes induced by oxidative stress play a critical role in cardiac dysfunction associated with ventricular remodeling and heart failure. We recently reported that leonurine attenuated hypoxia-induced cardiomyocyte damage. In this study, we investigated the mechanism of leonurine (originally from Herba leonuri but we synthesized it chemically it as also called SCM-198) (H₂O₂)-induced rat embryonic heart-derived H9c2 cells from apoptosis. Exposing H9c2 cells to H₂O₂ significantly decreased cell viability, and this was attenuated by pretreatment with leonurine for 4 h in a concentration-dependent manner. Meanwhile, leonurine was found to reduce intracellular reactive oxygen species (ROS) generation in H₂O₂-stimulated cell. Moreover, H9c2 cells stimulated by H₂O₂ was accompanied with apparent apoptotic characteristics, including fragmentation of DNA, apoptotic body formation, release of cytochrome c, translocation of Bax to mitochondria, loss of mitochondrial membrane potential (ΔΨ(m)) and activation of caspase 3. Furthermore, H₂O₂ also induced rapid and significant phosphorylation of the c-Jun-N-terminal kinase 1/2 (JNK1/2), which was inhibited SP600125 (a JNK1/2 inhibitor). All of these events were attenuated by leonurine pretreatment. Taken together, these results demonstrated that leonurine could protect H9c2 cells from H₂O₂-induced apoptosis via modulation of mitochondrial dysfunction associated with blocking the activation of JNK1/2.

  19. Peroxisome proliferator-activated receptor delta (PPARdelta) activation protects H9c2 cardiomyoblasts from oxidative stress-induced apoptosis.

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    Pesant, Matthieu; Sueur, Stéphanie; Dutartre, Patrick; Tallandier, Mireille; Grimaldi, Paul A; Rochette, Luc; Connat, Jean-Louis

    2006-02-01

    Activation of peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARgamma plays beneficial roles in cardiovascular disorders such as atherosclerosis and heart reperfusion. Although PPARalpha and gamma have been documented to reduce oxidative stress in the vasculature and the heart, the role of PPARdelta remains poorly studied. We focused on PPARdelta function in the regulation of oxidative stress-induced apoptosis in the rat cardiomyoblast cell line H9c2. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we showed that PPARdelta is the predominantly expressed isotype whereas PPARalpha was weakly detected. By performing cell viability assays, we also showed that the selective PPARdelta agonist GW501516 protected cells from H(2)O(2)-induced cell death. The protective effect of GW501516 was due to an inhibition of H(2)O(2)-triggered apoptosis as shown by annexin-V labeling, DNA fragmentation analysis, and caspase-3 activity measurement. We demonstrated by transient transfection of a dominant negative mutant of PPARdelta that the protection induced by GW501516 was totally dependent on PPARdelta. Semi-quantitative RT-PCR and Western blotting analysis demonstrated that GW501516 treatment upregulated catalase. Moreover, forced overexpression of catalase inhibited H(2)O(2)-triggered apoptosis, as evidenced by annexin-V labeling. Taken together, our results account for an important role of PPARdelta in inhibiting the onset of oxidative stress-induced apoptosis in H9c2 cells. PPARdelta appears to be a new therapeutic target for the regulation of heart reperfusion-associated oxidative stress and stimulation of enzymatic antioxidative defences.

  20. Glibenclamide Mimics Metabolic Effects of Metformin in H9c2 Cells.

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    Salani, Barbara; Ravera, Silvia; Fabbi, Patrizia; Garibaldi, Silvano; Passalacqua, Mario; Brunelli, Claudio; Maggi, Davide; Cordera, Renzo; Ameri, Pietro

    2017-01-01

    Sulfonylureas, such as glibenclamide, are antidiabetic drugs that stimulate beta-cell insulin secretion by binding to the sulfonylureas receptors (SURs) of adenosine triphosphate-sensitive potassium channels (KATP). Glibenclamide may be also cardiotoxic, this effect being ascribed to interference with the protective function of cardiac KATP channels for which glibenclamide has high affinity. Prompted by recent evidence that glibenclamide impairs energy metabolism of renal cells, we investigated whether this drug also affects the metabolism of cardiac cells. The cardiomyoblast cell line H9c2 was treated for 24 h with glibenclamide or metformin, a known inhibitor of the mitochondrial respiratory chain. Cell viability was evaluated by sulforodhamine B assay. ATP and AMP were measured according to the enzyme coupling method and oxygen consumption by using an amperometric electrode, while Fo-F1 ATP synthase activity assay was evaluated by chemiluminescent method. Protein expression was measured by western blot. Glibenclamide deregulated energy balance of H9c2 cardiomyoblasts in a way similar to that of metformin. It inhibited mitochondrial complexes I, II and III with ensuing impairment of oxygen consumption and ATP synthase activity, ATP depletion and increased AMPK phosphorylation. Furthermore, glibenclamide disrupted mitochondrial subcellular organization. The perturbation of mitochondrial energy balance was associated with enhanced anaerobic glycolysis, with increased activity of phosphofructo kinase, pyruvate kinase and lactic dehydrogenase. Interestingly, some additive effects of glibenclamide and metformin were observed. Glibenclamide deeply alters cell metabolism in cardiac cells by impairing mitochondrial organization and function. This may further explain the risk of cardiovascular events associated with the use of this drug, alone or in combination with metformin. © 2017 The Author(s). Published by S. Karger AG, Basel.

  1. Mitochondrial dysfunction in H9c2 cells during ischemia and amelioration with Tribulus terrestris L.

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    Reshma, P L; Sainu, Neethu S; Mathew, Anil K; Raghu, K G

    2016-05-01

    The present study investigates the protective effect of partially characterized Tribulus terrestris L. fruit methanol extract against mitochondrial dysfunction in cell based (H9c2) myocardial ischemia model. To induce ischemia, the cells were maintained in an ischemic buffer (composition in mM -137 NaCl, 12 KCl, 0.5 MgCl2, 0.9 CaCl2, 20 HEPES, 20 2-deoxy-d-glucose, pH-6.2) at 37°C with 0.1% O2, 5% CO2, and 95% N2 in a hypoxia incubator for 1h. Cells were pretreated with various concentrations of T. terrestris L. fruit methanol extract (10 and 25μg/ml) and Cyclosporin A (1μM) for 24h prior to the induction of ischemia. Different parameters like lactate dehydrogenase release, total antioxidant capacity, glutathione content and antioxidant enzymes were investigated. Studies were conducted on mitochondria by analyzing alterations in mitochondrial membrane potential, integrity, and dynamics (fission and fusion proteins - Mfn1, Mfn2, OPA1, Drp1 and Fis1). Various biochemical processes in mitochondria like activity of electron transport chain (ETC) complexes, oxygen consumption and ATP production was measured. Ischemia for 1h caused a significant (p≤0.05) increase in LDH leakage, decrease in antioxidant activity and caused mitochondrial dysfunction. T. terrestris L. fruit methanol extract pretreatment was found effective in safeguarding mitochondria via its antioxidant potential, mediated through various bioactives. HPLC of T. terrestris L. fruit methanol extract revealed the presence of ferulic acid, phloridzin and diosgenin. T. terrestris L. fruit ameliorate ischemic insult in H9c2 cells by safeguarding mitochondrial function. This validates the use of T. terrestris L. against heart disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a 'function follows form' model of differentiation.

    Science.gov (United States)

    Kankeu, Cynthia; Clarke, Kylie; Van Haver, Delphi; Gevaert, Kris; Impens, Francis; Dittrich, Anna; Roderick, H Llewelyn; Passante, Egle; Huber, Heinrich J

    2018-05-17

    The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes.

  3. Protective effect of Dendrobium officinale polysaccharides on H2O2-induced injury in H9c2 cardiomyocytes.

    Science.gov (United States)

    Zhao, Xiaoyan; Dou, Mengmeng; Zhang, Zhihao; Zhang, Duoduo; Huang, Chengzhi

    2017-10-01

    The preliminary studies have shown that Dendrobium officinale possessed therapeutic effects on hypertension and atherosclerosis. Studies also reported that Dendrobium officinale polysaccharides showed antioxidant capabilities. However, little is known about its effects on myocardial cells under oxidative stress. The present study was designed to study the protective effect of Dendrobium officinale polysaccharides against H 2 O 2 -induced oxidative stress in H9c2 cells. MTT assay was carried out to determine the cell viability of H9c2 cells when pretreated with Dendrobium officinale polysaccharides. Fluorescent microscopy measurements were performed for evaluating the apoptosis in H9c2 cells. Furthermore, effects of Dendrobium officinale polysaccharides on the activities of antioxidative indicators (malondialdehyde, superoxide dismutase), reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) levels were analyzed. Dendrobium officinale polysaccharides attenuated H 2 O 2 -induced cell death, as determined by the MTT assay. Dendrobium officinale polysaccharides decreased malondialdehyde levels, increased superoxide dismutase activities, and inhibited the generation of intracellular ROS. Moreover, pretreatment with Dendrobium officinale polysaccharides also inhibited apoptosis and increased the MMP levels in H9c2 cells. These results suggested the protective effects of Dendrobium officinale polysaccharides against H 2 O 2 -induced injury in H9c2 cells. The results also indicated the anti-oxidative capability of Dendrobium officinale polysaccharides. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  4. Autophagy plays an important role in Sunitinib-mediated cell death in H9c2 cardiac muscle cells

    International Nuclear Information System (INIS)

    Zhao Yuqin; Xue Tao; Yang Xiaochun; Zhu Hong; Ding Xiaofei; Lou Liming; Lu Wei; Yang Bo; He Qiaojun

    2010-01-01

    Sunitinib, which is a multitargeted tyrosine-kinase inhibitor, exhibits antiangiogenic and antitumor activity, and extends survival of patients with metastatic renal-cell carcinoma (mRCC) and gastrointestinal stromal tumors (GIST). This molecule has also been reported to be associated with cardiotoxicity at a high frequency, but the mechanism is still unknown. In the present study, we observed that Sunitinib showed high anti-proliferative effect on H9c2 cardiac muscle cells measured by PI staining and the MTT assay. But apoptotic markers (PARP cleavage, caspase 3 cleavage and chromatin condensation) were uniformly negative in H9c2 cells after Sunitinib treatment for 48 h, indicating that another cell death pathway may be involved in Sunitinib-induced cardiotoxicity. Here we found Sunitinib dramatically increased autophagic flux in H9c2 cells. Acidic vesicle fluorescence and high expression of LC3-II in H9c2 cells identified autophagy as a Sunitinib-induced process that might be associated with cytotoxicity. Furthermore, knocking down Beclin 1 by RNA-interference to block autophagy in H9c2 cells revealed that the death rate was decreased when treated with Sunitinib in comparison to control cells. These results confirmed that autophagy plays an important role in Sunitinib-mediated H9c2 cells cytotoxicity. Taken together, the data presented here strongly suggest that autophagy is associated with Sunitinib-induced cardiotoxicity, and that inhibition of autophagy constitutes a viable strategy for reducing Sunitinib-induced cardiomyocyte death thereby alleviating Sunitinib cardiotoxicity.

  5. Disruption of dopamine D1/D2 receptor complex is involved in the function of haloperidol in cardiac H9c2 cells.

    Science.gov (United States)

    Lencesova, L; Szadvari, I; Babula, P; Kubickova, J; Chovancova, B; Lopusna, K; Rezuchova, I; Novakova, Z; Krizanova, O; Novakova, M

    2017-12-15

    Haloperidol is an antipsychotic agent and acts as dopamine D2 receptor (D2R) antagonist, as a prototypical ligand of sigma1 receptors (Sig1R) and it increases expression of type 1 IP 3 receptors (IP 3 R1). However, precise mechanism of haloperidol action on cardiomyocytes through dopaminergic signaling was not described yet. This study investigated a role of dopamine receptors in haloperidol-induced increase in IP 3 R1 and Sig1R, and compared physiological effect of melperone and haloperidol on basic heart parameters in rats. We used differentiated NG-108 cells and H9c2 cells. Gene expression, Western blot and immunofluorescence were used to evaluate haloperidol-induced differences; proximity ligation assay (PLA) and immunoprecipitation to determine interactions of D1/D2 receptors. To evaluate cardiac parameters, Wistar albino male rats were used. We have shown that antagonism of D2R with either haloperidol or melperone results in upregulation of both, IP 3 R1 and Sig1R, which is associated with increased D2R, but reduced D1R expression. Immunofluorescence, immunoprecipitation and PLA support formation of heteromeric D1/D2 complexes in H9c2 cells. Treatment with haloperidol (but not melperone) caused decrease in systolic and diastolic blood pressure and significant increase in heart rate. Because D1R/D2R complexes can engage Gq-like signaling in other experimental systems, these results are consistent with the possibility that disruption of D1R/D2R complex in H9c2 cells might cause a decrease in IP 3 R1 activity, which in turn may account for the increase expression of IP 3 R and Sig1R. D2R is probably not responsible for changes in cardiac parameters, since melperone did not have any effect. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Polyphenol-rich apple (Malus domestica L.) peel extract attenuates arsenic trioxide induced cardiotoxicity in H9c2 cells via its antioxidant activity.

    Science.gov (United States)

    Vineetha, Vadavanath Prabhakaran; Girija, Seetharaman; Soumya, Rema Sreenivasan; Raghu, Kozhiparambil Gopalan

    2014-03-01

    Evidences suggest that apple peel has a wide range of polyphenols having antioxidant activity and its consumption has been linked with improved health benefits. Arsenic trioxide (ATO) is a very effective drug for the treatment of acute promyelocytic leukemia (APL) but it leads to cardiotoxicity mediated through alterations in various cardiac ion channels and by increasing the intracellular calcium level and reactive oxygen species (ROS). The aim of the present investigation was to study the effect of methanolic extract of apple peel (APME) and aqueous extract of apple peel (APAE) on ATO (5 μM) induced toxicity in the H9c2 cardiac myoblast cell line. We estimated the cellular status of innate antioxidant enzymes, level of ROS, mitochondrial superoxide, glutathione and intracellular calcium with ATO and apple peel extracts. Prior to the cell line based study, we had evaluated the antioxidant potential of apple peel extract by 1,1-diphenyl-2-picrylhydrazyl (DPPH), total reducing power (TRP), superoxide anion and hydroxyl radical scavenging activity, in addition to quantifying total phenolic and flavonoid content. Both the extracts showed considerable antioxidant activity in cell-free chemical assays. In addition, both APME and APAE prevented the alteration in antioxidant status induced by ATO in H9c2 cells. Significant differential alterations had been observed in the activity of lactate dehydrogenase, superoxide dismutase, catalase, glutathione, glutathione peroxidase, thioredoxin reductase, xanthine oxidase, calcium overload and caspase 3 activity with ATO. The overall result revealed the protective property of polyphenol-rich apple peel extract against ATO induced cardiac toxicity via its antioxidant activity.

  7. [Protective effect of taxifolin on H2O2-induced 
H9C2 cell pyroptosis].

    Science.gov (United States)

    Ye, Yanqiong; Wang, Xiaoli; Cai, Qian; Zhuang, Jian; Tan, Xiaohua; He, Wei; Zhao, Mingyi

    2017-12-28

    To explore the effect of taxifolin on H2O2-induced pyroptosis in H9C2 cells and the possible mechanisms.
 Methods: The H9C2 cells was divided into 3 groups: a control group, a hydrogen peroxide (H2O2)group and a taxifolin group. The morphology of H9C2 cells was observed by inverted phase contrast microscope. The mitochondrial membrane potential was measured by JC-1 staining and flow cytometry. The alteration of the level of reactive oxygen species (ROS) was detected by specific mitochondrial probe. The protein levels of cysteinyl aspartate specific proteinase-1 (caspase-1)was determined by Western blot. The mRNA levels of interleukin-18 (IL-18), interleukin-1a (IL-1a), interleukin-1b (IL-1b), absent in melanoma 2 (AIM2), apoptosis-associated apeck-like protein (ASC), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)and nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain-containing protein 4 (NLRC4) were determined by reverse transcription-polymerase chain reaction (RT-PCR).
 Results: Compared with the control group, the morphology of H9C2 cells obviously changed in the H2O2-treated group, which was guadually improved in the presence of taxifolin. Compared with the control group, the mitochondrial membrane potential was markedly decreased in the H2O2-treated cells, accompanied by the increase ofROS (both PH2O2 group, the mitochondrial membrane potential changes in the taxifolin group was increased while the ROS was decreased, with significant difference (both PH2O2-treated group were significantly increased (all PH2O2-induced H9C2 cell pyroptosis through inhibition of AIM2, NLRP3 and NLRC4 in flammasome.

  8. Acetylcholine Attenuates Hypoxia/ Reoxygenation-Induced Mitochondrial and Cytosolic ROS Formation in H9c2 Cells via M2 Acetylcholine Receptor

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    Yi Miao

    2013-02-01

    Full Text Available Background: The anti-infammatory and cardioprotective effect of acetylcholine (ACh has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R-induced oxidative stress remains obscure. Methods: In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS by measuring mitochondrial ROS (mtROS, mitochondrial DNA (mtDNA copy number, xanthine oxidase (XO and NADPH oxidase (NOX activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR expression. Results: 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group. Conclusions: ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.

  9. Chlorogenic acid analogues from Gynura nepalensis protect H9c2 cardiomyoblasts against H2O2-induced apoptosis.

    Science.gov (United States)

    Yu, Bang-Wei; Li, Jin-Long; Guo, Bin-Bin; Fan, Hui-Min; Zhao, Wei-Min; Wang, He-Yao

    2016-11-01

    Chlorogenic acid has shown protective effect on cardiomyocytes against oxidative stress-induced damage. Herein, we evaluated nine caffeoylquinic acid analogues (1-9) isolated from the leaves of Gynura nepalensis for their protective effect against H 2 O 2 -induced H9c2 cardiomyoblast damage and explored the underlying mechanisms. H9c2 cardiomyoblasts were exposed to H 2 O 2 (0.3 mmol/L) for 3 h, and cell viability was detected with MTT assay. Hoechst 33342 staining was performed to evaluate cell apoptosis. MMPs (mitochondrial membrane potentials) were measured using a JC-1 assay kit, and ROS (reactive oxygen species) generation was measured using CM-H 2 DCFDA. The expression levels of relevant proteins were detected using Western blot analysis. Exposure to H 2 O 2 markedly decreased the viability of H9c2 cells and catalase activity, and increased LDH release and intracellular ROS production; accompanied by a loss of MMP and increased apoptotic rate. Among the 9 chlorogenic acid analogues as well as the positive control drug epigallocatechin gallate (EGCG) tested, compound 6 (3,5-dicaffeoylquinic acid ethyl ester) was the most effective in protecting H9c2 cells from H 2 O 2 -induced cell death. Pretreatment with compound 6 (1.56-100 μmol/L) dose-dependently alleviated all the H 2 O 2 -induced detrimental effects. Moreover, exposure to H 2 O 2 significantly increased the levels of Bax, p53, cleaved caspase-8, and cleaved caspase-9, and decreased the level of Bcl-2, resulting in cell apoptosis. Exposure to H 2 O 2 also significantly increased the phosphorylation of p38, JNK and ERK in the H9c2 cells. Pretreatment with compound 6 (12.5 and 25 μmol/L) dose-dependently inhibited the H 2 O 2 -induced increase in the level of cleaved caspase-9 but not of cleaved caspase-8. It also dose-dependently suppressed the H 2 O 2 -induced phosphorylation of JNK and ERK but not that of p38. Compound 6 isolated from the leaves of Gynura nepalensis potently protects H9c2

  10. MicroRNA-145 Aggravates Hypoxia-Induced Injury by Targeting Rac1 in H9c2 Cells.

    Science.gov (United States)

    Wang, Ximing; Zhang, Yanxia; Wang, Hongshan; Zhao, Genshang; Fa, Xianen

    2017-01-01

    Myocardial infarction (MI) is a leading cause of morbidity and mortality. Here, we sought to explore the potential role and underlying mechanism of miR-145 in MI. H9c2 cells were cultured under persistent hypoxia to simulate MI. The hypoxia-induced injury was assessed on the basis of cell viability, migration, invasion and apoptosis. The expression of miR-145 was evaluated by qRT-PCR and the influence of aberrantly expressed miR-145 on H9c2 cells under hypoxia was also estimated. Utilizing bioinformatics methods, the target genes of miR-145 were verified by luciferase reporter assay. Then, effects of abnormally expressed target gene on miR-145 silenced H9c2 cells were assessed. Finally, the phosphorylation levels of key kinases in the phosphatidylinositol-3-kinase (PI3K)/AKT and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways were detected by Western blot analysis. Hypoxia remarkably lowered viability, migration and invasion but promoted cell apoptosis. Meantime, the miR-145 level was up-regulated in H9c2 cells under hypoxia. Following experiments suggested that hypoxia-induced injury was exacerbated by miR-145 overexpression while was alleviated by miR-145 silence. Rac1 was predicted and further validated to be a target gene of miR-145. The influence of miR-145 silencing on H9c2 cells under hypoxia could be reversed by down-regulation of Rac1. Additionally, the phosphorylation levels of PI3K, AKT, MAPK and ERK were all elevated in miR-145 silenced cells and these alterations were reversed by down-regulation of Rac1. miR-145 silencing could protect H9c2 cells against hypoxia-induced injury by targeting Rac1, in which PI3K/AKT and MAPK/ERK pathways might be involved. © 2017 The Author(s). Published by S. Karger AG, Basel.

  11. Effects of Downregulation of MicroRNA-181a on H2O2-Induced H9c2 Cell Apoptosis via the Mitochondrial Apoptotic Pathway

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    Lei Wang

    2014-01-01

    Full Text Available Glutathione peroxidase-1 (GPx1 is a pivotal intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. This study aims to identify a microRNA (miRNA that targets GPx1 to maintain redox homeostasis. Dual luciferase assays combined with mutational analysis and immunoblotting were used to validate the bioinformatically predicted miRNAs. We sought to select miRNAs that were responsive to oxidative stress induced by hydrogen peroxide (H2O2 in the H9c2 rat cardiomyocyte cell line. Quantitative real-time PCR (qPCR demonstrated that the expression of miR-181a in H2O2-treated H9c2 cells was markedly upregulated. The downregulation of miR-181a significantly inhibited H2O2-induced cellular apoptosis, ROS production, the increase in malondialdehyde (MDA levels, the disruption of mitochondrial structure, and the activation of key signaling proteins in the mitochondrial apoptotic pathway. Our results suggest that miR-181a plays an important role in regulating the mitochondrial apoptotic pathway in cardiomyocytes challenged with oxidative stress. MiR-181a may represent a potential therapeutic target for the treatment of oxidative stress-associated cardiovascular diseases.

  12. Protective effect of p-coumaric acid against doxorubicin induced toxicity in H9c2 cardiomyoblast cell lines

    Directory of Open Access Journals (Sweden)

    Sunitha M. Chacko

    2015-01-01

    Full Text Available Doxorubicin (Dox has been used for more than four decades to treat cancer, particularly solid tumours and haematological malignancies. However, the administration of this drug is a matter of concern in the clinical community, since Dox therapy is commonly associated with dose-dependent cardiotoxicity. Attempts at alleviating drug generated cardiac damage using naturally occurring compounds with radical scavenging property are a promising area of research. p-Coumaric acid (pCA is one such compound which has significant antiradical scavenging effect. This study aims to investigate the effect of pre and co-administration of pCA on mitigating or preventing Dox induced cardiotoxicity in vitro using H9c2 cardiomyoblast cell lines. Addition of pCA and Dox were performed for both treatment and control sets on H9c2 cells. Sulphorhodamine B assay was used to study the cytotoxic effect of pCA and Dox. The effect of the drug on cell morphology, cell viability and nuclear damage was studied using AO/EB and DAPI staining. ROS production was studied using DCFH-DA staining. Mitochondrial membrane potential and intracellular calcium levels were assessed by rhodamine 123 and Fura 2AM staining. pCA showed strong ABTS cation radical scavenging activity and FRAP activity in a dose dependent manner. The results showed that Dox has significant cytotoxic effect in a dose dependent manner while pCA, even at higher concentrations did not display any significant cytotoxicity on H9c2 cells. Both pre treatment and co- administration of pCA reduced the drug induced toxic effects on cell morphology and enhanced the number of viable cells in comparison to the Dox treated cells as evident from the AO/EB and DAPI staining images. The Dox induced ROS production was found to be significantly reduced in pCA pre-treated and co-administered cells. Dox induced changes in mitochondrial membrane potential and intracellular calcium levels were remarkably improved following pre and co

  13. Bauhinia championii Flavone Attenuates Hypoxia-Reoxygenation Induced Apoptosis in H9c2 Cardiomyocytes by Improving Mitochondrial Dysfunction.

    Science.gov (United States)

    Liao, Ping; Sun, Guibo; Zhang, Chan; Wang, Min; Sun, Yao; Zhou, Yuehan; Sun, Xiaobo; Jian, Jie

    2016-11-04

    This study aimed to determine the effects of Bauhinia championii flavone (BCF) on hypoxia-reoxygenation (H/R) induced apoptosis in H9c2 cardiomyocytes and to explore potential mechanisms. The H/R model in H9c2 cardiomyocytes was established by 6 h of hypoxia and 12 h of reoxygenation. Cell viability was detected by CCK-8 assay. Apoptotic rate was measured by Annexin V/PI staining. Levels of mitochondria-associated ROS, mitochondrial transmembrane potential (∆Ψm) and mitochondrial permeability transition pores (MPTP) opening were assessed by fluorescent probes. ATP production was measured by ATP assay kit. The release of cytochrome c, translocation of Bax, and related proteins were measured by western blotting. Our results showed that pretreatment with BCF significantly improved cell viability and attenuated the cardiomyocyte apoptosis caused by H/R. Furthermore, BCF increased ATP production and inhibited ROS-generating mitochondria, depolarization of ΔΨm, and MPTP opening. Moreover, BCF pretreatment decreased Bax mitochondrial translocation, cytochrome c release, and activation of caspase-3, as well as increased the expression of p-PI3K, p-Akt, and the ratio of Bcl-2 to Bax. Interestingly, a specific inhibitor of phosphatidylinositol 3-kinase, LY294002, partly reversed the anti-apoptotic effect of BCF. These observations indicated that BCF pretreatment attenuates H/R-induced myocardial apoptosis strength by improving mitochondrial dysfunction via PI3K/Akt signaling pathway.

  14. Protective Effect of Hibiscus Sabdariffa on Doxorubicin-induced Cytotoxicity in H9c2 Cardiomyoblast Cells.

    Science.gov (United States)

    Hosseini, Azar; Bakhtiari, Elham; Mousavi, Seyed Hadi

    2017-01-01

    Doxorubicin (DOX) is an effective anticancer drug. But its clinical application is limited, because DOX induces apoptosis in cardiomyocytes and it leads to permanent degenerative cardiomyopathy and heart failure. Recent trainings showed that Hibiscus sabdariffa exhibit pharmacological actions such as potent antioxidant. So, in this study we explored the protective effect of H. sabdariffa extract on doxorubicin-induced cytotoxicity in H9c2 cells. Cell viability was quantified by MTT assay. Apoptotic cells were determined using PI staining of DNA fragmentation by flowcytometry (sub-G1 peak). Cells were cultured with 5 μM DOX for 24 h to create the cell damage. H9c2 cells were pretreated with different concentrations (7.81-500 μg/mL) of H. sabdariffa extract (HSE) for 2 h before DOX treatment in all trials. Pretreatment with HSE increased cell viability at concentration of 31.25-500 μg/mL. Compared to control cells, apoptosis was induced in DOX treated cells after 24 h, (sabdariffa could exert the cardioprotective effects on DOX-induced toxicity partly by antiapoptotic activity.

  15. Odanacatib Inhibits Resistin-induced Hypertrophic H9c2 Cardiomyoblast Cells Through LKB1/AMPK Pathway

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    Xian Zheng

    2017-08-01

    Full Text Available ABSTRACT Odanacatib (ODN is a selective inhibitor of cathepsin K. The cysteine protease cathepsin K has been implicated in cardiac hypertrophy. Resistine is an adipokine which is identified to promote cardiac hypertrophy. Here, we hypothesize that ODN mitigates resistin-induced myocyte hypertrophy. Cell surface area and protein synthesis were measured after treatment with resistin and ODN in H9c2 cells. The expression of cardiomyocyte hypertrophy marker BNP and β-MHC was detected by RT-qPCR. The expression and phosphorylation of AMPK and LKB1 were analyzed with Western blot. Resistin could significantly increase cardiomyocyte cell surface area, protein synthesis, and embryonic gene BNP and β-MHC expression, inhibit phosphorylation of AMPK and LKB1. ODN could significantly reverse the effects of resistin. Collectively, our data suggest that ODN can inhibit cardiomyocyte hypertrophy induced by resistin and the underlying mechanism may be involved in LKB1/AMPK pathway.

  16. TanshinoneIIA and cryptotanshinone protect against hypoxia-induced mitochondrial apoptosis in H9c2 cells.

    Directory of Open Access Journals (Sweden)

    Hyou-Ju Jin

    Full Text Available Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA and cryptotanshinone (CT on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 μΜ 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1α expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak. TIIA and CT (0.3 and 3 μΜ, in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins.

  17. The protective effect of lycopene on hypoxia/reoxygenation-induced endoplasmic reticulum stress in H9C2 cardiomyocytes.

    Science.gov (United States)

    Gao, Yang; Jia, Pengyu; Shu, WenQi; Jia, Dalin

    2016-03-05

    Nowadays, drugs protecting ischemia/reperfusion (I/R) myocardium become more suitable for clinic. It has been confirmed lycopene has various protections, but lacking the observation of its effect on endoplasmic reticulum stress (ERS)-mediated apoptosis caused by hypoxia/reoxygenation (H/R). This study aims to clarify the protective effect of lycopene on ERS induced by H/R in H9C2 cardiomyocytes. Detect the survival rate, lactic dehydrogenase (LDH) activity, apoptosis ratio, glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP), c-Jun-N-terminal protein Kinase (JNK) and Caspase-12 mRNA and protein expression and phosphorylation of JNK (p-JNK) protein expression. LDH activity, apoptosis ratio and GRP78 protein expression increase in the H/R group, reduced by lycopene. The survival rate reduces in the H/R and thapsigargin (TG) groups; lycopene and 4-phenyl butyric acid (4-PBA) can improve it caused by H/R, lycopene also can improve it caused by TG. The apoptosis ratio, the expression of GRP78, CHOP and Caspase-12 mRNA and protein and p-JNK protein increase in the H/R and TG groups, weaken in the lycopene+H/R, 4-PBA+H/R and lycopene+TG groups. There is no obvious change in the expression of JNK mRNA or protein. Hence, our results provide the evidence that 10 μM lycopene plays an obviously protective effect on H/R H9C2 cardiomyocytes, realized through reducing ERS and apoptosis. The possible mechanism may be related to CHOP, p-JNK and Caspase-12 pathways. Copyright © 2016. Published by Elsevier B.V.

  18. Therapeutic concentrations of mitoxantrone elicit energetic imbalance in H9c2 cells as an earlier event.

    Science.gov (United States)

    Rossato, Luciana Grazziotin; Costa, Vera Marisa; Vilas-Boas, Vânia; de Lourdes Bastos, Maria; Rolo, Anabela; Palmeira, Carlos; Remião, Fernando

    2013-12-01

    Mitoxantrone (MTX) is a chemotherapeutic agent that emerged as an alternative to anthracycline therapy. However, MTX also causes late cardiotoxicity, being oxidative stress and mitochondrial-impaired function proposed as possible mechanisms. This work aimed to investigate the relevance of these mechanisms to the MTX toxicity in H9c2 cells, using therapeutic concentrations. The observed cytotoxicity of MTX was time and concentration dependent in both lactate dehydrogenase leakage assay and MTT reduction assay. Two therapeutic concentrations (100 nM and 1 μM) and three time points were selected (24, 48, and 96 h) for further studies. Both MTX concentrations caused a significant increase in caspase-3 activity, which was not prevented by inhibiting MTX CYP450-metabolism. Significant decreases were observed in the total and reduced glutathione levels only in MTX 100 nM at 96 h; however, neither alterations in oxidized glutathione nor increases in the malondialdehyde levels were observed at any time or concentrations tested. On the other hand, changes in the intracellular ATP levels, mitochondrial membrane potential, and intracellular calcium levels were observed in both concentrations and all time tested. Noteworthy, decreased levels of ATP-synthase expression and activity and increases in the reactive species generation were observed at 96 h in both working concentrations. However, the radical scavenger N-acetylcysteine or the mitochondrial function enhancer L-carnitine did not prevent MTX cytotoxicity. Thus, this work evidenced the early MTX-induced energetic crisis as a possible key factor in the cell injury.

  19. Tribulus terrestris (Linn.) Attenuates Cellular Alterations Induced by Ischemia in H9c2 Cells Via Antioxidant Potential.

    Science.gov (United States)

    Reshma, P L; Lekshmi, V S; Sankar, Vandana; Raghu, K G

    2015-06-01

    Tribulus terrestris L. was evaluated for its cardioprotective property against myocardial ischemia in a cell line model. Initially, methanolic extract was prepared and subjected to sequential extraction with various solvents. The extract with high phenolic content (T. terrestris L. ethyl acetate extract-TTME) was further characterized for its chemical constituents and taken forward for evaluation against cardiac ischemia. HPLC analysis revealed the presence of phenolic compounds like caffeic acid (12.41 ± 0.22 mg g(-1)), chlorogenic acid (0.52 ± 0.06 mg g(-1)) and 4-hydroxybenzoic acid (0.60 ± 0.08 mg g(-1)). H9c2 cells were pretreated with TTME (10, 25, 50 and 100 µg/ml) for 24 h before the induction of ischemia. Then ischemia was induced by exposing cells to ischemia buffer, in a hypoxic chamber, maintained at 0.1% O2, 95% N2 and 5% CO2, for 1 h. A significant (p ≤ 0.05) increase in reactive oxygen species generation (56%), superoxide production (18%), loss of plasma membrane integrity, dissipation of transmembrane potential, permeability transition pore opening and apoptosis had been observed during ischemia. However, pretreatment with TTME was found to significantly (p ≤ 0.05) attenuate the alterations caused by ischemia. The overall results of this study partially reveal the scientific basis of the use of T. terrestris L. in the traditional system of medicine for heart diseases. Copyright © 2015 John Wiley & Sons, Ltd.

  20. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    International Nuclear Information System (INIS)

    Si, Lina; Shi, Jin; Gao, Wenqun; Zheng, Min; Liu, Lingjuan; Zhu, Jing; Tian, Jie

    2014-01-01

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  1. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Si, Lina; Shi, Jin; Gao, Wenqun [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Zheng, Min [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Liu, Lingjuan; Zhu, Jing [Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Tian, Jie, E-mail: jietian@cqmu.edu.cn [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China)

    2014-07-18

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  2. Polysaccharide from Angelica sinensis protects H9c2 cells against oxidative injury and endoplasmic reticulum stress by activating the ATF6 pathway.

    Science.gov (United States)

    Niu, Xiaowei; Zhang, Jingjing; Ling, Chun; Bai, Ming; Peng, Yu; Sun, Shaobo; Li, Yingdong; Zhang, Zheng

    2018-01-01

    Objectives Angelica sinensis exerts various pharmacological effects, such as antioxidant and anti-apoptotic activity. This study aimed to investigate the active ingredients in A. sinensis with antioxidant properties and whether A. sinensis polysaccharide (ASP) protects H9c2 cells against oxidative and endoplasmic reticulum (ER) stress. Methods The ingredients of A. sinensis and their targets and related pathways were determined using web-based databases. Markers of oxidative stress, cell viability, apoptosis, and ER stress-related signalling pathways were measured in H9c2 cells treated with hydrogen peroxide (H 2 O 2 ) and ASP. Results The ingredient-pathway-disease network showed that A. sinensis exerted protective effects against oxidative injury through its various active ingredients on regulation of multiple pathways. Subsequent experiments showed that ASP pretreatment significantly decreased H 2 O 2 -induced cytotoxicity and apoptosis in H9c2 cells. ASP pretreatment inhibited H 2 O 2 -induced reactive oxygen species generation, lactic dehydrogenase release, and malondialdehyde production. ASP exerted beneficial effects by inducing activating transcription factor 6 (ATF6) and increasing ATF6 target protein levels, which in turn attenuated ER stress and increased antioxidant activity. Conclusions Our findings indicate that ASP, a major water-soluble component of A. sinensis, exerts protective effects against H 2 O 2 -induced injury in H9c2 cells by activating the ATF6 pathway, thus ameliorating ER and oxidative stress.

  3. The effect of Astragalus polysaccharides on attenuation of diabetic cardiomyopathy through inhibiting the extrinsic and intrinsic apoptotic pathways in high glucose -stimulated H9C2 cells.

    Science.gov (United States)

    Sun, Shuqin; Yang, Shuo; Dai, Min; Jia, Xiujuan; Wang, Qiyan; Zhang, Zheng; Mao, Yongjun

    2017-06-13

    Apoptosis plays a critical role in the progression of diabetic cardiomyopathy (DC). Astragalus polysaccharides (APS), an extract of astragalus membranaceus (AM), is an effective cardioprotectant. Currently, little is known about the detailed mechanisms underlying cardioprotective effects of APS. The aims of this study were to investigate the potential effects and mechanisms of APS on apoptosis employing a model of high glucose induction of apoptosis in H9C2 cells. A model of high glucose induction of H9C2 cell apoptosis was adopted in this research. The cell viabilities were analyzed by MTT assay, and the apoptotic response was quantified by flow cytometry. The expression levels of the apoptosis related proteins were determined by Real-time PCR and western blotting. Incubation of H9C2 cells with various concentrations of glucose (i.e., 5.5, 12.5, 25, 33 and 44 mmol/L) for 24 h revealed that cell viability was reduced by high glucose dose-dependently. Pretreatment of cells with APS could inhibit high glucose-induced H9C2 cell apoptosis by decreasing the expressions of caspases and the release of cytochrome C from mitochondria to cytoplasm. Further experiments also showed that APS could modulate the ratio of Bcl-2 to Bax in mitochondria. APS decreases high glucose-induced H9C2 cell apoptosis by inhibiting the expression of pro-apoptotic proteins of both the extrinsic and intrinsic pathways and modulating the ratio of Bcl-2 to Bax in mitochondria.

  4. Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection

    Science.gov (United States)

    Almami, Ibtesam; Dickenson, John M; Hargreaves, Alan J; Bonner, Philip L R

    2014-01-01

    BACKGROUND AND PURPOSE Tissue transglutaminase (TG2) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte-like H9c2 cells. EXPERIMENTAL APPROACH H9c2 cells were extracted following stimulation with phorbol-12-myristate-13-acetate (PMA) and forskolin. Transglutaminase activity was determined using an amine incorporating and a protein crosslinking assay. The presence of TG isoforms (TG1, 2, 3) was determined using Western blot analysis. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H2O2-induced oxidative stress in H9c2 cells. KEY RESULTS Western blotting showed TG2 >> TG1 protein expression but no detectable TG3. The amine incorporating activity of TG2 in H9c2 cells increased in a time and concentration-dependent manner following stimulation with PMA and forskolin. PMA and forskolin-induced TG2 activity was blocked by PKC (Ro 31-8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors respectively. The PMA- and forskolin-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Immunocytochemistry revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (β-tubulin) and novel (α-actinin) protein substrates for TG2. Pretreatment with PMA and forskolin reversed H2O2-induced decrease in MTT reduction and release of LDH. TG2 inhibitors R283 and Z-DON blocked PMA- and forskolin-induced cytoprotection. CONCLUSIONS AND IMPLICATIONS TG2 activity was stimulated via PKA- and PKC-dependent signalling pathways in H9c2 cells These results suggest a role for TG2 in cytoprotection induced by these kinases. PMID:24821315

  5. hHGF overexpression in myoblast sheets enhances their angiogenic potential in rat chronic heart failure.

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    Antti Siltanen

    2011-04-01

    Full Text Available After severe myocardial infarction (MI, heart failure results from ischemia, fibrosis, and remodeling. A promising therapy to enhance cardiac function and induce therapeutic angiogenesis via a paracrine mechanism in MI is myoblast sheet transplantation. We hypothesized that in a rat model of MI-induced chronic heart failure, this therapy could be further improved by overexpression of the antiapoptotic, antifibrotic, and proangiogenic hepatocyte growth factor (HGF in the myoblast sheets. We studied the ability of wild type (L6-WT and human HGF-expressing (L6-HGF L6 myoblast sheet-derived paracrine factors to stimulate cardiomyocyte, endothelial cell, or smooth muscle cell migration in culture. Further, we studied the autocrine effect of hHGF-expression on myoblast gene expression profiles by use of microarray analysis. We induced MI in Wistar rats by left anterior descending coronary artery (LAD ligation and allowed heart failure to develop for 4 weeks. Thereafter, we administered L6-WT (n = 15 or L6-HGF (n = 16 myoblast sheet therapy. Control rats (n = 13 underwent LAD ligation and rethoracotomy without therapy, and five rats underwent a sham operation in both surgeries. We evaluated cardiac function with echocardiography at 2 and 4 weeks after therapy, and analyzed cardiac angiogenesis and left ventricular architecture from histological sections at 4 weeks. Paracrine mediators from L6-HGF myoblast sheets effectively induced migration of cardiac endothelial and smooth muscle cells but not cardiomyocytes. Microarray data revealed that hHGF-expression modulated myoblast gene expression. In vivo, L6-HGF sheet therapy effectively stimulated angiogenesis in the infarcted and non-infarcted areas. Both L6-WT and L6-HGF therapies enhanced cardiac function and inhibited remodeling in a similar fashion. In conclusion, L6-HGF therapy effectively induced angiogenesis in the chronically failing heart. Cardiac function, however, was not further

  6. A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

    Science.gov (United States)

    Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

    2016-05-01

    The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Araloside C Prevents Hypoxia/Reoxygenation-Induced Endoplasmic Reticulum Stress via Increasing Heat Shock Protein 90 in H9c2 Cardiomyocytes

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    Yuyang Du

    2018-04-01

    Full Text Available Araloside C (AsC is a cardioprotective triterpenoid compound that is mainly isolated from Aralia elata. This study aims to determine the effects of AsC on hypoxia-reoxygenation (H/R-induced apoptosis in H9c2 cardiomyocytes and its underlying mechanisms. Results demonstrated that pretreatment with AsC (12.5 μM for 12 h significantly suppressed the H/R injury in H9c2 cardiomyocytes, including improving cell viability, attenuating the LDH leakage and preventing cardiomyocyte apoptosis. AsC also inhibited H/R-induced ER stress by reducing the activation of ER stress pathways (PERK/eIF2α and ATF6, and decreasing the expression of ER stress-related apoptotic proteins (CHOP and caspase-12. Moreover, AsC greatly improved the expression level of HSP90 compared with that in the H/R group. The use of HSP90 inhibitor 17-AAG and HSP90 siRNA blocked the above suppression effect of AsC on ER stress-related apoptosis caused by H/R. Taken together, AsC could reduce H/R-induced apoptosis possibly because it attenuates ER stress-dependent apoptotic pathways by increasing HSP90 expression.

  8. A polysaccharide of Dendrobium officinale ameliorates H2O2-induced apoptosis in H9c2 cardiomyocytes via PI3K/AKT and MAPK pathways.

    Science.gov (United States)

    Zhang, Jing-Yi; Guo, Ying; Si, Jin-Ping; Sun, Xiao-Bo; Sun, Gui-Bo; Liu, Jing-Jing

    2017-11-01

    Dendrobium officinale is one valuable traditional Chinese medicine, which has skyscraping medicinal value. Polysaccharide is the main active ingredient in D. officinale; its antioxidant activity is a hot research topic nowadays. Oxidative stress plays an important role in the pathological progress of a variety of cardiovascular disease, as one of key factors of cardiomyocyte apoptosis. This research adopts a model of H 2 O 2 induction-H9c2 cardiomyocytes apoptosis, aiming to study the effect of Dendrobium officinale Polysaccharide (DOP-GY) for cardiomyocyte apoptosis caused by oxidative stress and its possible mechanism. Our results showed that pretreatment of DOP-GY (low dose: 6.25μg/mL, medium dose: 12.5μg/mL, high dose: 25μg/mL) followed by a 2h incubation with 200μM H 2 O 2 elevated the survival rate, cutted the LDH leakage, reduced lipid peroxidation damage, improved the activity of the endogenous antioxidant enzymes. In addition, the pretreatment of DOP-GY significantly inhibited the production of ROS, declined of the mitochondrial membrane potential, down-regulated pro-apoptosis protein and up-regulated anti-apoptosis protein. The protective effect was correlated with the PI3K/Akt and MAPK signal pathway. Collectively, these observations suggest that DOY-GY has the potential to exert cardioprotective effects against H 2 O 2 -induced H9c2 cardiomyocyte apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Cardioprotective effect of breviscapine: inhibition of apoptosis in H9c2 cardiomyocytes via the PI3K/Akt/eNOS pathway following simulated ischemia/reperfusion injury.

    Science.gov (United States)

    Wang, Jun; Ji, Shu-Yun; Liu, Si-Zhu; Jing, Rui; Lou, Wei-Juan

    2015-09-01

    Breviscapine (BE) is a standardized Chinese herbal medicine extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. It has been widely used to treat cardiovascular and cerebrovascular diseases. However, there are no reports on the protective effects and underlying molecular mechanisms of BE action on myocardial ischemia/reperfusion (MI/R)-induced cardiomyocyte apoptosis. In the present study, we aimed to confirm the cardioprotective effect of BE from MI/R injury in vivo, and investigate the potential molecular mechanisms against simulated ischemia/reperfusion (SI/R)-induced cardiomyocyte apoptosis in vitro. The rat model of MI/R injury was induced by 30 min of transient vessel occlusion followed by 3 h of reperfusion. BE significantly reduced the myocardium infarct size and production of cardiac troponin (cTnl) in serum. In an in vitro experiment, H9c2 cardiomyocytes were incubated with vehicle or ischemic buffer during hypoxia; then, they were reoxygenated with or without BE. BE markedly improved the cell viability and decreased lactate dehydrogenase (LDH) release. We confirmed the anti-apoptotic effect of BE with the Hoechst 33258 staining assay, and this effect was associated with an increase in Bcl-2 and a decrease in active caspase-3 expression. Western blot analysis also showed that BE increased the phosphorylation of Akt and eNOS in H9c2 cells, and the protective effects of BE were partially inhibited by the phosphatidylinositol 3'-kinase (PI3K) specific inhibitor LY294002. Our results suggested that BE could provide significant cardioprotection against MI/R injury, and the potential mechanisms might involve suppression of cardiomyocyte apoptosis through activating the PI3K/Akt/eNOS signaling pathway.

  10. Luteolin Prevents H2O2-Induced Apoptosis in H9C2 Cells through Modulating Akt-P53/Mdm2 Signaling Pathway

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    Hong Chang

    2016-01-01

    Full Text Available Introduction. Luteolin, a falconoid compound in many Chinese herbs and formula, plays important roles in cardiovascular diseases. The underlying mechanism of luteolin remains to be further elaborated. Methods. A model of hydrogen peroxide- (H2O2- induced H9C2 cells apoptosis was established. Cell viabilities were examined with an MTT assay. 2′,7′-Dichlorofluorescin diacetate (DCFH-DA and flow cytometry were used to detect ROS level and apoptosis rate, respectively. The expressions of signaling proteins related to apoptosis were analyzed by western blot and mRNA levels were detected by real-time polymerase chain reaction (PCR. Quercetin was applied as positive drug. Results. Incubation with various concentrations of H2O2 (0, 50, 100, and 200 μM for 1 h caused dose-dependent loss of cell viability and 100 μM H2O2 reduced the cell viability to approximately 50%. Treatments with luteolin and quercetin protected cells from H2O2-induced cytotoxicity and reduced cellular ROS level and apoptosis rate. Moreover, luteolin could downregulate the expressions of Bax, caspase-8, cleaved-caspase-3, and p53 in apoptotic signaling pathway. Further study showed that the expressions of Akt, Bcl-2, and Mdm2 were upregulated by luteolin. Conclusion. Luteolin protects H9C2 cells from H2O2-induced apoptosis. The protective and antiapoptotic effects of luteolin could be mediated by regulating the Akt-P53/Mdm2 apoptotic pathway.

  11. Astragaloside IV Inhibits Oxidative Stress-Induced Mitochondrial Permeability Transition Pore Opening by Inactivating GSK-3β via Nitric Oxide in H9c2 Cardiac Cells

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    Yonggui He

    2012-01-01

    Full Text Available Objective. This study aimed to investigate whether astragaloside IV modulates the mitochondrial permeability transition pore (mPTP opening through glycogen synthase kinase 3β (GSK-3β in H9c2 cells. Methods. H9c2 cells were exposed to astragaloside IV for 20 min. GSK-3β (Ser9, Akt (Ser473, and VASP (Ser239 activities were determined with western blot. The mPTP opening was evaluated by measuring mitochondrial membrane potential (ΔΨm. Nitric oxide (NO generation was measured by 4-amino-5-methylamino-2′, 7′-difluorofluorescein (DAF-FM diacetate. Fluorescence images were obtained with confocal microscopy. Results. Astragaloside IV significantly enhanced GSK-3β phosphorylation and prevented H2O2-induced loss of ΔΨm. These effects of astragaloside IV were reversed by the phosphatidylinositol 3-kinase (PI3K inhibitor LY294002, the NO sensitive guanylyl cyclase selective inhibitor ODQ, and the PKG inhibitor KT5823. Astragaloside IV activated Akt and PKG. Astragaloside IV was also shown to increase NO production, an effect that was reversed by L-NAME and LY294002. Astragaloside IV applied at reperfusion reduced cell death caused by simulated ischemia/reperfusion, indicating that astragaloside IV can prevent reperfusion injury. Conclusions. These data suggest that astragaloside IV prevents the mPTP opening and reperfusion injury by inactivating GSK-3β through the NO/cGMP/PKG signaling pathway. NOS is responsible for NO generation and is activated by the PI3K/Akt pathway.

  12. Vitamin C and sodium bicarbonate enhance the antioxidant ability of H9C2 cells and induce HSPs to relieve heat stress.

    Science.gov (United States)

    Yin, Bin; Tang, Shu; Sun, Jiarui; Zhang, Xiaohui; Xu, Jiao; Di, Liangjiao; Li, Zhihong; Hu, Yurong; Bao, Endong

    2018-02-13

    Heat stress is exacerbated by global warming and affects human and animal health, leading to heart damage caused by imbalances in reactive oxygen species (ROS) and the antioxidant system, acid-base chemistry, electrolytes and respiratory alkalosis. Vitamin C scavenges excess ROS, and sodium bicarbonate maintains acid-base and electrolyte balance, and alleviates respiratory alkalosis. Herein, we explored the ability of vitamin C alone and in combination with equimolar sodium bicarbonate (Vitamin C-Na) to stimulate endogenous antioxidants and heat shock proteins (HSPs) to relieve heat stress in H9C2 cells. Control, vitamin C (20 μg/ml vitamin C for 16 h) and vitamin C-Na (20 μg/ml vitamin C-Na for 16 h) groups were heat-stressed for 1, 3 or 5 h. Granular and vacuolar degeneration, karyopyknosis and damage to nuclei and mitochondria were clearly reduced in treatment groups, as were apoptosis, lactate dehydrogenase activity and ROS and malondialdehyde levels, while superoxide dismutase activity was increased. Additionally, CRYAB, Hsp27, Hsp60 and Hsp70 mRNA levels were upregulated at 3 h (p < 0.01), and protein levels were increased for CRYAB at 0 h (p < 0.05) and 1 h (p < 0.01), and for Hsp70 at 3 and 5 h (p < 0.01). Thus, pre-treatment with vitamin C or vitamin C-Na might protect H9C2 cells against heat damage by enhancing the antioxidant ability and upregulating CRYAB and Hsp70.

  13. The protective effect of lipid emulsion in preventing bupivacaine-induced mitochondrial injury and apoptosis of H9C2 cardiomyocytes.

    Science.gov (United States)

    Chen, Zhe; Jin, Zhousheng; Xia, Yun; Zhao, Shishi; Xu, Xuzhong; Papadimos, Thomas J; Wang, Quanguang

    2017-11-01

    Lipid emulsion (LE) has been shown to be effective in the resuscitation of bupivacaine-induced cardiac arrest, but the precise mechanism of this action has not been fully elucidated. Pursuant to this lack of information on the mechanism in which LE protects the myocardium during bupivacaine-induced toxicity, we explored mitochondrial function and cell apoptosis. H9C2 cardiomyocytes were used in study. Cells were randomly divided in different groups and were cultivated 6 h, 12 h, and 24 h. The mitochondria were extracted and mitochondrial ATP content was measured, as was mitochondrial membrane potential, the concentration of calcium ion (Ca2+), and the activity of Ca2+-ATP enzyme (Ca2+-ATPase). Cells from groups Bup1000, LE group, and Bup1000LE were collected to determine cell viability, cell apoptosis, and electron microscopy scanning of mitochondrial ultrastructure (after 24 h). We found that LE can reverse the inhibition of the mitochondrial function induced by bupivacaine, regulate the concentration of calcium ion in mitochondria, resulting in the protection of myocardial cells from toxicity induced by bupivacaine.

  14. TSA protects H9c2 cells against thapsigargin-induced apoptosis related to endoplasmic reticulum stress-mediated mitochondrial injury.

    Science.gov (United States)

    Li, Zhiping; Liu, Yan; Dai, Xinlun; Zhou, Qiangqiang; Liu, Xueli; Li, Zeyu; Chen, Xia

    2017-05-01

    Endoplasmic reticulum stress (ERS) activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. Recently, TSA has shown protective effects on ERS and its mechanisms related to ER pathway has been previously characterized. However, whether TSA exerts its protective role via metabolic events remain largely undefined. Objectives : To explore the possible involvement of the metabolic changes during ERS and to better understand how TSA influence mitochondrial function to facilitate cellular adaptation. Results : TSA is an inhibitor of histone deacetylase which could significantly inhibit H9c2 cell apoptosis induced by Thapsigargin (TG). It also intervene the decrease of mitochondrial membrane potential. By immunofluorescence staining, we have shown that GRP78 was concentrated in the perinuclear region and co-localized with ER. However, treatments with TG and TSA could let it overlap with the mitochondrial marker MitoTracker. Cellular fractionation also confirmed the location of GRP78 in mitochondrion. TSA decreases ERS-induced cell apoptosis and mitochondrial injury may related to enhance the location of GRP78 in mitochondrion.

  15. Remote loading of doxorubicin into liposomes by transmembrane pH gradient to reduce toxicity toward H9c2 cells

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    Mohamed Alyane

    2016-03-01

    Full Text Available The use of doxorubicin (DOX is limited by its dose-dependent cardiotoxicity. Entrapped DOX in liposome has been shown to reduce cardiotoxicity. Results showed that about 92% of the total drug was encapsulated in liposome. The release experiments showed a weak DOX leakage in both culture medium and in PBS, more than 98% and 90% of the encapsulated DOX respectively was still retained in liposomes after 24 h of incubation. When the release experiments were carried out in phosphate buffer pH5.3, the leakage of DOX from liposomes reached 37% after 24 h of incubation. Evaluation of cellular uptake of the liposomal DOX indicated the possible endocytosis of liposomes because the majority of visible fluorescence of DOX was mainly in the cytoplasm, whereas the nuclear compartment showed a weak intensity. When using unloaded fluorescent-liposomes, the fluorescence was absent in nuclei suggests that liposomes cannot cross the nuclear membrane. MTT assay and measurement of LDH release suggest that necrosis is the form of cellular death predominates in H9c2 cells exposed to high doses of DOX, while for weak doses apoptosis could be the predominate form. Entrapped DOX reduced significantly DOX toxicity after 3 and 6 h of incubation, but after 20 h entrapped DOX is more toxic than free one.

  16. Inhibition of Na+ channel currents in rat myoblasts by 4-aminopyridine

    International Nuclear Information System (INIS)

    Lu Boxun; Liu Linyun; Liao Lei; Zhang Zhihong; Mei Yanai

    2005-01-01

    Our previous study revealed that 4-aminopyridine (4-AP), a specific blocker of A-type current, could also inhibit inward Na + currents (I Na ) with a state-independent mechanism in rat cerebellar granule cells. In the present study, we report an inhibitory effect of 4-AP on voltage-gated and tetrodotoxin (TTX)-sensitive I Na recorded from cultured rat myoblasts. 4-AP inhibited I Na amplitude in a dose-dependent manner between the concentrations of 0.5 and 10 mM without significant alteration in the activation or inactivation kinetics of the channel. By comparison to the 4-AP-induced inhibitory effect on cerebellum neurons, the inhibitory effect on myoblasts was enhanced through repetitive pulse and inflected by changing frequency. Specifically, the lower the frequency of pulse, the higher the inhibition observed, suggesting that block manner is inversely use-dependent. Moreover, experiments adding 4-AP to the intracellular solution indicate that the inhibitory effects are localized inside the cell. Additionally, 4-AP significantly modifies the properties of steady-state activation and inactivation kinetics of the channel. Our data suggest that the K + channel blocker 4-AP inhibits both neuron and myoblast Na + channels via different mechanisms. These findings may also provide information regarding 4-AP-induced pharmacological and toxicological effects in clinical use and experimental research

  17. Combination of Nigella sativa with Glycyrrhiza glabra and Zingiber officinale augments their protective effects on doxorubicin-induced toxicity in h9c2 cells.

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    Hosseini, Azar; Shafiee-Nick, Reza; Mousavi, Seyed Hadi

    2014-12-01

    The use of doxorubicin (DOX) is limited by its dose-dependent cardio toxicity in which reactive Oxygen Species (ROS) play an important role in the pathological process. The aim of this study was to evaluate the protective effect of three medicinal plants, Nigella sativa (N), Glycyrrhiza glabra (G) and Zingiber officinale (Z), and their combination (NGZ), against DOX-induced apoptosis and death in H9c2 cells. The cells were incubated with different concentrations of each extract or NGZ for 4 hr which continued in the presence or absence of 5µM doxorubicin for 24 hr. Cell viability and the apoptotic rate were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) and propidium iodide (PI) staining assays, respectively. The level of ROS and lipid peroxidation were measured by fluorimetric methods. Treatment with doxorubicin increased ROS generation, enhanced malondialdehyde (MDA) formation, and induced apoptosis. Co-treatment of the cells with each herb extract increased viability of cells dose-dependently with a maximum protection effect of about 30%, and their potencies were N>G>Z. The combination of the threshold dose of each extract (NGZ) produced a similar effect, which was increased dose-dependently to a maximum protection of 70%. These effects were correlated with the effects of NGZ on ROS and MDA. All of the extracts have some protective effects against DOX-induced toxicity in cardiomyocytes with similar efficacies, but with different potencies. However, NGZ produced much higher protective effect via reducing oxidative stress and inhibiting of apoptotic induction processes. Further investigations are needed to determine the effects of NGZ on DOX chemotherapy.

  18. Ursolic Acid-enriched herba cynomorii extract induces mitochondrial uncoupling and glutathione redox cycling through mitochondrial reactive oxygen species generation: protection against menadione cytotoxicity in h9c2 cells.

    Science.gov (United States)

    Chen, Jihang; Wong, Hoi Shan; Ko, Kam Ming

    2014-01-27

    Herba Cynomorii (Cynomorium songaricum Rupr., Cynomoriaceae) is one of the most commonly used 'Yang-invigorating' tonic herbs in Traditional Chinese Medicine (TCM). An earlier study in our laboratory has demonstrated that HCY2, an ursolic acid-enriched fraction derived from Herba Cynomorii, increased mitochondrial ATP generation capacity (ATP-GC) and induced mitochondrial uncoupling as well as a cellular glutathione response, thereby protecting against oxidant injury in H9c2 cells. In this study, we demonstrated that pre-incubation of H9c2 cells with HCY2 increased mitochondrial reactive oxygen species (ROS) generation in these cells, which is likely an event secondary to the stimulation of the mitochondrial electron transport chain. The suppression of mitochondrial ROS by the antioxidant dimethylthiourea abrogated the HCY2-induced enhancement of mitochondrial uncoupling and glutathione reductase (GR)-mediated glutathione redox cycling, and also protected against menadione-induced cytotoxicity. Studies using specific inhibitors of uncoupling protein and GR suggested that the HCY2-induced mitochondrial uncoupling and glutathione redox cycling play a determining role in the cytoprotection against menadione-induced oxidant injury in H9c2 cells. Experimental evidence obtained thus far supports the causal role of HCY2-induced mitochondrial ROS production in eliciting mitochondrial uncoupling and glutathione antioxidant responses, which offer cytoprotection against oxidant injury in H9c2 cells.

  19. Long non-coding RNA TUG1 inhibits apoptosis and inflammatory response in LPS-treated H9c2 cells by down-regulation of miR-29b.

    Science.gov (United States)

    Zhang, Haifang; Li, Hui; Ge, Ang; Guo, Enyu; Liu, Shuxia; Zhang, Lijuan

    2018-05-01

    Myocarditis is an important cause for cardiovascular morbidity and mortality in children and adults. The lncRNA taurine up-regulated gene 1 (TUG1) plays important roles in cell apoptosis and inflammation in tumor and liver injury. The present study aimed to investigate the role of TUG1 in LPS-injured H9c2 cells and explore the underlying molecular mechanism. H9c2 cells were stimulated with LPS to induce inflammatory injury. The expression of TUG1 was altered by transient transfections. Cell viability and apoptotic cell rates were detected by CCK-8 assay and flow cytometry assay, respectively. Inflammatory response was determined by detecting levels of inflammatory cytokines using qRT-PCR and ELISA. Furthermore, western blot analysis was conducted to assess the expression levels of core factors related with apoptosis and activations of NF-κB and JAK/STAT signaling pathways. LPS exposure reduced cell viability but enhanced cell apoptosis and inflammation in H9c2 cells. Moreover, TUG1 expression was down-regulated in LPS-injured H9c2 cells. TUG1 overexpression attenuated LPS-induced injuries in H9c2 cells, evidenced by augmented cell viability, declined apoptotic cell rates and decreased levels of pro-apoptotic factors and inflammatory cytokines. Inversely, TUG1 inhibition exerted the opposite effects. More importantly, TUG1 negatively modulated the expression of miR-29b and miR-29b mimic blocked the effect of TUG1 overexpression on cell viability, apoptosis, inflammation and inactivation of NF-κB and JAK/STAT signaling pathways in LPS-stimulated H9c2 cells. This study demonstrated that TUG1 played the anti-apoptotic and anti-inflammatory roles in LPS-injured H9c2 cells via down-regulating miR-29b and inhibiting NF-κB and JAK/STAT pathways. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  20. 5-AIQ inhibits H{sub 2}O{sub 2}-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β signaling pathway in H9c2 cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun; Jang, Yong Chang [Department of Applied Biochemistry, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Yi, Kyu Yang [Bio-Organic Science Division, Korea Research Institute of Chemical Technology, Daejeon, Chungnam, 305-600 (Korea, Republic of); Chung, Hun-Jong [Industrial Medicine Department, Chungju Hospital, Konkuk Medical School, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Park, Jong Seok [Department of Biomedical Laboratory Science, Taegu Health College, Taegu 702-722 (Korea, Republic of); Kim, Bokyung [Department of Physiology, Konkuk Medical School, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Feng, Zhong-Ping [Department of Physiology, College of Medicine, University of Toronto, Toronto, Ont., Canada M5S 1A8 (Canada); Shin, Hwa-Sup, E-mail: hsshin@kku.ac.kr [Department of Applied Biochemistry, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of)

    2013-04-01

    Poly(adenosine 5′-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H{sub 2}O{sub 2}-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H{sub 2}O{sub 2}-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H{sub 2}O{sub 2}-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3β (GSK-3β), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3β activation. In addition, inhibiting the Akt/GSK-3β pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H{sub 2}O{sub 2}-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3β pathway. - Highlights: ► 5-AIQ, a PARP inhibitor, decreased H{sub 2}O{sub 2}-induced H9c2 cell death and apoptosis. ► 5-AIQ upregulated antioxidant Mn-SOD and catalase, while decreasing ROS production. ► 5-AIQ decreased H{sub 2}O{sub 2}-induced increase in cleaved caspase-3 and Bax and decrease in Bcl2. ► 5-AIQ activated Akt and GSK-3

  1. Role of transglutaminase 2 in A1 adenosine receptor- and β2-adrenoceptor-mediated pharmacological pre- and post-conditioning against hypoxia-reoxygenation-induced cell death in H9c2 cells.

    Science.gov (United States)

    Vyas, Falguni S; Nelson, Carl P; Dickenson, John M

    2018-01-15

    Pharmacologically-induced pre- and post-conditioning represent attractive therapeutic strategies to reduce ischaemia/reperfusion injury during cardiac surgery and following myocardial infarction. We have previously reported that transglutaminase 2 (TG2) activity is modulated by the A 1 adenosine receptor and β 2 -adrenoceptor in H9c2 cardiomyoblasts. The primary aim of this study was to determine the role of TG2 in A 1 adenosine receptor and β 2 -adrenoceptor-induced pharmacological pre- and post-conditioning in the H9c2 cells. H9c2 cells were exposed to 8h hypoxia (1% O 2 ) followed by 18h reoxygenation, after which cell viability was assessed by monitoring mitochondrial reduction of MTT, lactate dehydrogenase release and caspase-3 activation. N 6 -cyclopentyladenosine (CPA; A 1 adenosine receptor agonist), formoterol (β 2 -adrenoceptor agonist) or isoprenaline (non-selective β-adrenoceptor agonist) were added before hypoxia/reoxygenation (pre-conditioning) or at the start of reoxygenation following hypoxia (post-conditioning). Pharmacological pre- and post-conditioning with CPA and isoprenaline significantly reduced hypoxia/reoxygenation-induced cell death. In contrast, formoterol did not elicit protection. Pre-treatment with pertussis toxin (G i/o -protein inhibitor), DPCPX (A 1 adenosine receptor antagonist) or TG2 inhibitors (Z-DON and R283) attenuated the A 1 adenosine receptor-induced pharmacological pre- and post-conditioning. Similarly, pertussis toxin, ICI 118,551 (β 2 -adrenoceptor antagonist) or TG2 inhibition attenuated the isoprenaline-induced cell survival. Knockdown of TG2 using small interfering RNA (siRNA) attenuated CPA and isoprenaline-induced pharmacological pre- and post-conditioning. Finally, proteomic analysis following isoprenaline treatment identified known (e.g. protein S100-A6) and novel (e.g. adenine phosphoribosyltransferase) protein substrates for TG2. These results have shown that A 1 adenosine receptor and β 2 -adrenoceptor

  2. Comparison of arrhythmogenicity and proinflammatory activity induced by intramyocardial or epicardial myoblast sheet delivery in a rat model of ischemic heart failure.

    Directory of Open Access Journals (Sweden)

    Tommi Pätilä

    Full Text Available Although cell therapy of the failing heart by intramyocardial injections of myoblasts to results in regenerative benefit, it has also been associated with undesired and prospectively fatal arrhythmias. We hypothesized that intramyocardial injections of myoblasts could enhance inflammatory reactivity and facilitate electrical cardiac abnormalities that can be reduced by epicardial myoblast sheet delivery. In a rat model of ischemic heart failure, myoblast therapy either by intramyocardial injections or epicardial cell sheets was given 2 weeks after occlusion of the coronary artery. Ventricular premature contractions (VPCs were assessed, using an implanted three-lead electrocardiograph at 1, 7, and 14 days after therapy, and 16-point epicardial electropotential mapping (EEPM was used to evaluate ventricular arrhythmogenicity under isoproterenol stress. Cardiac functioning was assessed by echocardiography. Both transplantation groups showed therapeutic benefit over sham therapy. However, VPCs were more frequent in the Injection group on day 1 and day 14 after therapy than in animals receiving epicardial or sham therapy (p < 0.05 and p < 0.01, respectively. EEPM under isoproterenol stress showed macroreentry at the infarct border area, leading to ventricular tachycardias in the Injection group, but not in the myoblast sheet- or sham-treated groups (p = 0.045. Both transplantation types modified the myocardial cytokine expression profile. In animals receiving epicardial myoblast therapy, selective reductions in the expressions of interferon gamma, interleukin (IL-1β and IL12 were observed, accompanied by reduced infiltration of inflammatory CD11b- and CD68-positive leukocytes, compared with animals receiving myoblasts as intramyocardial injections. Intramyocardial myoblast delivery was associated with enhanced inflammatory and immunomodulatory reactivity and increased frequency of VPCs. In comparison to intramyocardial injection, the epicardial

  3. The Opening of ATP-Sensitive K+ Channels Protects H9c2 Cardiac Cells Against the High Glucose-Induced Injury and Inflammation by Inhibiting the ROS-TLR4-Necroptosis Pathway

    Directory of Open Access Journals (Sweden)

    Weijie Liang

    2017-02-01

    Full Text Available Background/Aims: Hyperglycemia activates multiple signaling molecules, including reactive oxygen species (ROS, toll-like receptor 4 (TLR4, receptor-interacting protein 3 (RIP3, a kinase promoting necroptosis, which mediate hyperglycemia-induced cardiac injury. This study explored whether inhibition of ROS-TLR4-necroptosis pathway contributed to the protection of ATP-sensitive K+ (KATP channel opening against high glucose-induced cardiac injury and inflammation. Methods: H9c2 cardiac cells were treated with 35 mM glucose (HG to establish a model of HG-induced insults. The expression of RIP3 and TLR4 were tested by western blot. Generation of ROS, cell viability, mitochondrial membrane potential (MMP and secretion of inflammatory cytokines were measured as injury indexes. Results: HG increased the expression of TLR4 and RIP3. Necrostatin-1 (Nec-1, an inhibitor of necroptosis or TAK-242 (an inhibitor of TLR4 co-treatment attenuated HG-induced up-regulation of RIP3. Diazoxide (DZ, a mitochondrial KATP channel opener or pinacidil (Pin, a non-selective KATP channel opener or N-acetyl-L-cysteine (NAC, a ROS scavenger pre-treatment blocked the up-regulation of TLR4 and RIP3. Furthermore, pre-treatment with DZ or Pin or NAC, or co-treatment with TAK-242 or Nec-1 attenuated HG-induced a decrease in cell viability, and increases in ROS generation, MMP loss and inflammatory cytokines secretion. However, 5-hydroxy decanoic acid (5-HD, a mitochondrial KATP channel blocker or glibenclamide (Gli, a non-selective KATP channel blocker pre-treatment did not aggravate HG-induced injury and inflammation. Conclusion: KATP channel opening protects H9c2 cells against HG-induced injury and inflammation by inhibiting ROS-TLR4-necroptosis pathway.

  4. A novel compound DT-010 protects against doxorubicin-induced cardiotoxicity in zebrafish and H9c2 cells by inhibiting reactive oxygen species-mediated apoptotic and autophagic pathways.

    Science.gov (United States)

    Tang, Fan; Zhou, Xinhua; Wang, Liang; Shan, Luchen; Li, Chuwen; Zhou, Hefeng; Lee, Simon Ming-Yuen; Hoi, Maggie Pui-Man

    2018-02-05

    Doxorubicin (Dox) is an effective anti-cancer agent but limited by its cardiotoxicity, thus the search for pharmacological agents for enhancing anti-cancer activities and protecting against cardiotoxicity has been a subject of great interest. We have previously reported the synergistic anti-cancer effects of a novel compound DT-010. In the present study, we further investigated the cardioprotective effects of DT-010 in zebrafish embryos in vivo and the molecular underlying mechanisms in H9c2 cardiomyocytes in vitro. We showed that DT-010 prevented the Dox-induced morphological distortions in the zebrafish heart and the associated cardiac impairments, and especially improved ventricular functions. By using H9c2 cells model, we showed that DT-010 directly inhibited the generation of reactive oxygen species by Dox and protected cell death and cellular damage. We further observed that DT-010 protected against Dox-induced myocardiopathy via inhibiting downstream molecular pathways in response to oxidative stress, including reactive oxygen species-mediated MAPK signaling pathways ERK and JNK, and apoptotic pathways involving the activation of caspase 3, caspase 7, and PARP signaling. Recent studies also suggest the importance of alterations in cardiac autophagy in Dox cardiotoxicity. We further showed that DT-010 could inhibit the induction of autophagosomes formation by Dox via regulating the upstream Akt/AMPK/mTOR signaling. Since Dox-induced cardiotoxicity is multifactorial, our results suggest that multi-functional agent such as DT-010 might be an effective therapeutic agent for combating cardiotoxicity associated with chemotherapeutic agents such as Dox. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

    Science.gov (United States)

    Valentine, Cathleen D.; Haggie, Peter M.

    2011-01-01

    The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β1- and β2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β1- and β2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β2AR confinement. For both β1- and β2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β1- or β2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes. PMID:21680711

  6. Confinement of β(1)- and β(2)-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae.

    Science.gov (United States)

    Valentine, Cathleen D; Haggie, Peter M

    2011-08-15

    The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β(1)- and β(2)AR, are structurally similar but mediate distinct signaling responses. Scaffold protein-mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β(1)- and β(2)AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)-domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β(2)AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β(2)AR confinement. For both β(1)- and β(2)AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β(1)- or β(2)AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes.

  7. Use of 5-Bromodeoxyuridine and irradiation for the estimation of the myoblast and myocyte content of primary rat heart cell cultures

    International Nuclear Information System (INIS)

    Masse, M.J.O.; Harary, I.

    1980-01-01

    A method for killing dividing cells was adapted for the elimination of dividing heart muscle cells (myoblasts) in cultures. We have used this method to demonstrate their presence and to estimate their number as well as the number of nondividing heart muscle cells (myocytes) in the neo-natal rat heart. Cells were cultivated in BUdR (5-bromodeoxyuridine) 10 -4 M for 3 days and then irradiated with long uv light. The selective elimination of dividing cells led to a loss of myosin Ca 2+ -activated ATPase in the cultures. The percent of ATPase left after irradiation was 32% of the control in cultures derived from 1-day postnatal rats and 48% in cultures from 4-day postnatal rats. This reflects an in vivo shift of myoblasts to myocytes in the muscle cell population as the rat ages

  8. Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5'AMP-activated protein kinase (AMPK). Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer technique.

    Science.gov (United States)

    Sambandam, Nandakumar; Steinmetz, Michael; Chu, Angel; Altarejos, Judith Y; Dyck, Jason R B; Lopaschuk, Gary D

    2004-07-01

    Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD has several Ser/Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD (Ad.MCD) and constitutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using an adenoviral gene delivery approach in order to examine if MCD is regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein (Ad.GFP). MCD activity increased 40- to 50-fold in Ad.MCD + Ad.GFP cells when compared with Ad.GFP control. Co-expressing AMPK with MCD further augmented MCD expression and activity in Ad.MCD + Ad.CA-AMPK cells compared with the Ad.MCD + Ad.GFP control. Subcellular fractionation further revealed that 54.7 kDa isoform of MCD expression was significantly higher in cytosolic fractions of Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +Ad.GFP control. However, the MCD activities in cytosolic fractions were not different between the two groups. Interestingly, in the mitochondrial fractions, MCD activity significantly increased in Ad.MCD + Ad.CA-AMPK cells when compared with Ad.MCD + Ad.GFP cells. Using phosphoserine and phosphothreonine antibodies, no phosphorylation of MCD by AMPK was observed. The increase in MCD activity in mitochondria-rich fractions of Ad.MCD + Ad.CA-AMPK cells was accompanied by an increase in the level of the 50.7 kDa isoform of MCD protein in the mitochondria. This differential regulation of MCD expression and activity in the mitochondria by AMPK may potentially regulate malonyl-CoA levels at sites nearby CPT-I on the mitochondria.

  9. Effects of Ghrelin on Triglyceride Accumulation and Glucose Uptake in Primary Cultured Rat Myoblasts under Palmitic Acid-Induced High Fat Conditions

    Directory of Open Access Journals (Sweden)

    Lingling Han

    2015-01-01

    Full Text Available This study aimed to study the effects of acylated ghrelin on glucose and triglyceride metabolism in rat myoblasts under palmitic acid- (PA- induced high fat conditions. Rat myoblasts were treated with 0, 10−11, 10−9, or 10−7 M acylated ghrelin and 0.3 mM PA for 12 h. Triglyceride accumulation was determined by Oil-Red-O staining and the glycerol phosphate dehydrogenase-peroxidase enzymatic method, and glucose uptake was determined by isotope tracer. The glucose transporter 4 (GLUT4, AMP-activated protein kinase (AMPK, acetyl-CoA carboxylase (ACC, and uncoupling protein 3 (UCP3 were assessed by RT-PCR and western blot. Compared to 0.3 mM PA, ghrelin at 10−9 and 10−7 M reduced triglyceride content (5.855 ± 0.352 versus 5.030 ± 0.129 and 4.158 ± 0.254 mM, P<0.05 and prevented PA-induced reduction of glucose uptake (1.717 ± 0.264 versus 2.233 ± 0.333 and 2.333 ± 0.273 10−2 pmol/g/min, P<0.05. The relative protein expression of p-AMPKα/AMPKα, UCP3, and p-ACC under 0.3 mM PA was significantly reduced compared to controls (all P<0.05, but those in the 10−9 and 10−7 M ghrelin groups were significantly protected from 0.3 mM PA (all P<0.05. In conclusion, acylated ghrelin reduced PA-induced triglyceride accumulation and prevented the PA-induced decrease in glucose uptake in rat myoblasts. These effects may involve fatty acid oxidation.

  10. FGF-2 Transcriptionally Down-Regulates the Expression of BNIP3L via PI3K/Akt/FoxO3a Signaling and Inhibits Necrosis and Mitochondrial Dysfunction Induced by High Concentrations of Hydrogen Peroxide in H9c2 Cells

    Directory of Open Access Journals (Sweden)

    Qian Chen

    2016-12-01

    Full Text Available Background/Aims: Cardiovascular disease is a growing major global public health problem. Necrosis is one of the main forms of cardiomyocyte death in heart disease. Oxidative stress is regarded as one of the key regulators of cardiac necrosis, which eventually leads to cardiovascular disease. Many pharmacological and in vitro studies have suggested that FGF-2 can act directly on cardiomyocytes to maintain the integrity and function of the myocardium and prevent damage during oxidative stress. However, the mechanisms by which FGF-2 rescues the myocardium from oxidative stress damage in cardiovascular disease remain unclear. The present study explored the protective effects of FGF-2 in the H2O2-induced necrosis of H9C2 cardiomyocytes as well as the possible signaling pathways involved. Methods: Necrosis of H9c2 cardiomyocytes was induced by H2O2 and assessed using a Cell Counting Kit-8 (CCK8 assay and flow cytometry analysis. The cells were pretreated with the PI3K/Akt inhibitor Wortmannin to investigate the possible involvement of the PI3K/Akt pathway in the protection by FGF-2. The levels of Akt, p-Akt, FoxO3a, p-FoxO3a, and BNIP3L were detected by Western blot. Chromatin immuno-precipitation (ChIP analysis was used to test whether FoxO3a binds directly to the BNIP3L promoter region. A luciferase assay was used to study the effects of FoxO3a on BNIP3L gene promoter activity. Mitochondrial ΔΨM was quantified using tetramethylrhodamine methyl ester perchlorate (TMRM. The mitochondrial oxygen consumption rate (OCR was assessed with a Seahorse XF24 Analyzer. Results: Treatment with H2O2 decreased the phosphorylation of Akt and FoxO3a, and it induced the nuclear localization of FoxO3a and the necrosis of H9c2 cells. These effects of H2O2 were abrogated by pretreatment with FGF-2. Furthermore, the protective effects of FGF-2 were abolished by the PI3K/Akt inhibitor Wortmannin. ChIP analyses indicated that FoxO3a binds directly to the BNIP3L promoter

  11. Myoblast fusion in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Haralalka, Shruti [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Abmayr, Susan M., E-mail: sma@stowers.org [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, MO 66160 (United States)

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  12. Myoblast fusion in Drosophila

    International Nuclear Information System (INIS)

    Haralalka, Shruti; Abmayr, Susan M.

    2010-01-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  13. Absence of muscle regeneration after implantation of a collagen matrix seeded with myoblasts

    NARCIS (Netherlands)

    van Wachem, PB; Brouwer, LA; van Luyn, MJA

    Collagens are widely used as biomaterials for e.g. soft tissue reconstruction. The present study was aimed at reconstruction of abdominal wall muscle using processed dermal sheep collagen (DSC) and myoblast seeding. Myoblasts were harvested from foetal quadriceps muscle of an inbred rat strain,

  14. Syncytin-1 in differentiating human myoblasts

    DEFF Research Database (Denmark)

    Bjerregard, Bolette; Ziomkiewicz, Iwona; Schulz, Alexander

    2014-01-01

    Myoblasts fuse to form myotubes, which mature into skeletal muscle fibres. Recent studies indicate that an endogenous retroviral fusion gene, syncytin-1, is important for myoblast fusions in man. We have now expanded these data by examining the immunolocalization of syncytin in human myoblasts...... fusions. Thus, syncytin is involved in human myoblast fusions and is localized in areas of contact between fusing cells. Moreover, syncytin and caveolin-3 might interact at the level of the sarcolemma....

  15. Taurine ameliorated homocysteine-induced H9C2 cardiomyocyte apoptosis by modulating endoplasmic reticulum stress.

    Science.gov (United States)

    Zhang, Zhimin; Zhao, Lianyou; Zhou, Yanfen; Lu, Xuanhao; Wang, Zhengqiang; Wang, Jipeng; Li, Wei

    2017-05-01

    Homocysteine (Hcy)-triggered endoplasmic reticulum (ER) stress-mediated endothelial cell apoptosis has been suggested as a cause of Hcy-dependent vascular injury. However, whether ER stress is the molecular mechanism linking Hcy and cardiomyocytes death is unclear. Taurine has been reported to exert cardioprotective effects via various mechanisms. However, whether taurine protects against Hcy-induced cardiomyocyte death by attenuating ER stress is unknown. This study aimed to evaluate the opposite effects of taurine on Hcy-induced cardiomyocyte apoptosis and their underlying mechanisms. Our results demonstrated that low-dose or short-term Hcy treatment increased the expression of glucose-regulated protein 78 (GRP78) and activated protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6), which in turn prevented apoptotic cell death. High-dose Hcy or prolonged Hcy treatment duration significantly up-regulated levels of C/EBP homologous protein (CHOP), cleaved caspase-12, p-c-Jun N-terminal kinase (JNK), and then triggered apoptotic events. High-dose Hcy also resulted in a decrease in mitochondrial membrane potential (Δψm) and an increase in cytoplasmic cytochrome C and the expression of cleaved caspase-9. Pretreatment of cardiomyocytes with sodium 4-phenylbutyric acid (an ER stress inhibitor) significantly inhibited Hcy-induced apoptosis. Furthermore, blocking the PERK pathway partly alleviated Hcy-induced ER stress-modulated cardiomyocyte apoptosis, and down-regulated the levels of Bax and cleaved caspase-3. Experimental taurine pretreatment inhibited the expression of ER stress-related proteins, and protected against apoptotic events triggered by Hcy-induced ER stress. Taken together, our results suggest that Hcy triggered ER stress in cardiomyocytes, which was the crucial molecular mechanism mediating Hcy-induced cardiomyocyte apoptosis, and the adverse effect of Hcy could be prevented by taurine.

  16. Defective myoblasts identified in Duchenne muscular dystrophy.

    OpenAIRE

    Blau, H M; Webster, C; Pavlath, G K

    1983-01-01

    A defect in the proliferative capacity of satellite cells, mononucleated precursors of mature muscle fibers, was found in clonal analyses of cells cultured from Duchenne muscular dystrophy (DMD) patients. The total yield of myoblasts per gram of muscle biopsy was decreased to 5% of normal. Of the DMD myoblast clones obtained, a large proportion contained a morphological class of flat distended cells that had an increased generation time and ceased to proliferate beyond 100-1,000 cells but cou...

  17. Inhibition of galectin-3 ameliorates the consequences of cardiac lipotoxicity in a rat model of diet-induced obesity

    Directory of Open Access Journals (Sweden)

    Gema Marín-Royo

    2018-02-01

    Full Text Available Obesity is accompanied by metabolic alterations characterized by insulin resistance and cardiac lipotoxicity. Galectin-3 (Gal-3 induces cardiac inflammation and fibrosis in the context of obesity; however, its role in the metabolic consequences of obesity is not totally established. We have investigated the potential role of Gal-3 in the cardiac metabolic disturbances associated with obesity. In addition, we have explored whether this participation is, at least partially, acting on mitochondrial damage. Gal-3 inhibition in rats that were fed a high-fat diet (HFD for 6 weeks with modified citrus pectin (MCP; 100 mg/kg/day attenuated the increase in cardiac levels of total triglyceride (TG. MCP treatment also prevented the increase in cardiac protein levels of carnitine palmitoyl transferase IA, mitofusin 1, and mitochondrial complexes I and II, reactive oxygen species accumulation and decrease in those of complex V but did not affect the reduction in 18F-fluorodeoxyglucose uptake observed in HFD rats. The exposure of cardiac myoblasts (H9c2 to palmitic acid increased the rate of respiration, mainly due to an increase in the proton leak, glycolysis, oxidative stress, β-oxidation and reduced mitochondrial membrane potential. Inhibition of Gal-3 activity was unable to affect these changes. Our findings indicate that Gal-3 inhibition attenuates some of the consequences of cardiac lipotoxicity induced by a HFD since it reduced TG and lysophosphatidyl choline (LPC levels. These reductions were accompanied by amelioration of the mitochondrial damage observed in HFD rats, although no improvement was observed regarding insulin resistance. These findings increase the interest for Gal-3 as a potential new target for therapeutic intervention to prevent obesity-associated cardiac lipotoxicity and subsequent mitochondrial dysfunction.

  18. Protection by 6-aminonicotinamide against oxidative stress in cardiac cells

    DEFF Research Database (Denmark)

    Hofgaard, Johannes P; Sigurdardottir, Kristin Sigridur; Treiman, Marek

    2006-01-01

    necrosis following global ischemia in an isolated rat heart, apparently by limiting the oxidative injury component. We therefore explored the antioxidative potential of 6AN in a model using H9C2(2-1) rat cardiac myoblasts exposed to H2O2 stress. Dependent on the specific protocol, 6AN pretreatment for 6...

  19. The use of cell-sheet technique eliminates arrhythmogenicity of skeletal myoblast-based therapy to the heart with enhanced therapeutic effects.

    Science.gov (United States)

    Narita, Takuya; Shintani, Yasunori; Ikebe, Chiho; Kaneko, Masahiro; Harada, Narumi; Tshuma, Nomathamsanqa; Takahashi, Kunihiko; Campbell, Niall G; Coppen, Steven R; Yashiro, Kenta; Sawa, Yoshiki; Suzuki, Ken

    2013-09-20

    Clinical application of skeletal myoblast transplantation has been curtailed due to arrhythmogenicity and inconsistent therapeutic benefits observed in previous studies. However, these issues may be solved by the use of a new cell-delivery mode. It is now possible to generate "cell-sheets" using temperature-responsive dishes without artificial scaffolds. This study aimed to validate the safety and efficacy of epicardial placement of myoblast-sheets (myoblast-sheet therapy) in treating heart failure. After coronary artery ligation in rats, the same numbers of syngeneic myoblasts were transplanted by intramyocardial injection or cell-sheet placement. Continuous radio-telemetry monitoring detected increased ventricular arrhythmias, including ventricular tachycardia, after intramyocardial injection compared to the sham-control, while these were abolished in myoblast-sheet therapy. This effect was conjunct with avoidance of islet-like cell-cluster formation that disrupts electrical conduction, and with prevention of increased arrhythmogenic substrates due to exaggerated inflammation. Persistent ectopic donor cells were found in the lung only after intramyocardial injection, strengthening the improved safety of myoblast-sheet therapy. In addition, myoblast-sheet therapy enhanced cardiac function, corresponding to a 9.2-fold increase in donor cell survival, compared to intramyocardial injection. Both methods achieved reduced infarct size, decreased fibrosis, attenuated cardiomyocyte hypertrophy, and increased neovascular formation, in association with myocardial upregulation of a group of relevant molecules. The pattern of these beneficial changes was similar between two methods, but the degree was more substantial after myoblast-sheet therapy. The cell-sheet technique enhanced safety and therapeutic efficacy of myoblast-based therapy, compared to the current method, thereby paving the way for clinical application. Copyright © 2012 Elsevier Ireland Ltd. All rights

  20. Marked heterogeneity in growth characteristics of myoblast clonal cultures and myoblast mixed cultures obtained from the same individual.

    Science.gov (United States)

    Maier, Andrea B; Cohen, Ron; Blom, Joke; van Heemst, Diana; Westendorp, Rudi G J

    2012-01-01

    Sarcopenia is defined as an age-related decrease in skeletal muscle mass and function while adjacent satellite cells are unable to compensate for this loss. However, myoblast cultures can be established even in the presence of sarcopenia. It is yet unknown whether satellite cells from failing muscle in older age are equally affected, as human satellite cells have been assessed using myoblast mixed cultures and not by using myoblast clonal cultures. We questioned to what extent myoblast mixed cultures reflect the in vivo characteristics of single satellite cells from adult skeletal muscle. We established a myoblast mixed culture and three myoblast clonal cultures out of the same muscle biopsy and cultured these cells for 100 days. Replicative capacity and oxidative stress resistance were compared. We found marked heterogeneity between the myoblast clonal cultures that all had a significantly lower replicative capacity when compared to the mixed culture. Replicative capacity of the clonal cultures was inversely related to the β-galactosidase activity after exposure to oxidative stress. Addition of L-carnosine enhanced the remaining replicative capacity in all cultures with a concomitant marginal decrease in β-galactosidase activity. It is concluded that myoblast mixed cultures in vitro do not reflect the marked heterogeneity between single isolated satellite cells. The consequences of the heterogeneity on muscle performance remain to be established. Copyright © 2011 S. Karger AG, Basel.

  1. Use of Flow Focusing Technique for Microencapsulation of Myoblasts.

    Science.gov (United States)

    Ciriza, J; Saenz del Burgo, L; Hernández, R M; Orive, G; Pedraz, J L

    2017-01-01

    Alginate cell microencapsulation implies the immobilization of cells within a polymeric membrane that allows the bidirectional diffusion of nutrients and oxygen inside the microcapsules and the release of waste and therapeutic molecules outside them. This technology has been applied to several cell types and it has been extensively described with pancreatic islets. However, other cells such as myoblasts are being currently studied and showing high interest. Moreover, different systems and approaches have been developed for cell encapsulation such as electrostatic extrusion and Flow focusing technology. When Flow focusing technology is applied for myoblast encapsulation, several factors should be considered, such as the pressure, the flow of the system, or the diameter size of the nebulizer, which will determine the final diameter size and shape of the microcapsules containing the myoblasts. Finally, viability of encapsulated myoblasts needs to be assessed before further studies are performed.

  2. Mast cell tryptase stimulates myoblast proliferation; a mechanism relying on protease-activated receptor-2 and cyclooxygenase-2

    Directory of Open Access Journals (Sweden)

    Côté Claude H

    2011-10-01

    Full Text Available Abstract Background Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2. The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade. Methods Proliferation of L6 rat skeletal myoblasts stimulated with PAR-2 agonists (tryptase, trypsin and SLIGKV was assessed. The specificity of the tryptase effect was evaluated with a specific inhibitor, APC-366. Western blot analyses were used to evaluate the expression and functionality of PAR-2 receptor and to assess the expression of COX-2. COX-2 activity was evaluated with a commercial activity assay kit and by measurement of PGF2α production. Proliferation assays were also performed in presence of different prostaglandins (PGs. Results Tryptase increased L6 myoblast proliferation by 35% above control group and this effect was completely inhibited by APC-366. We confirmed the expression of PAR-2 receptor in vivo in skeletal muscle cells and in satellite cells and in vitro in L6 cells, where PAR-2 was found to be functional. Trypsin and SLIGKV increased L6 cells proliferation by 76% and 26% above control, respectively. COX-2 activity was increased following stimulation with PAR-2 agonist but its expression remained unchanged. Inhibition of COX-2 activity by NS-398 abolished the stimulation of cell proliferation induced by tryptase and trypsin. Finally, 15-deoxy-Δ-12,14-prostaglandin J2 (15Δ-PGJ2, a product of COX-2-derived prostaglandin D2, stimulated myoblast proliferation, but not PGE2 and PGF2α. Conclusions Taken together, our data show that tryptase can stimulate myoblast proliferation and this effect is part of a signaling cascade dependent on PAR-2 activation and on the downstream

  3. Mechanical stimuli on C2C12 myoblasts affect myoblast differentiation, focal adhesion kinase phosphorylation and galectin-1 expression

    DEFF Research Database (Denmark)

    Grossi, Alberto Blak; Lametsch, Rene; Karlsson, Anders H

    2011-01-01

    Mechanical forces are crucial in the regulation of cell morphology and function. At the cellular level, these forces influence myoblast differentiation and fusion. In this study we applied mechanical stimuli to embryonic muscle cells using magnetic microbeads, a method shown to apply stress...... by mechanical stimulation including Galectin-1, Annexin III, and RhoGDI. In this study we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms...... during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of Galectin-1, in mechanically stimulated myoblasts. A potential involvement of Galectin-1 in myoblast differentiation is presented....

  4. Analysis of differential lipofection efficiency in primary and established myoblasts.

    Science.gov (United States)

    Pampinella, Francesca; Lechardeur, Delphine; Zanetti, Elena; MacLachlan, Ian; Benharouga, Mohammed; Lukacs, Gergely L; Vitiello, Libero

    2002-02-01

    In this study we have compared the process of lipid-mediated transfection in primary and established myoblasts, in an attempt to elucidate the mechanisms responsible for the scarce transfectability of the former. We determined the metabolic stability of cytoplasmically injected and lipofected DNA in primary and established myoblasts and carried out a comparative time course analysis of luciferase reporter-gene expression and DNA stability. The efficiency of the transcription-translation machinery of the two cell types was compared by intranuclear injection of naked plasmid DNA encoding luciferase. Subcellular colocalization of fluorescein-labeled lipopolyplexes with specific endosomal and lysosomal markers was performed by confocal microscopy to monitor the intracellular trafficking of plasmid DNA during transfection. The metabolic stability of plasmid DNA was similar in primary and established myoblasts after both lipofection and cytoplasmic injection. In both cell types, lipofection had no detectable effect on the rate of cell proliferation. Confocal analysis showed that nuclear translocation of transfected DNA coincided with localization in a compartment devoid of endosome- or lysosome-specific marker proteins. The residency time of plasmid DNA in this compartment differed for primary and established myoblasts. Our findings suggest that the lower transfectability of primary myoblasts is mostly due to a difference in the intracellular delivery pathway that correlates with more rapid delivery of internalized complex to the lysosomal compartment.

  5. Isolation of human foetal myoblasts and its application for microencapsulation

    Science.gov (United States)

    Li, Anna Aihua; Bourgeois, Jacqueline; Potter, Murray; Chang, Patricia L

    2008-01-01

    Abstract Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells. These characteristics make them desirable for recombinant modification and use in microencapsulated cellular gene therapeutics. We have established a system in vitro to obtain a pure population of primary human foetal myoblasts under several rounds of selection with non-collagen coated plates and identified by desmin staining. These primary myoblasts presented good proliferation ability and better differentiation characteristics in monolayer and after microencapsulation compared to murine myoblast C2C12 cells based on creatine phosphokinase (CPK), major histocompatibility complex (MHC) and multi-nucleated myotubule determination. The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18–24 hrs. Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity. They should be useful for cell-based therapy in human clinical applications with microencapsulation technology. PMID:18366454

  6. Abelson tyrosine-protein kinase 2 Regulates Myoblast Proliferation and Controls Muscle Fiber Length

    OpenAIRE

    Burden, Steven; Lee, Jennifer

    2017-01-01

    Muscle fiber length is nearly uniform within a muscle but widely different among muscles. Here, we show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm and other muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of available myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but expansion of the diaphragm ...

  7. The critical role of myostatin in differentiation of sheep myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chenxi [College of Life Science and Technology, Xinjiang University, Urumqi (China); Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Li, Wenrong; Zhang, Xuemei; Zhang, Ning; He, Sangang; Huang, Juncheng [Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Laboratory of Grass-fed Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Urumqi (China); Animal Biotechnological Research Center, Xinjiang Academy of Animal Science, Urumqi (China); Ge, Yubin [The State Engineering Laboratory of AIDS Vaccine, College of Life Science, Jilin University, Changchun (China); Liu, Mingjun, E-mail: xjlmj2004@yahoo.com.cn [Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Laboratory of Grass-fed Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Urumqi (China); Animal Biotechnological Research Center, Xinjiang Academy of Animal Science, Urumqi (China)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Identification of the effective and specific shRNA to knockdown MSTN. Black-Right-Pointing-Pointer Overexpression of MSTN reversibly suppressed myogenic differentiation. Black-Right-Pointing-Pointer shRNA knockdown of endogenous MSTN promoted ovine myoblast differentiation. Black-Right-Pointing-Pointer MSTN inhibits myogenic differentiation through down-regulation of MyoD and Myogenin and up-regulation of Smad3. Black-Right-Pointing-Pointer Provides a promise for the generation of transgenic sheep to improve meat productivity. -- Abstract: Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

  8. The critical role of myostatin in differentiation of sheep myoblasts

    International Nuclear Information System (INIS)

    Liu, Chenxi; Li, Wenrong; Zhang, Xuemei; Zhang, Ning; He, Sangang; Huang, Juncheng; Ge, Yubin; Liu, Mingjun

    2012-01-01

    Highlights: ► Identification of the effective and specific shRNA to knockdown MSTN. ► Overexpression of MSTN reversibly suppressed myogenic differentiation. ► shRNA knockdown of endogenous MSTN promoted ovine myoblast differentiation. ► MSTN inhibits myogenic differentiation through down-regulation of MyoD and Myogenin and up-regulation of Smad3. ► Provides a promise for the generation of transgenic sheep to improve meat productivity. -- Abstract: Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

  9. Marked heterogeneity in growth characteristics of myoblast clonal cultures and myoblast mixed cultures obtained from the same individual

    NARCIS (Netherlands)

    Maier, Andrea B.; Cohen, Ron; Blom, Joke; Van Heemst, Diana; Westendorp, Rudi G.J.

    Background: Sarcopenia is defined as an age-related decrease in skeletal muscle mass and function while adjacent satellite cells are unable to compensate for this loss. However, myoblast cultures can be established even in the presence of sarcopenia. Objective: It is yet unknown whether satellite

  10. Intercellular adhesion molecule-1 augments myoblast adhesion and fusion through homophilic trans-interactions.

    Science.gov (United States)

    Pizza, Francis X; Martin, Ryan A; Springer, Evan M; Leffler, Maxwell S; Woelmer, Bryce R; Recker, Isaac J; Leaman, Douglas W

    2017-07-11

    The overall objective of the study was to identify mechanisms through which intercellular adhesion molecule-1 (ICAM-1) augments the adhesive and fusogenic properties of myogenic cells. Hypotheses were tested using cultured myoblasts and fibroblasts, which do not constitutively express ICAM-1, and myoblasts and fibroblasts forced to express full length ICAM-1 or a truncated form lacking the cytoplasmic domain of ICAM-1. ICAM-1 mediated myoblast adhesion and fusion were quantified using novel assays and cell mixing experiments. We report that ICAM-1 augments myoblast adhesion to myoblasts and myotubes through homophilic trans-interactions. Such adhesive interactions enhanced levels of active Rac in adherent and fusing myoblasts, as well as triggered lamellipodia, spreading, and fusion of myoblasts through the signaling function of the cytoplasmic domain of ICAM-1. Rac inhibition negated ICAM-1 mediated lamellipodia, spreading, and fusion of myoblasts. The fusogenic property of ICAM-1-ICAM-1 interactions was restricted to myogenic cells, as forced expression of ICAM-1 by fibroblasts did not augment their fusion to ICAM-1+ myoblasts/myotubes. We conclude that ICAM-1 augments myoblast adhesion and fusion through its ability to self-associate and initiate Rac-mediated remodeling of the actin cytoskeleton.

  11. Transdifferentiation of myoblasts into osteoblasts - possible use for bone therapy.

    Science.gov (United States)

    Lin, Daphne P L; Carnagarin, Revathy; Dharmarajan, Arun; Dass, Crispin R

    2017-12-01

    Transdifferentiation is defined as the conversion of one cell type to another and is an ever-expanding field with a growing number of cells found to be capable of such a process. To date, the fact remains that there are limited treatment options for fracture healing, osteoporosis and bone repair post-destruction by bone tumours. Hence, this review focuses on the transdifferentiation of myoblast to osteoblast as a means to further understand the transdifferentiation process and to investigate a potential therapeutic option if successful. The potent osteoinductive effects of the bone morphogenetic protein-2 are largely implicated in the transdifferentiation of myoblast to osteoblast. Bone morphogenetic protein-2-induced activation of the Smad1 protein ultimately results in JunB synthesis, the first transcriptional step in myoblast dedifferentiation. The upregulation of the activating protein-1 binding activity triggers the transcription of the runt-related transcription factor 2 gene, a transcription factor that plays a major role in osteoblast differentiation. This potential transdifferentiation treatment may be utilised for dental implants, fracture healing, osteoporosis and bone repair post-destruction by bone tumours. © 2017 Royal Pharmaceutical Society.

  12. CD36 is required for myoblast fusion during myogenic differentiation

    International Nuclear Information System (INIS)

    Park, Seung-Yoon; Yun, Youngeun; Kim, In-San

    2012-01-01

    Highlights: ► CD36 expression was induced during myogenic differentiation. ► CD36 expression was localized in multinucleated myotubes. ► The expression of myogenic markers is attenuated in CD36 knockdown C2C12 cells. ► Knockdown of CD36 significantly inhibited myotube formation during differentiation. -- Abstract: Recently, CD36 has been found to be involved in the cytokine-induced fusion of macrophage. Myoblast fusion to form multinucleated myotubes is required for myogenesis and muscle regeneration. Because a search of gene expression database revealed the attenuation of CD36 expression in the muscles of muscular dystrophy patients, the possibility that CD36 could be required for myoblast fusion was investigated. CD36 expression was markedly up-regulated during myoblast differentiation and localized in multinucleated myotubes. Knockdown of endogenous CD36 significantly decreased the expression of myogenic markers as well as myotube formation. These results support the notion that CD36 plays an important role in cell fusion during myogenic differentiation. Our finding will aid the elucidation of the common mechanism governing cell-to-cell fusion in various fusion models.

  13. Ductile electroactive biodegradable hyperbranched polylactide copolymers enhancing myoblast differentiation.

    Science.gov (United States)

    Xie, Meihua; Wang, Ling; Guo, Baolin; Wang, Zhong; Chen, Y Eugene; Ma, Peter X

    2015-12-01

    Myotube formation is crucial to restoring muscular functions, and biomaterials that enhance the myoblast differentiation into myotubes are highly desirable for muscular repair. Here, we report the synthesis of electroactive, ductile, and degradable copolymers and their application in enhancing the differentiation of myoblasts to myotubes. A hyperbranched ductile polylactide (HPLA) was synthesized and then copolymerized with aniline tetramer (AT) to produce a series of electroactive, ductile and degradable copolymers (HPLAAT). The HPLA and HPLAAT showed excellent ductility with strain to failure from 158.9% to 42.7% and modulus from 265.2 to 758.2 MPa. The high electroactivity of the HPLAAT was confirmed by UV spectrometer and cyclic voltammogram measurements. These HPLAAT polymers also showed improved thermal stability and controlled biodegradation rate compared to HPLA. Importantly, when applying these polymers for myotube formation, the HPLAAT significantly improved the proliferation of C2C12 myoblasts in vitro compared to HPLA. Furthermore, these polymers greatly promoted myogenic differentiation of C2C12 cells as measured by quantitative analysis of myotube number, length, diameter, maturation index, and gene expression of MyoD and TNNT. Together, our study shows that these electroactive, ductile and degradable HPLAAT copolymers represent significantly improved biomaterials for muscle tissue engineering compared to HPLA. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. CD36 is required for myoblast fusion during myogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Seung-Yoon [Department of Biochemistry, College of Medicine, Dongguk University and Medical Institute of Dongguk University, Gyeongju 780-714 (Korea, Republic of); Yun, Youngeun [Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, In-San, E-mail: iskim@knu.ac.kr [Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Biomedical Research Institute, Korea Institute Science and Technology, Seoul (Korea, Republic of)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer CD36 expression was induced during myogenic differentiation. Black-Right-Pointing-Pointer CD36 expression was localized in multinucleated myotubes. Black-Right-Pointing-Pointer The expression of myogenic markers is attenuated in CD36 knockdown C2C12 cells. Black-Right-Pointing-Pointer Knockdown of CD36 significantly inhibited myotube formation during differentiation. -- Abstract: Recently, CD36 has been found to be involved in the cytokine-induced fusion of macrophage. Myoblast fusion to form multinucleated myotubes is required for myogenesis and muscle regeneration. Because a search of gene expression database revealed the attenuation of CD36 expression in the muscles of muscular dystrophy patients, the possibility that CD36 could be required for myoblast fusion was investigated. CD36 expression was markedly up-regulated during myoblast differentiation and localized in multinucleated myotubes. Knockdown of endogenous CD36 significantly decreased the expression of myogenic markers as well as myotube formation. These results support the notion that CD36 plays an important role in cell fusion during myogenic differentiation. Our finding will aid the elucidation of the common mechanism governing cell-to-cell fusion in various fusion models.

  15. Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length

    OpenAIRE

    Lee, Jennifer K; Hallock, Peter T; Burden, Steven J

    2017-01-01

    Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myobla...

  16. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

    Science.gov (United States)

    Chaturvedi, Vishal; Dye, Danielle E; Kinnear, Beverley F; van Kuppevelt, Toin H; Grounds, Miranda D; Coombe, Deirdre R

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

  17. Combination of Nigella sativa with Glycyrrhiza glabra and Zingiber officinale augments their protective effects on doxorubicin-induced toxicity in h9c2 cells

    Directory of Open Access Journals (Sweden)

    Azar Hosseini

    2015-12-01

    Conclusion: All of the extracts have some protective effects against DOX-induced toxicity in cardiomyocytes with similar efficacies, but with different potencies. However, NGZ produced much higher protective effect via reducing oxidative stress and inhibiting of apoptotic induction processes. Further investigations are needed to determine the effects of NGZ on DOX chemotherapy.  

  18. Sirtuin-3 (SIRT3) protein attenuates doxorubicin-induced oxidative stress and improves mitochondrial respiration in H9c2 cardiomyocytes

    Science.gov (United States)

    Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electro...

  19. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  20. Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology

    International Nuclear Information System (INIS)

    Shafey, Dina; Cote, Patrice D.; Kothary, Rashmi

    2005-01-01

    Dosage of the survival motor neuron (SMN) protein has been directly correlated with the severity of disease in patients diagnosed with spinal muscular atrophy (SMA). It is also clear that SMA is a neurodegenerative disorder characterized by the degeneration of the α-motor neurons in the anterior horn of the spinal cord and atrophy of the associated skeletal muscle. What is more controversial is whether it is neuronal and/or muscle-cell-autonomous defects that are responsible for the disease per se. Although motor neuron degeneration is generally accepted as the primary event in SMA, intrinsic muscle defects in this disease have not been ruled out. To gain a better understanding of the influence of SMN protein dosage in muscle, we have generated a hypomorphic series of myoblast (C2C12) stable cell lines with variable Smn knockdown. We show that depletion of Smn in these cells resulted in a decrease in the number of nuclear 'gems' (gemini of coiled bodies), reduced proliferation with no increase in cell death, defects in myoblast fusion, and malformed myotubes. Importantly, the severity of these abnormalities is directly correlated with the decrease in Smn dosage. Taken together, our work supports the view that there is an intrinsic defect in skeletal muscle cells of SMA patients and that this defect contributes to the overall pathogenesis in this devastating disease

  1. Myoblast replication is reduced in the IUGR fetus despite maintained proliferative capacity in vitro.

    Science.gov (United States)

    Soto, Susan M; Blake, Amy C; Wesolowski, Stephanie R; Rozance, Paul J; Barthel, Kristen B; Gao, Bifeng; Hetrick, Byron; McCurdy, Carrie E; Garza, Natalia G; Hay, William W; Leinwand, Leslie A; Friedman, Jacob E; Brown, Laura D

    2017-03-01

    Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass and insulin resistance, suggesting muscle growth may be restricted by molecular events that occur during fetal development. To explore the basis of restricted fetal muscle growth, we used a sheep model of progressive placental insufficiency-induced IUGR to assess myoblast proliferation within intact skeletal muscle in vivo and isolated myoblasts stimulated with insulin in vitro Gastrocnemius and soleus muscle weights were reduced by 25% in IUGR fetuses compared to those in controls (CON). The ratio of PAX7+ nuclei (a marker of myoblasts) to total nuclei was maintained in IUGR muscle compared to CON, but the fraction of PAX7+ myoblasts that also expressed Ki-67 (a marker of cellular proliferation) was reduced by 23%. Despite reduced proliferation in vivo, fetal myoblasts isolated from IUGR biceps femoris and cultured in enriched media in vitro responded robustly to insulin in a dose- and time-dependent manner to increase proliferation. Similarly, insulin stimulation of IUGR myoblasts upregulated key cell cycle genes and DNA replication. There were no differences in the expression of myogenic regulatory transcription factors that drive commitment to muscle differentiation between CON and IUGR groups. These results demonstrate that the molecular machinery necessary for transcriptional control of proliferation remains intact in IUGR fetal myoblasts, indicating that in vivo factors such as reduced insulin and IGF1, hypoxia and/or elevated counter-regulatory hormones may be inhibiting muscle growth in IUGR fetuses. © 2017 Society for Endocrinology.

  2. Novel Endogenous, Insulin-Stimulated Akt2 Protein Interaction Partners in L6 Myoblasts.

    Directory of Open Access Journals (Sweden)

    Michael Caruso

    Full Text Available Insulin resistance and Type 2 diabetes are marked by an aberrant response in the insulin signaling network. The phosphoinositide-dependent serine/threonine kinase, Akt2, plays a key role in insulin signaling and glucose uptake, most notably within skeletal muscle. Protein-protein interaction regulates the functional consequence of Akt2 and in turn, Akt2's role in glucose uptake. However, only few insulin-responsive Akt2 interaction partners have been identified in skeletal muscle cells. In the present work, rat L6 myoblasts, a widely used insulin sensitive skeletal muscle cell line, were used to examine endogenous, insulin-stimulated Akt2 protein interaction partners. Akt2 co-immunoprecipitation was coupled with 1D-SDS-PAGE and fractions were analyzed by HPLC-ESI-MS/MS to reveal Akt2 protein-protein interactions. The pull-down assay displayed specificity for the Akt2 isoform; Akt1 and Akt3 unique peptides were not detected. A total of 49 were detected with a significantly increased (47 or decreased (2 association with Akt2 following insulin administration (n = 4; p<0.05. Multiple pathways were identified for the novel Akt2 interaction partners, such as the EIF2 and ubiquitination pathways. These data suggest that multiple new endogenous proteins may associate with Akt2 under basal as well as insulin-stimulated conditions, providing further insight into the insulin signaling network. Data are available via ProteomeXchange with identifier PXD002557.

  3. Group I Paks Promote Skeletal Myoblast Differentiation In Vivo and In Vitro

    DEFF Research Database (Denmark)

    Joseph, Giselle A; Lu, Min; Radu, Maria

    2017-01-01

    fusion in Drosophila We report that both Pak1 and Pak2 are activated during mammalian myoblast differentiation. One pathway of activation is initiated by N-cadherin ligation and involves the cadherin coreceptor Cdo with its downstream effector, Cdc42. Individual genetic deletion of Pak1 and Pak2 in mice....... Furthermore, primary myoblasts lacking Pak1 and Pak2 display delayed expression of myogenic differentiation markers and myotube formation. These results identify Pak1 and Pak2 as redundant regulators of myoblast differentiation in vitro and in vivo and as components of the promyogenic Ncad/Cdo/Cdc42 signaling...

  4. Identification of gene expression modifications in myostatin-stimulated myoblasts

    International Nuclear Information System (INIS)

    Yang Wei; Zhang Yong; Ma Guoda; Zhao Xinyi; Chen Yan; Zhu Dahai

    2005-01-01

    Myostatin belongs to the transforming growth factor beta superfamily and has been shown to function as an inhibitor of skeletal muscle proliferation and differentiation. To gain insight into the molecular mechanisms of myostatin function during myogenesis, differential display reverse transcription PCR was employed to identify altered gene expressions associated with myostatin inhibitory function in chicken fetal myoblasts (CFMs). In this work, we have identified seven up-regulated and 12 down-regulated genes in myostatin stimulated CFMs. Those genes are involved in myogenic differentiation, cell architecture, energy metabolism, signal transduction, and apoptosis. The down-regulation of muscle creatine kinase B, troponin C, and myosin regulatory light chain is in agreement with the myostatin negative role in myocyte differentiation. In addition, the expression alteration of skeletal muscle-specific cardiac ankyrin repeat protein and the bcl-2 related anti-apoptotic protein Nr-13 suggests possible unique roles for myostatin in regulating myogenesis by controlling cofactors participated transcriptional regulation and apoptosis

  5. Differentiation-Associated Downregulation of Poly(ADP-Ribose Polymerase-1 Expression in Myoblasts Serves to Increase Their Resistance to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Gábor Oláh

    Full Text Available Poly(ADP-ribose polymerase 1 (PARP-1, the major isoform of the poly (ADP-ribose polymerase family, is a constitutive nuclear and mitochondrial protein with well-recognized roles in various essential cellular functions such as DNA repair, signal transduction, apoptosis, as well as in a variety of pathophysiological conditions including sepsis, diabetes and cancer. Activation of PARP-1 in response to oxidative stress catalyzes the covalent attachment of the poly (ADP-ribose (PAR groups on itself and other acceptor proteins, utilizing NAD+ as a substrate. Overactivation of PARP-1 depletes intracellular NAD+ influencing mitochondrial electron transport, cellular ATP generation and, if persistent, can result in necrotic cell death. Due to their high metabolic activity, skeletal muscle cells are particularly exposed to constant oxidative stress insults. In this study, we investigated the role of PARP-1 in a well-defined model of murine skeletal muscle differentiation (C2C12 and compare the responses to oxidative stress of undifferentiated myoblasts and differentiated myotubes. We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes. This alteration correlated with an increased resistance to oxidative stress of the myotubes, as measured by MTT and LDH assays. Mitochondrial function, assessed by measuring mitochondrial membrane potential, was preserved under oxidative stress in myotubes compared to myoblasts. Moreover, basal respiration, ATP synthesis, and the maximal respiratory capacity of mitochondria were higher in myotubes than in myoblasts. Inhibition of the catalytic activity of PARP-1 by PJ34 (a phenanthridinone PARP inhibitor exerted greater protective effects in undifferentiated myoblasts than in differentiated myotubes. The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6. Forced overexpression of PARP1 in C2C12 myotubes sensitized the cells to oxidant

  6. One-Step Purification of Human Skeletal Muscle Myoblasts and Subsequent Expansion Using Laminin-Coated Surface.

    Science.gov (United States)

    Chowdhury, Shiplu Roy; binti Ismail, Annis; Chee, Sia Chye; bin Laupa, Mohd Suffian; binti Jaffri, Fadhlun; Saberi, Salfarina Ezrina Mohmad; Idrus, Ruszymah Bt Hj

    2015-11-01

    Skeletal myoblasts have been extensively used to study muscle growth and differentiation, and were recently tested for their application as cell therapy and as a gene delivery system to treat muscle and nonmuscle diseases. However, contamination of fibroblasts in isolated cells from skeletal muscle is one of the long-standing problems for routine expansion. This study aimed to establish a simple one-step process to purify myoblasts and maintain their purity during expansion. Mixed cells were preplated serially on laminin- and collagen type I-coated surfaces in a different array for 5, 10, and 15 min. Immunocytochemical staining with antibodies specific to myoblasts was performed to evaluate myoblast attachment efficiency, purity, and yield. It was found that laminin-coated surface favors the attachment of myoblasts. Highest myoblast purity of 78.9% ± 6.8% was achieved by 5 min of preplating only on the laminin-coated surface with a yield of 56.9% ± 3.3%. Primary cells, isolated from skeletal muscle (n = 4), confirm the enhancement of purity through preplating on laminin-coated surface for 5 min. Subsequent expansion after preplating enhanced myoblast purity due to an increase in myoblast growth than fibroblasts. Myoblast purity of ∼ 98% was achieved when another preplating was performed during passaging. In conclusion, myoblasts can be purified and efficiently expanded in one step by preplating on laminin-coated surface, which is a simple and robust technique.

  7. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Gojo, Satoshi [Department of Cardiac Support, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, Osam, E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2013-02-08

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.

  8. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-01-01

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases

  9. [Human myoblast culture as muscle stem cells in medical and biological studies].

    Science.gov (United States)

    Terekhov, S M; Krokhina, T B; Shishkin, S S; Krakhmaleva, I N; Zakharov, S F; Ershova, E S

    2001-01-01

    The method for obtaining human myoblast culture has been modified to consider the specific histological localization of the satellite cells as well as their growth properties; the cultivation conditions have been selected to grow up to 150000 cells/cm2. At high densities, the cells remain mononuclear and preserve their typical myoblast morphology as well as the capacity for fusion and the formation of myotubes. By contrast to fibroblasts, up to 80% of the cells in the myoblast culture were positive in the acid phosphatase test, which indicates their stem nature. The obtained myoblast cultures were used in the clinical tests of cell-mediated gene therapy of Duchenne's muscular dystrophy as well as in the bioassay for the effects of biologically active compounds.

  10. Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length.

    Science.gov (United States)

    Lee, Jennifer K; Hallock, Peter T; Burden, Steven J

    2017-12-12

    Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but as a consequence of expansion of the diaphragm muscle, the diaphragm central tendon is reduced in size, likely contributing to reduced stamina of Abl2 mutant mice. Ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon of Abl2 +/- mice. Specialized tendon cells, resembling tendon cells at myotendinous junctions, form at the ends of these muscle islands, suggesting that myofibers induce differentiation of tendon cells, which reciprocally regulate myofiber length and orientation.

  11. Elastic hydrogel substrate supports robust expansion of murine myoblasts and enhances their engraftment

    International Nuclear Information System (INIS)

    Ding, Ke; Yang, Zhong; Xu, Jian-zhong; Liu, Wen-ying; Zeng, Qiang; Hou, Fang; Lin, Sen

    2015-01-01

    The application of satellite cell-derived myoblasts in regenerative medicine has been restricted by the rapid loss of stemness during in vitro cell expansion using traditional culture systems. However, studies published in the past decade have highlighted the influence of substrate elasticity on stem cell fate and revealed that culture on a soft hydrogel substrate can promote self-renewal and prolong the regenerative potential of muscle stem cells. Whether hydrogel substrates have similar effects after long-term robust expansion remains to be determined. Herein we prepared an elastic chitosan/beta-glycerophosphate/collagen hydrogel mimicking the soft microenvironment of muscle tissues for use as the substrate for satellite cell culture and investigated its influence on long-term cell expansion. After 20 passages in culture, satellite cell-derived myoblasts cultured on our hydrogel substrate exhibited significant improvements in proliferation capability, cell viability, colony forming frequency, and potential for myogenic differentiation compared to those cultured on a routine rigid culture surface. Immunochemical staining and western blot analysis both confirmed that myoblasts cultured on the hydrogel substrate expressed higher levels of several differentiation-related markers, including Pax7, Pax3, and SSEA-1, and a lower level of MyoD compared to myoblasts cultured on rigid culture plates (all p<0.05). After transplantation into the tibialis anterior of nude mice, myoblasts that had been cultured on the hydrogel substrate demonstrated a significantly greater engraftment efficacy than those cultured on the traditional surface. Collectively, these results indicate that the elastic hydrogel substrate supported robust expansion of murine myoblasts and enhanced their engraftment in vivo. - Highlights: • An elastic hydrogel was designed to mimic the pliable muscle tissue microenvironment. • Myoblasts retained their stemness in long-term culture on the elastic

  12. Estrogens enhance myoblast differentiation in facioscapulohumeral muscular dystrophy by antagonizing DUX4 activity

    OpenAIRE

    Teveroni, Emanuela; Pellegrino, Marsha; Sacconi, Sabrina; Calandra, Patrizia; Cascino, Isabella; Farioli-Vecchioli, Stefano; Puma, Angela; Garibaldi, Matteo; Morosetti, Roberta; Tasca, Giorgio; Ricci, Enzo; Trevisan, Carlo Pietro; Galluzzi, Giuliana; Pontecorvi, Alfredo; Crescenzi, Marco

    2017-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is characterized by extreme variability in symptoms, with females being less severely affected than males and presenting a higher proportion of asymptomatic carriers. The sex-related factors involved in the disease are not known. Here, we have utilized myoblasts isolated from FSHD patients (FSHD myoblasts) to investigate the effect of estrogens on muscle properties. Our results demonstrated that...

  13. Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

    Science.gov (United States)

    Masselink, Wouter; Masaki, Megumi; Sieiro, Daniel; Marcelle, Christophe; Currie, Peter D

    2017-10-15

    The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud. Copyright © 2017. Published by Elsevier Inc.

  14. Elastic hydrogel substrate supports robust expansion of murine myoblasts and enhances their engraftment

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Ke, E-mail: dk1118@yeah.net [Department of Pediatric Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072 (China); Yang, Zhong [Department of Clinical Hematology, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Xu, Jian-zhong, E-mail: xjzspine@163.com [Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Liu, Wen-ying; Zeng, Qiang; Hou, Fang [Department of Pediatric Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072 (China); Lin, Sen [Department of Anatomy and Histology & Embryology, Chengdu Medical College, Chengdu 610500 (China)

    2015-09-10

    The application of satellite cell-derived myoblasts in regenerative medicine has been restricted by the rapid loss of stemness during in vitro cell expansion using traditional culture systems. However, studies published in the past decade have highlighted the influence of substrate elasticity on stem cell fate and revealed that culture on a soft hydrogel substrate can promote self-renewal and prolong the regenerative potential of muscle stem cells. Whether hydrogel substrates have similar effects after long-term robust expansion remains to be determined. Herein we prepared an elastic chitosan/beta-glycerophosphate/collagen hydrogel mimicking the soft microenvironment of muscle tissues for use as the substrate for satellite cell culture and investigated its influence on long-term cell expansion. After 20 passages in culture, satellite cell-derived myoblasts cultured on our hydrogel substrate exhibited significant improvements in proliferation capability, cell viability, colony forming frequency, and potential for myogenic differentiation compared to those cultured on a routine rigid culture surface. Immunochemical staining and western blot analysis both confirmed that myoblasts cultured on the hydrogel substrate expressed higher levels of several differentiation-related markers, including Pax7, Pax3, and SSEA-1, and a lower level of MyoD compared to myoblasts cultured on rigid culture plates (all p<0.05). After transplantation into the tibialis anterior of nude mice, myoblasts that had been cultured on the hydrogel substrate demonstrated a significantly greater engraftment efficacy than those cultured on the traditional surface. Collectively, these results indicate that the elastic hydrogel substrate supported robust expansion of murine myoblasts and enhanced their engraftment in vivo. - Highlights: • An elastic hydrogel was designed to mimic the pliable muscle tissue microenvironment. • Myoblasts retained their stemness in long-term culture on the elastic

  15. Skeletal muscle myoblasts possess a stretch-responsive local angiotensin signalling system.

    Science.gov (United States)

    Johnston, Adam P W; Baker, Jeff; De Lisio, Michael; Parise, Gianni

    2011-06-01

    A paucity of information exists regarding the presence of local renin-angiotensin systems (RASs) in skeletal muscle and associated muscle stem cells. Skeletal muscle and muscle stem cells were isolated from C57BL/6 mice and examined for the presence of a local RAS using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), Western blotting and liquid chromatography-mass spectrometry (LC-MS). Furthermore, the effect of mechanical stimulation on RAS member gene expression was analysed. Whole skeletal muscle, primary myoblasts and C2C12 derived myoblasts and myotubes differentially expressed members of the RAS including angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II (Ang II) type 1 (AT(1)) and type 2 (AT(2)). Renin transcripts were never detected, however, mRNA for the 'renin-like' enzyme cathepsin D was observed and Ang I and Ang II were identified in cell culture supernatants from proliferating myoblasts. AT(1) appeared to co-localise with polymerised actin filaments in proliferating myoblasts and was primarily found in the nucleus of terminally differentiated myotubes. Furthermore, mechanical stretch of proliferating and differentiating C2C12 cells differentially induced mRNA expression of angiotensinogen, AT(1) and AT(2). Proliferating and differentiated muscle stem cells possess a local stress-responsive RAS in vitro. The precise function of a local RAS in myoblasts remains unknown. However, evidence presented here suggests that Ang II may be a regulator of skeletal muscle myoblasts.

  16. MicroRNA-27a promotes myoblast proliferation by targeting myostatin

    International Nuclear Information System (INIS)

    Huang, Zhiqing; Chen, Xiaoling; Yu, Bing; He, Jun; Chen, Daiwen

    2012-01-01

    Highlights: ► We identified a myogenic role for miR-27a and a new target, myostatin. ► The miR-27a was confirmed to target myostatin 3′UTR. ► miR-27a is upregulated and myostatin is downregulated during myoblast proliferation. ► miR-27a promotes myoblast proliferation by reducing the expression of myostatin. -- Abstract: MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3′UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.

  17. MicroRNA-27a promotes myoblast proliferation by targeting myostatin

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Zhiqing; Chen, Xiaoling; Yu, Bing; He, Jun [Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014 (China); Chen, Daiwen, E-mail: dwchen@sicau.edu.cn [Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014 (China)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer We identified a myogenic role for miR-27a and a new target, myostatin. Black-Right-Pointing-Pointer The miR-27a was confirmed to target myostatin 3 Prime UTR. Black-Right-Pointing-Pointer miR-27a is upregulated and myostatin is downregulated during myoblast proliferation. Black-Right-Pointing-Pointer miR-27a promotes myoblast proliferation by reducing the expression of myostatin. -- Abstract: MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3 Prime UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.

  18. Role of an inward rectifier K+ current and of hyperpolarization in human myoblast fusion

    Science.gov (United States)

    Liu, J-H; Bijlenga, P; Fischer-Lougheed, J; Occhiodoro, T; Kaelin, A; Bader, C R; Bernheim, L

    1998-01-01

    The role of K+ channels and membrane potential in myoblast fusion was evaluated by examining resting membrane potential and timing of expression of K+ currents at three stages of differentiation of human myogenic cells: undifferentiated myoblasts, fusion-competent myoblasts (FCMBs), and freshly formed myotubes. Two K+ currents contribute to a hyperpolarization of myoblasts prior to fusion: IK(NI), a non-inactivating delayed rectifier, and IK(IR), an inward rectifier. IK(NI) density is low in undifferentiated myoblasts, increases in FCMBs and declines in myotubes. On the other hand, IK(IR) is expressed in 28 % of the FCMBs and in all myotubes. IK(IR) is reversibly blocked by Ba2+ or Cs+. Cells expressing IK(IR) have resting membrane potentials of −65 mV. A block by Ba2+ or Cs+ induces a depolarization to a voltage determined by IK(NI) (−32 mV). Cs+ and Ba2+ ions reduce myoblast fusion. It is hypothesized that the IK(IR)-mediated hyperpolarization allows FCMBs to recruit Na+, K+ and T-type Ca2+ channels which are present in these cells and would otherwise be inactivated. FCMBs, rendered thereby capable of firing action potentials, could amplify depolarizing signals and may accelerate fusion. PMID:9705997

  19. FSHD myoblasts fail to downregulate intermediate filament protein vimentin during myogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Lipinski M.

    2011-10-01

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is an autosomal dominant hereditary neuromuscular disorder. The clinical features of FSHD include weakness of the facial and shoulder girdle muscles followed by wasting of skeletal muscles of the pelvic girdle and lower extremities. Although FSHD myoblasts grown in vitro can be induced to differentiate into myotubes by serum starvation, the resulting FSHD myotubes have been shown previously to be morphologically abnormal. Aim. In order to find the cause of morphological anomalies of FSHD myotubes we compared in vitro myogenic differentiation of normal and FSHD myoblasts at the protein level. Methods. We induced myogenic differentiation of normal and FSHD myoblasts by serum starvation. We then compared protein extracts from proliferating myoblasts and differentiated myotubes using SDS-PAGE followed by mass spectrometry identification of differentially expressed proteins. Results. We demonstrated that the expression of vimentin was elevated at the protein and mRNA levels in FSHD myotubes as compared to normal myotubes. Conclusions. We demonstrate for the first time that in contrast to normal myoblasts, FSHD myoblasts fail to downregulate vimentin after induction of in vitro myogenic differentiation. We suggest that vimentin could be an easily detectable marker of FSHD myotubes

  20. Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

    Directory of Open Access Journals (Sweden)

    Julio Alonso-Padilla

    2015-01-01

    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease.Genetically engineered parasitic strains are used for high throughput screening (HTS of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6 and was validated against a series of known anti-trypanosomatid drugs.We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.

  1. The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse

    DEFF Research Database (Denmark)

    Vasyutina, Elena; Martarelli, Benedetta; Brakebusch, Cord

    2009-01-01

    Rac1 and Cdc42 are small G-proteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. We used conditional mutagenesis in mice to demonstrate that Rac1 and Cdc42 are essential for myoblast fusion in vivo and in vitro. The deficit in fusion of Rac1 or Cdc42 mut...... genetic analysis demonstrates thus that the function of Rac in myoblast fusion is evolutionarily conserved from insects to mammals and that Cdc42, a molecule hitherto not implicated in myoblast fusion, is essential for the fusion of murine myoblasts....

  2. Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts

    Directory of Open Access Journals (Sweden)

    Sachchida Nand Pandey

    2015-05-01

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is believed to be caused by aberrant expression of double homeobox 4 (DUX4 due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01 and primary (4.7 fold, p < 0.01 FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells.

  3. Substrate stiffness affects skeletal myoblast differentiation in vitro

    Directory of Open Access Journals (Sweden)

    Sara Romanazzo, Giancarlo Forte, Mitsuhiro Ebara, Koichiro Uto, Stefania Pagliari, Takao Aoyagi, Enrico Traversa and Akiyoshi Taniguchi

    2012-01-01

    Full Text Available To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.

  4. Impact of Hepatocyte Growth Factor on Skeletal Myoblast Transplantation Late after Myocardial Infarction

    Directory of Open Access Journals (Sweden)

    Stacy B. O'blenes

    2013-01-01

    Full Text Available In clinical studies, skeletal myoblast (SKMB transplantation late after myocardial infarction (MI has minimal impact on left ventricular (LV function. This may be related to our previous observation that the extent of SKMB engraftment is minimal in chronic MI when compared to acute MI, which correlates with decreased hepatocyte growth factor (HGF expression, an important regulator of SKMB function. Here, we investigated delivery of exogenous HGF as a strategy for augmenting SKMB engraftment late after MI. Rats underwent SKMB transplantation 4 weeks after coronary ligation. HGF or vehicle control was delivered intravenously during the subsequent 2 weeks. LV function was assessed by MRI before and 2 weeks after SKMB transplantation. We evaluated HGF delivery, SKMB engraftment, and expression of genes associated with post-MI remodeling. Serum HGF was 6.2 ± 2.4 ng/mL after 2 weeks of HGF infusion (n = 7, but undetectable in controls (n = 7. LV end-diastolic volume and ejection fraction did not improve with HGF treatment (321 ± 27 mm 3 , 42% ± 2% vs. 285 ± 33 mm 3 , 43% ± 2%, HGF vs. control. MIs were larger in HGF-treated animals (50 ± 7 vs. 30 ± 6 mm 3 , P = 0.046, but the volume of engrafted SKMBs or percentage of MIs occupied by SKMBs did not increase with HGF (1.7 ± 0.3 mm 3 , 4.7% ± 1.9% vs. 1.4 ± 0.4 mm 3 , 5.3% ± 1.6%, HGF vs. control. Expression of genes associated with post-infarction remodeling was not altered by HGF. Delivery of exogenous HGF failed to augment SKMB engraftment and functional recovery in chronic MI. Expression of genes associated with LV remodeling was not altered by HGF. Alternative strategies to enhance engraftment of SKMB must be explored to optimize the clinical efficacy of SKMB transplantation.

  5. The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion.

    Science.gov (United States)

    Estrada, Beatriz; Maeland, Anne D; Gisselbrecht, Stephen S; Bloor, James W; Brown, Nicholas H; Michelson, Alan M

    2007-07-15

    Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where--as in myoblast fusion--membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells are unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane.

  6. Live cell imaging reveals marked variability in myoblast proliferation and fate

    Science.gov (United States)

    2013-01-01

    Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

  7. Bioenergetic Profile Experiment using C2C12 Myoblast Cells

    Science.gov (United States)

    Nicholls, David G.; Darley-Usmar, Victor M.; Wu, Min; Jensen, Per Bo; Rogers, George W.; Ferrick, David A.

    2010-01-01

    The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity. Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment. Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR. In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell. This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and

  8. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) activates promyogenic signaling pathways, thereby promoting myoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sang-Jin; Go, Ga-Yeon; Yoo, Miran; Kim, Yong Kee [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Seo, Dong-Wan [College of Pharmacy, Dankook University, Cheonan 330-714 (Korea, Republic of); Kang, Jong-Sun [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon 440-746 (Korea, Republic of); Bae, Gyu-Un, E-mail: gbae@sookmyung.ac.kr [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

    2016-01-29

    Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) regulates postnatal myogenesis by alleviating myostatin activity, but the molecular mechanisms by which it regulates myogenesis are not fully understood. In this study, we investigate molecular mechanisms of PPARβ/δ in myoblast differentiation. C2C12 myoblasts treated with a PPARβ/δ agonist, GW0742 exhibit enhanced myotube formation and muscle-specific gene expression. GW0742 treatment dramatically activates promyogenic kinases, p38MAPK and Akt, in a dose-dependent manner. GW0742-stimulated myoblast differentiation is mediated by p38MAPK and Akt, since it failed to restore myoblast differentiation repressed by inhibition of p38MAPK and Akt. In addition, GW0742 treatment enhances MyoD-reporter activities. Consistently, overexpression of PPARβ/δ enhances myoblast differentiation accompanied by elevated activation of p38MAPK and Akt. Collectively, these results suggest that PPARβ/δ enhances myoblast differentiation through activation of promyogenic signaling pathways. - Highlights: • A PPARβ/δ agonist, GW0742 promotes myoblast differentiation. • GW0742 activates both p38MAPK and Akt activation in myogenic differentiation. • GW0742 enhances MyoD activity for myogenic differentiation. • Overexpression of PPARβ/δ enhances myoblast differentiation via activating promyogenic signaling pathways. • This is the first finding for agonistic mechanism of PPARβ/δ in myogenesis.

  9. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) activates promyogenic signaling pathways, thereby promoting myoblast differentiation

    International Nuclear Information System (INIS)

    Lee, Sang-Jin; Go, Ga-Yeon; Yoo, Miran; Kim, Yong Kee; Seo, Dong-Wan; Kang, Jong-Sun; Bae, Gyu-Un

    2016-01-01

    Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) regulates postnatal myogenesis by alleviating myostatin activity, but the molecular mechanisms by which it regulates myogenesis are not fully understood. In this study, we investigate molecular mechanisms of PPARβ/δ in myoblast differentiation. C2C12 myoblasts treated with a PPARβ/δ agonist, GW0742 exhibit enhanced myotube formation and muscle-specific gene expression. GW0742 treatment dramatically activates promyogenic kinases, p38MAPK and Akt, in a dose-dependent manner. GW0742-stimulated myoblast differentiation is mediated by p38MAPK and Akt, since it failed to restore myoblast differentiation repressed by inhibition of p38MAPK and Akt. In addition, GW0742 treatment enhances MyoD-reporter activities. Consistently, overexpression of PPARβ/δ enhances myoblast differentiation accompanied by elevated activation of p38MAPK and Akt. Collectively, these results suggest that PPARβ/δ enhances myoblast differentiation through activation of promyogenic signaling pathways. - Highlights: • A PPARβ/δ agonist, GW0742 promotes myoblast differentiation. • GW0742 activates both p38MAPK and Akt activation in myogenic differentiation. • GW0742 enhances MyoD activity for myogenic differentiation. • Overexpression of PPARβ/δ enhances myoblast differentiation via activating promyogenic signaling pathways. • This is the first finding for agonistic mechanism of PPARβ/δ in myogenesis.

  10. Myomaker, Regulated by MYOD, MYOG and miR-140-3p, Promotes Chicken Myoblast Fusion

    Directory of Open Access Journals (Sweden)

    Wen Luo

    2015-11-01

    Full Text Available The fusion of myoblasts is an important step during skeletal muscle differentiation. A recent study in mice found that a transmembrane protein called Myomaker, which is specifically expressed in muscle, is critical for myoblast fusion. However, the cellular mechanism of its roles and the regulatory mechanism of its expression remain unclear. Chicken not only plays an important role in meat production but is also an ideal model organism for muscle development research. Here, we report that Myomaker is also essential for chicken myoblast fusion. Forced expression of Myomaker in chicken primary myoblasts promotes myoblast fusion, whereas knockdown of Myomaker by siRNA inhibits myoblast fusion. MYOD and MYOG, which belong to the family of myogenic regulatory factors, can bind to a conserved E-box located proximal to the Myomaker transcription start site and induce Myomaker transcription. Additionally, miR-140-3p can inhibit Myomaker expression and myoblast fusion, at least in part, by binding to the 3ʹ UTR of Myomaker in vitro. These findings confirm the essential roles of Myomaker in avian myoblast fusion and show that MYOD, MYOG and miR-140-3p can regulate Myomaker expression.

  11. Spatial and functional restriction of regulatory molecules during mammalian myoblast fusion

    International Nuclear Information System (INIS)

    Pavlath, Grace K.

    2010-01-01

    Myoblast fusion is a highly regulated process that is key for forming skeletal muscle during development and regeneration in mammals. Much remains to be understood about the molecular regulation of myoblast fusion. Some molecules that influence mammalian muscle fusion display specific cellular localization during myogenesis. Such molecules can be localized to the contact region between two fusing cells either in both cells or only in one of the cells. How distinct localization of molecules contributes to fusion is not clear. Further complexity exists as other molecules are functionally restricted to myoblasts at later stages of myogenesis to regulate their fusion with multinucleated myotubes. This review examines these three categories of molecules and discusses how spatial and functional restriction may contribute to the formation of a multinucleated cell. Understanding how and why molecules become restricted in location or function is likely to provide further insights into the mechanisms regulating mammalian muscle fusion.

  12. Myoblast transplantation for heart repair: A review of the state of the field

    Institute of Scientific and Technical Information of China (English)

    Howard J. Leonhardt; Michael Brown

    2006-01-01

    Over 200 humans have been treated with myoblast transplantation for heart muscle repair since June 2000. Bioheart sponsored percutaneous delivery studies began in May 2001 in Europe. Approximately one third of the patients have exhibited substantial improvement in left ventricular ejection fraction (LVEF) of over 30% and two heart failure class improvements. Over 80% of the patients have exhibited one heart failure class improvement with moderate improvement of LVEF. Clinical trials seem to demonstrate a marked reduction in emergency hospitalizations in myoblast treated patients. Many years of careful studies have lead to randomized controlled studies that are enrolling patients now at numerous centers worldwide. A firm conclusion on the safety and efficacy of myoblast transplantation cannot be determined until these randomized studies are completed. Final results from randomized controlled studies should be available soon. (J Geriatr Cardiol 2006;3:165-7.)

  13. Bone Marrow Mesenchymal Stromal Cells Stimulate Skeletal Myoblast Proliferation through the Paracrine Release of VEGF

    Science.gov (United States)

    Chellini, Flaminia; Mazzanti, Benedetta; Nistri, Silvia; Nosi, Daniele; Saccardi, Riccardo; Quercioli, Franco; Zecchi-Orlandini, Sandra; Formigli, Lucia

    2012-01-01

    Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies. PMID:22815682

  14. HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

    Science.gov (United States)

    González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

    2017-10-10

    The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving

  15. Short-hairpin Mediated Myostatin Knockdown Resulted in Altered Expression of Myogenic Regulatory Factors with Enhanced Myoblast Proliferation in Fetal Myoblast Cells of Goats.

    Science.gov (United States)

    Kumar, Rohit; Singh, Satyendra Pal; Mitra, Abhijit

    2018-01-02

    Myostatin (MSTN) is a well-known negative regulator of skeletal muscle development. Reduced expression due to natural mutations in the coding region and knockout as well as knockdown of MSTN results in an increase in the muscle mass. In the present study, we demonstrated as high as 60 and 52% downregulation (p < 0.01) of MSTN mRNA and protein in the primary fetal myoblast cells of goats using synthetic shRNAs (n = 3), without any interferon response. We, for the first time, evaluated the effect of MSTN knockdown on the expression of MRFs (namely, MyoD, Myf5), follistatin (FST), and IGFs (IGF-1 & IGF-2) in goat myoblast cells. MSTN knockdown caused an upregulation (p < 0.05) of MyoD and downregulation (p < 0.01) of MYf5 and FST expression. Moreover, we report up to ∼four fold (p < 0.001) enhanced proliferation in myoblasts after four days of culture. The anti-MSTN shRNA demonstrated in the present study could be used for the production of transgenic goats to increase the muscle mass.

  16. Novel lncRNAs in myogenesis: a miR-31 overlapping transcript controls myoblast differentiation.

    KAUST Repository

    Ballarino, Monica; Cazzella, Valentina; D'Andrea, Daniel; Grassi, Luigi; Bisceglie, Lavinia; Cipriano, Andrea; Santini, Tiziana; Pinnarò , Chiara; Morlando, Mariangela; Tramontano, Anna; Bozzoni, Irene

    2014-01-01

    Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.

  17. Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.

    Science.gov (United States)

    Mars, Tomaz; Strazisar, Marusa; Mis, Katarina; Kotnik, Nejc; Pegan, Katarina; Lojk, Jasna; Grubic, Zoran; Pavlin, Mojca

    2015-04-01

    Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.

  18. Analysis of Mitochondrial Network Morphology in Cultured Myoblasts from Patients with Mitochondrial Disorders

    Czech Academy of Sciences Publication Activity Database

    Sládková, J.; Spáčilová, J.; Čapek, Martin; Tesařová, M.; Hansíková, H.; Honzík, T.; Martínek, J.; Zámečník, J.; Kostková, O.; Zeman, J.

    2015-01-01

    Roč. 39, č. 5 (2015), s. 340-350 ISSN 0191-3123 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LH13028 Institutional support: RVO:67985823 Keywords : cristae * Fiji * image analysis * mitochondrial disorders * myoblasts * ultrastructure Subject RIV: EA - Cell Biology Impact factor: 0.828, year: 2015

  19. Novel lncRNAs in myogenesis: a miR-31 overlapping transcript controls myoblast differentiation.

    KAUST Repository

    Ballarino, Monica

    2014-12-15

    Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.

  20. Developmental Programming in Response to Intrauterine Growth Restriction Impairs Myoblast Function and Skeletal Muscle Metabolism

    Science.gov (United States)

    Yates, D. T.; Macko, A. R.; Nearing, M.; Chen, X.; Rhoads, R. P.; Limesand, S. W.

    2012-01-01

    Fetal adaptations to placental insufficiency alter postnatal metabolic homeostasis in skeletal muscle by reducing glucose oxidation rates, impairing insulin action, and lowering the proportion of oxidative fibers. In animal models of intrauterine growth restriction (IUGR), skeletal muscle fibers have less myonuclei at birth. This means that myoblasts, the sole source for myonuclei accumulation in fibers, are compromised. Fetal hypoglycemia and hypoxemia are complications that result from placental insufficiency. Hypoxemia elevates circulating catecholamines, and chronic hypercatecholaminemia has been shown to reduce fetal muscle development and growth. We have found evidence for adaptations in adrenergic receptor expression profiles in myoblasts and skeletal muscle of IUGR sheep fetuses with placental insufficiency. The relationship of β-adrenergic receptors shifts in IUGR fetuses because Adrβ2 expression levels decline and Adrβ1 expression levels are unaffected in myofibers and increased in myoblasts. This adaptive response would suppress insulin signaling, myoblast incorporation, fiber hypertrophy, and glucose oxidation. Furthermore, this β-adrenergic receptor expression profile persists for at least the first month in IUGR lambs and lowers their fatty acid mobilization. Developmental programming of skeletal muscle adrenergic receptors partially explains metabolic and endocrine differences in IUGR offspring, and the impact on metabolism may result in differential nutrient utilization. PMID:22900186

  1. Black ginseng activates Akt signaling, thereby enhancing myoblast differentiation and myotube growth

    Directory of Open Access Journals (Sweden)

    Soo-Yeon Lee

    2018-01-01

    Conclusion: BG enhances myoblast differentiation and myotube hypertrophy by activating Akt/mTOR/p70S6k axis. Thus, our study demonstrates that BG has promising potential to treat or prevent muscle loss related to aging or other pathological conditions, such as diabetes.

  2. Biodistribution studies of 99mTc-labeled myoblasts in a murine model of muscular dystrophy

    International Nuclear Information System (INIS)

    Colombo, F.R.; Torrente, Y.; Casati, R.; Benti, R.; Corti, S.; Salani, S.; D'Angelo, M.G.; DeLiso, A.; Scarlato, G.; Bresolin, N.; Gerundini, P.

    2001-01-01

    The purpose of this study was twofold: first, to evaluate the myoblast labeling of various 99m Tc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding 99m Tc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt). One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [ 99m Tc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin

  3. Developmental Programming in Response to Intrauterine Growth Restriction Impairs Myoblast Function and Skeletal Muscle Metabolism

    Directory of Open Access Journals (Sweden)

    D. T. Yates

    2012-01-01

    Full Text Available Fetal adaptations to placental insufficiency alter postnatal metabolic homeostasis in skeletal muscle by reducing glucose oxidation rates, impairing insulin action, and lowering the proportion of oxidative fibers. In animal models of intrauterine growth restriction (IUGR, skeletal muscle fibers have less myonuclei at birth. This means that myoblasts, the sole source for myonuclei accumulation in fibers, are compromised. Fetal hypoglycemia and hypoxemia are complications that result from placental insufficiency. Hypoxemia elevates circulating catecholamines, and chronic hypercatecholaminemia has been shown to reduce fetal muscle development and growth. We have found evidence for adaptations in adrenergic receptor expression profiles in myoblasts and skeletal muscle of IUGR sheep fetuses with placental insufficiency. The relationship of β-adrenergic receptors shifts in IUGR fetuses because Adrβ2 expression levels decline and Adrβ1 expression levels are unaffected in myofibers and increased in myoblasts. This adaptive response would suppress insulin signaling, myoblast incorporation, fiber hypertrophy, and glucose oxidation. Furthermore, this β-adrenergic receptor expression profile persists for at least the first month in IUGR lambs and lowers their fatty acid mobilization. Developmental programming of skeletal muscle adrenergic receptors partially explains metabolic and endocrine differences in IUGR offspring, and the impact on metabolism may result in differential nutrient utilization.

  4. Cardiac cell therapy: overexpression of connexin43 in skeletal myoblasts and prevention of ventricular arrhythmias

    NARCIS (Netherlands)

    Fernandes, Sarah; van Rijen, Harold V. M.; Forest, Virginie; Evain, Stéphane; Leblond, Anne-Laure; Mérot, Jean; Charpentier, Flavien; de Bakker, Jacques M. T.; Lemarchand, Patricia

    2009-01-01

    Cell-based therapies have great potential for the treatment of cardiovascular diseases. Recently, using a transgenic mouse model Roell et al. reported that cardiac engraftment of connexin43 (Cx43)-overexpressing myoblasts in vivo prevents post-infarct arrhythmia, a common cause of death in patients

  5. DRAGON, a GPI-anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts.

    Science.gov (United States)

    Kanomata, Kazuhiro; Kokabu, Shoichiro; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

    2009-06-01

    Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

  6. Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length

    Science.gov (United States)

    Lee, Jennifer K; Hallock, Peter T

    2017-01-01

    Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but as a consequence of expansion of the diaphragm muscle, the diaphragm central tendon is reduced in size, likely contributing to reduced stamina of Abl2 mutant mice. Ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon of Abl2+/− mice. Specialized tendon cells, resembling tendon cells at myotendinous junctions, form at the ends of these muscle islands, suggesting that myofibers induce differentiation of tendon cells, which reciprocally regulate myofiber length and orientation. PMID:29231808

  7. Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts

    Czech Academy of Sciences Publication Activity Database

    Filipová, A.; Diaz-Garcia, D.; Bezrouk, A.; Čížková, D.; Havelek, R.; Vávrová, J.; Dayanithi, Govindan; Řezáčová, M.

    2015-01-01

    Roč. 39, č. 8 (2015), s. 943-953 ISSN 1065-6995 Institutional support: RVO:68378041 Keywords : cell line * ionizing radiation * multiple cilia * myoblast * primary cilium * serum starvation stress Subject RIV: FP - Other Medical Disciplines Impact factor: 1.663, year: 2015

  8. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells.

    Science.gov (United States)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-02-08

    Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Function of skeletal muscle tissue formed after myoblast transplantation into irradiated mouse muscles.

    Science.gov (United States)

    Wernig, A; Zweyer, M; Irintchev, A

    2000-01-15

    1. Pretreatment of muscles with ionising radiation enhances tissue formation by transplanted myoblasts but little is known about the effects on muscle function. We implanted myoblasts from an expanded, male-donor-derived, culture (i28) into X-ray irradiated (16 Gy) or irradiated and damaged soleus muscles of female syngeneic mice (Balb/c). Three to 6 months later the isometric contractile properties of the muscles were studied in vitro, and donor nuclei were visualised in muscle sections with a Y chromosome-specific DNA probe. 2. Irradiated sham-injected muscles had smaller masses than untreated solei and produced less twitch and tetanic force (all by about 18 %). Injection of 106 myoblasts abolished these deficiencies and innervation appeared normal. 3. Cryodamage of irradiated solei produced muscle remnants with few (1-50) or no fibres. Additional myoblast implantation led to formation of large muscles (25 % above normal) containing numerous small-diameter fibres. Upon direct electrical stimulation, these muscles produced considerable twitch (53 % of normal) and tetanic forces (35 % of normal) but innervation was insufficient as indicated by weak nerve-evoked contractions and elevated ACh sensitivity. 4. In control experiments on irradiated muscles, reinnervation was found to be less complete after botulinum toxin paralysis than after nerve crush indicating that proliferative arrest of irradiated Schwann cells may account for the observed innervation deficits. 5. Irradiation appears to be an effective pretreatment for improving myoblast transplantation. The injected cells can even produce organised contractile tissue replacing whole muscle. However, impaired nerve regeneration limits the functional performance of the new muscle.

  10. TIPS to manipulate myogenesis: retention of myoblast differentiation capacity using microsphere culture

    Directory of Open Access Journals (Sweden)

    N Parmar

    2015-07-01

    Full Text Available Cell therapy is an emerging option for regenerating skeletal muscle. Improved delivery methods for anchorage-dependent myoblasts are likely to improve integration and function of transplanted muscle cells. Highly porous microspheres, produced using thermally induced phase separation (TIPS, have features ideally suited for minimally invasive cell delivery. The purpose of this study was to investigate, for the first time, the use of TIPS microspheres as highly porous microcarriers for manipulation of human skeletal muscle myoblasts (HSMM under defined culture conditions. HSMM cells readily attached to the surface of poly (DL-lactide-co-glycolide (PLGA TIPS microcarriers, where they were induced to continue proliferating or to be driven towards differentiation whilst under static-dynamic culture conditions for 7 days. Switching from proliferation medium to differentiation medium for 7 days, resulted in increased protein expression of skeletal muscle cell contractile apparatus components, MyoD and skeletal muscle myosin heavy chain, compared with cells cultured on conventional culture plasticware for the same duration (p < 0.001. Growth of myoblasts on the surface of the microcarriers and their migration following simulated delivery, caused no change to the proliferative capacity of cells over 7 days. Results from this study demonstrate that TIPS microspheres provide an ideal vehicle for the expansion and delivery of myoblasts for therapeutic applications. Transplantation of myoblasts anchored to a substrate, rather than in suspension, will reduce the amount of ex vivo manipulation required during preparation of the product and allows cells to be delivered in a more natural state. This will improve the ability to control cell dosage and increase the likelihood of efficacy.

  11. Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

    Directory of Open Access Journals (Sweden)

    Sindhu Subramaniam

    Full Text Available Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2. Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation. Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for

  12. Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

    Science.gov (United States)

    Subramaniam, Sindhu; Sreenivas, Prethish; Cheedipudi, Sirisha; Reddy, Vatrapu Rami; Shashidhara, Lingadahalli Subrahmanya; Chilukoti, Ravi Kumar; Mylavarapu, Madhavi; Dhawan, Jyotsna

    2014-01-01

    Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological

  13. An integrated strategy for analyzing the unique developmental programs of different myoblast subtypes.

    Directory of Open Access Journals (Sweden)

    2006-02-01

    Full Text Available An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets--based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes--provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true-positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides

  14. Isolation and Characterization of Human Myoblast Culture In Vitro for Technologies of Cell and Gene Therapy of Skeletal Muscle Pathologies.

    Science.gov (United States)

    Tabakov, V Yu; Zinov'eva, O E; Voskresenskaya, O N; Skoblov, M Yu

    2018-03-01

    We analyzed cultures of 5 independent myoblast lines from human skeletal muscles. It was shown that the content of desmin-positive cells in cultures at early passages exceeds 90%. Typical morphofunctional signs of myogenic differentiation disturbances were identified and their dynamics was studied. Signs of alternative adipogenic and chondrogenic differentiation of cells were revealed. Based on these data, limitations for the use of myoblast cultures of certain passages for biomedical research and cell therapy were evaluated.

  15. Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy.

    Science.gov (United States)

    Muratore, Massimo; Srsen, Vlastimil; Waterfall, Martin; Downes, Andrew; Pethig, Ronald

    2012-09-01

    Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 μm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.

  16. Proliferation of Human Primary Myoblasts Is Associated with Altered Energy Metabolism in Dependence on Ageing In Vivo and In Vitro

    Directory of Open Access Journals (Sweden)

    Reedik Pääsuke

    2016-01-01

    Full Text Available Background. Ageing is associated with suppressed regenerative potential of muscle precursor cells due to decrease of satellite cells and suppressive intramuscular milieu on their activation, associated with ageing-related low-grade inflammation. The aim of the study was to characterize the function of oxidative phosphorylation (OXPHOS, glycolysis, adenylate kinase (AK, and creatine kinase (CK mediated systems in young and older individuals. Materials and Methods. Myoblasts were cultivated from biopsies taken by transcutaneous conchotomy from vastus lateralis muscle in young (20–29 yrs, n=7 and older (70–79 yrs, n=7 subjects. Energy metabolism was assessed in passages 2 to 6 by oxygraphy and enzyme analysis. Results. In myoblasts of young and older subjects the rate of OXPHOS decreased during proliferation from passages 2 to 6. The total activities of CK and AK decreased. Myoblasts of passage 2 cultivated from young muscle showed higher rate of OXPHOS and activities of CK and AK compared to myoblasts from older subjects while hexokinase and pyruvate kinase were not affected by ageing. Conclusions. Proliferation of myoblasts in vitro is associated with downregulation of OXPHOS and energy storage and transfer systems. Ageing in vivo exerts an impact on satellite cells which results in altered metabolic profile in favour of the prevalence of glycolytic pathways over mitochondrial OXPHOS of myoblasts.

  17. The Satellite Cell Niche Regulates the Balance between Myoblast Differentiation and Self-Renewal via p53.

    Science.gov (United States)

    Flamini, Valentina; Ghadiali, Rachel S; Antczak, Philipp; Rothwell, Amy; Turnbull, Jeremy E; Pisconti, Addolorata

    2018-03-13

    Satellite cells are adult muscle stem cells residing in a specialized niche that regulates their homeostasis. How niche-generated signals integrate to regulate gene expression in satellite cell-derived myoblasts is poorly understood. We undertook an unbiased approach to study the effect of the satellite cell niche on satellite cell-derived myoblast transcriptional regulation and identified the tumor suppressor p53 as a key player in the regulation of myoblast quiescence. After activation and proliferation, a subpopulation of myoblasts cultured in the presence of the niche upregulates p53 and fails to differentiate. When satellite cell self-renewal is modeled ex vivo in a reserve cell assay, myoblasts treated with Nutlin-3, which increases p53 levels in the cell, fail to differentiate and instead become quiescent. Since both these Nutlin-3 effects are rescued by small interfering RNA-mediated p53 knockdown, we conclude that a tight control of p53 levels in myoblasts regulates the balance between differentiation and return to quiescence. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Injectable scaffold materials differ in their cell instructive effects on primary human myoblasts

    DEFF Research Database (Denmark)

    Hejbøl, Eva Kildall; Sellathurai, Jeeva; Nair, Prabha Damodaran

    2017-01-01

    Scaffolds are materials used for delivery of cells for regeneration of tissues. They support three-dimensional organization and improve cell survival. For the repair of small skeletal muscles, injections of small volumes of cells are attractive, and injectable scaffolds for delivery of cells offer...... a minimally invasive technique. In this study, we examined in vitro the cell instructive effects of three types of injectable scaffolds, fibrin, alginate, and poly(lactic-co-glycolic acid)-based microparticles on primary human myoblasts. The myoblast morphology and progression in the myogenic program differed......, depending on the type of scaffold material. In alginate gel, the cells obtained a round morphology, they ceased to proliferate, and entered quiescence. In the fibrin gels, differentiation was promoted, and myotubes were observed within a few days in culture, while poly(lactic-co-glycolic acid...

  19. Heavy ion irradiation induces autophagy in irradiated C2C12 myoblasts and their bystander cells

    International Nuclear Information System (INIS)

    Hino, Mizuki; Tajika, Yuki; Hamada, Nobuyuki

    2010-01-01

    Autophagy is one of the major processes involved in the degradation of intracellular materials. Here, we examined the potential impact of heavy ion irradiation on the induction of autophagy in irradiated C2C12 mouse myoblasts and their non-targeted bystander cells. In irradiated cells, ultrastructural analysis revealed the accumulation of autophagic structures at various stages of autophagy (id est (i.e.) phagophores, autophagosomes and autolysosomes) within 20 min after irradiation. Multivesicular bodies (MVBs) and autolysosomes containing MVBs (amphisomes) were also observed. Heavy ion irradiation increased the staining of microtubule-associated protein 1 light chain 3 and LysoTracker Red (LTR). Such enhanced staining was suppressed by an autophagy inhibitor 3-methyladenine. In addition to irradiated cells, bystander cells were also positive with LTR staining. Altogether, these results suggest that heavy ion irradiation induces autophagy not only in irradiated myoblasts but also in their bystander cells. (author)

  20. New perspectives in the treatment of damaged myocardium using autologous skeletal myoblasts

    International Nuclear Information System (INIS)

    Rigatelli, Gianluca; Rossini, Katia; Vindigni, Vincenzo; Mazzoleni, Francesco; Rigatelli, Giorgio; Carraro, Ugo

    2004-01-01

    Autologous skeletal myoblast transplantation may be used to ameliorate the healing process following myocardium infarct and, hopefully, cardiomyopathies. Despite successful animal experimentation, several issues need to be addressed in clinical settings, i.e., the impact of the delivery route, the extent of short- and long-term survival, and differentiation of the injected skeletal myoblasts. The authors offer some new hypotheses resulting from basic research, i.e., where and when to inject the myogenic cells, whatever their source, how to decrease new myofiber atrophy and improve their regeneration. Although these new hypotheses still need to be tested in humans, they may be decisive for future experimental studies and will lead to making endovascular cell implantation a more effective way to treat ischemic heart disease and failure

  1. β‐Taxilin participates in differentiation of C2C12 myoblasts into myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Sakane, Hiroshi; Makiyama, Tomohiko; Nogami, Satoru; Horii, Yukimi [Department of Molecular and Cell Biology, Graduate school of Medicine, Dokkyo Medical University, 880 Kitakobayashi, Mibu-town, Tochigi 321-0293 (Japan); Akasaki, Kenji [Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0292 (Japan); Shirataki, Hiromichi, E-mail: hiro-sh@dokkyomed.ac.jp [Department of Molecular and Cell Biology, Graduate school of Medicine, Dokkyo Medical University, 880 Kitakobayashi, Mibu-town, Tochigi 321-0293 (Japan)

    2016-07-15

    Myogenesis is required for the development of skeletal muscle. Accumulating evidence indicates that the expression of several genes are upregulated during myogenesis and these genes play pivotal roles in myogenesis. However, the molecular mechanism underlying myogenesis is not fully understood. In this study, we found that β-taxilin, which is specifically expressed in the skeletal muscle and heart tissues, was progressively expressed during differentiation of C2C12 myoblasts into myotubes, prompting us to investigate the role of β-taxilin in myogenesis. In C2C12 cells, knockdown of β-taxilin impaired the fusion of myoblasts into myotubes, and decreased the diameter of myotubes. We also found that β-taxilin interacted with dysbindin, a coiled-coil-containing protein. Knockdown of dysbindin conversely promoted the fusion of myoblasts into myotubes and increased the diameter of myotubes in C2C12 cells. Furthermore, knockdown of dysbindin attenuated the inhibitory effect of β-taxilin depletion on myotube formation of C2C12 cells. These results demonstrate that β-taxilin participates in myogenesis through suppressing the function of dysbindin to inhibit the differentiation of C2C12 myoblasts into myotubes. - Highlights: • β‐Taxilin is progressively expressed during differentiation of C2C12 cell. • Knockdown of β-taxilin impaired C2C12 myotube formation. • β‐Taxilin interacted with dysbindin. • Knockdown of dysbindin promoted C2C12 myotube formation. • The function of β-taxilin in C2C12 myotube formation depends on dysbindin.

  2. PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

    Directory of Open Access Journals (Sweden)

    Aiko Amagai

    2012-01-01

    Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

  3. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Dunham, Ian [EMBL-European Bioinformatics Institute (EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom); Murai, Kasumi [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Jones, Philip H., E-mail: phj20@cam.ac.uk [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom)

    2011-07-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  4. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    International Nuclear Information System (INIS)

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko; Dunham, Ian; Murai, Kasumi; Jones, Philip H.

    2011-01-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  5. Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts.

    Science.gov (United States)

    Sin, Jon; Andres, Allen M; Taylor, David J R; Weston, Thomas; Hiraumi, Yoshimi; Stotland, Aleksandr; Kim, Brandon J; Huang, Chengqun; Doran, Kelly S; Gottlieb, Roberta A

    2016-01-01

    Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

  6. The cardioprotective efficacy of TVP1022 in a rat model of ischaemia/reperfusion.

    Science.gov (United States)

    Ertracht, Offir; Liani, Esti; Bachner-Hinenzon, Noa; Bar-Am, Orit; Frolov, Luba; Ovcharenko, Elena; Awad, Huda; Blum, Shany; Barac, Yaron; Amit, Tamar; Adam, Dan; Youdim, Moussa; Binah, Ofer

    2011-06-01

    Because myocardial infarction is a major cause of morbidity and mortality worldwide, protecting the heart from the ischaemia and reperfusion (I/R) damage is the focus of intense research. Based on our in vitro findings showing that TVP1022 (the S-enantiomer of rasagiline, an anti-Parkinsonian drug) possesses cardioprotective effects, in the present study we investigated the hypothesis that TVP1022 can attenuate myocardial damage in an I/R model in rats. The model consisted of 30-min occlusion of the left anterior descending artery followed by 4 or 24 h reperfusion. In addition, we investigated the possible mechanisms of cardioprotection in H9c2 cells and neonatal rat ventricular myocytes (NRVM) exposed to oxidative stress induced by H(2) O(2) . TVP1022 (20 and 40 mg·kg(-1) ) administered 5 min before reperfusion followed by an additional dose 4 h after reperfusion reduced the infarct size and attenuated the decline in ventricular function. TVP1022 also attenuated I/R-induced deterioration in cardiac mitochondrial integrity evaluated by mitochondrial swelling capacity. In vitro, using H9c2 cells and NRVM, TVP1022 attenuated both serum free- and H(2) O(2) -induced damage, preserved mitochondrial membrane potential and Bcl-2 levels, inhibited mitochondrial cytochrome c release and the increase in cleaved caspase 9 and 3 levels, and enhanced the phosphorylation of protein kinase C and glycogen synthase kinase-3β. TVP1022 provided cardioprotection in a model of myocardial infarction, and therefore should be considered as a novel adjunctive therapy for attenuating myocardial damage resulting from I/R injuries. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  7. Leonurine (SCM-198) improves cardiac recovery in rat during chronic infarction.

    Science.gov (United States)

    Liu, XinHua; Pan, LiLong; Gong, QiHai; Zhu, YiZhun

    2010-12-15

    Leonurine, an alkaloid typically found in Herba leonuri, is known to have both antioxidant and cardioprotective properties. In the present study, we investigated the cardioprotective mechanism of leonurine the in vivo rat model of chronic myocardial ischemia and in vitro H9c2 cardiac myocyte model of oxidative stress. Myocardial ischemia was induced by ligating the left anterior descending coronary artery. Rats were divided into sham, myocardial ischemia+saline, and myocardial ischemia+leonurine (15 mg/kg/day). Cardiac function was recorded by catheterization. Apoptosis-related factor vascular endothelial growth factor (VEGF), survivin, Bcl-2 and Bax and pro-survival signaling pathways Akt, hypoxia inducible factor (HIF)-1α were measured by Western blotting or RT-PCR. Our results showed leonurine significantly improved myocardial function as evidenced by the decreased left ventricle end-diastolic pressure and the increased +dP/dt. Interestingly, leonurine increased the phosphorylation of Akt, the protein and gene expression of Bcl-2, but it reduced the protein and gene expression of Bax in vivo. Meanwhile leonurine significantly increased Akt phosphorylation in a concentration-dependent manner in H9c2 cardiac myocyte induced by oxidative stress in vitro, which was abolished by a phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. Furthermore, leonurine not only increased the expression of HIF-1α but also the expression of survivin and VEGF. The results of present study demonstrated, for the first time that leonurine has potent anti-apoptotic effects after chronic myocardial ischemia mediated by activating the PI3K/Akt signaling pathway. Angiogenic mechanisms may be partially responsible for such an effect, which needs to be studied further. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

    Science.gov (United States)

    Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

    2016-07-01

    Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.

  9. Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia-reperfusion injury in rat heart.

    Science.gov (United States)

    Granata, Riccarda; Trovato, Letizia; Gallo, Maria Pia; Destefanis, Silvia; Settanni, Fabio; Scarlatti, Francesca; Brero, Alessia; Ramella, Roberta; Volante, Marco; Isgaard, Jorgen; Levi, Renzo; Papotti, Mauro; Alloatti, Giuseppe; Ghigo, Ezio

    2009-07-15

    The hypothalamic neuropeptide growth hormone-releasing hormone (GHRH) stimulates GH synthesis and release in the pituitary. GHRH also exerts proliferative effects in extrapituitary cells, whereas GHRH antagonists have been shown to suppress cancer cell proliferation. We investigated GHRH effects on cardiac myocyte cell survival and the underlying signalling mechanisms. Reverse transcriptase-polymerase chain reaction analysis showed GHRH receptor (GHRH-R) mRNA in adult rat ventricular myocytes (ARVMs) and in rat heart H9c2 cells. In ARVMs, GHRH prevented cell death and caspase-3 activation induced by serum starvation and by the beta-adrenergic receptor agonist isoproterenol. The GHRH-R antagonist JV-1-36 abolished GHRH survival action under both experimental conditions. GHRH-induced cardiac cell protection required extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide-3 kinase (PI3K)/Akt activation and adenylyl cyclase/cAMP/protein kinase A signalling. Isoproterenol strongly upregulated the mRNA and protein of the pro-apoptotic inducible cAMP early repressor, whereas GHRH completely blocked this effect. Similar to ARVMs, in H9c2 cardiac cells, GHRH inhibited serum starvation- and isoproterenol-induced cell death and apoptosis through the same signalling pathways. Finally, GHRH improved left ventricular recovery during reperfusion and reduced infarct size in Langendorff-perfused rat hearts, subjected to ischaemia-reperfusion (I/R) injury. These effects involved PI3K/Akt signalling and were inhibited by JV-1-36. Our findings suggest that GHRH promotes cardiac myocyte survival through multiple signalling mechanisms and protects against I/R injury in isolated rat heart, indicating a novel cardioprotective role of this hormone.

  10. Maintenance of DNA repair capacity in differentiating rat muscle cells in vitro

    International Nuclear Information System (INIS)

    Koval, T.M.; Kaufman, S.J.

    1981-01-01

    Unscheduled DNA synthesis was measured at several times during the differentiation of cultured rat skeletal muscle cells in response to exposures to 254 nm UV light. There was no change in the amount of repair DNA synthesis as the cells fuse and differentiate from postmitotic prefusion myoblasts to multinucleated contracting myotubes. (author)

  11. A novel in vitro model for studying quiescence and activation of primary isolated human myoblasts

    DEFF Research Database (Denmark)

    Sellathurai, Jeeva; Cheedipudi, Sirisha; Dhawan, Jyotsna

    2013-01-01

    term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may...... be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis...

  12. Cellular and molecular studies of the effects of a selective COX-2 inhibitor celecoxib in the cardiac cell line H9c2 and their correlation with death mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Sakane, K.K. [Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba, São José dos Campos, SP (Brazil); Monteiro, C.J.; Silva, W.; Silva, A.R. [Núcleo de Pesquisa em Ciências Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, MG (Brazil); Santos, P.M. [Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba, São José dos Campos, SP (Brazil); Lima, K.F. [Núcleo de Pesquisa em Ciências Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, MG (Brazil); Moraes, K.C.M. [Instituto de Biociências, Departamento de Biologia, Universidade Estadual Paulista ‘‘Júlio de Mesquita Filho’’, Rio Claro, SP (Brazil)

    2013-11-29

    Cardiovascular disease is one of the leading causes of death worldwide, and evidence indicates a correlation between the inflammatory process and cardiac dysfunction. Selective inhibitors of cyclooxygenase-2 (COX-2) enzyme are not recommended for long-term use because of potentially severe side effects to the heart. Considering this and the frequent prescribing of commercial celecoxib, the present study analyzed cellular and molecular effects of 1 and 10 µM celecoxib in a cell culture model. After a 24-h incubation, celecoxib reduced cell viability in a dose-dependent manner as also demonstrated in MTT assays. Furthermore, reverse transcription-polymerase chain reaction analysis showed that the drug modulated the expression level of genes related to death pathways, and Western blot analyses demonstrated a modulatory effect of the drug on COX-2 protein levels in cardiac cells. In addition, the results demonstrated a downregulation of prostaglandin E2 production by the cardiac cells incubated with celecoxib, in a dose-specific manner. These results are consistent with the decrease in cell viability and the presence of necrotic processes shown by Fourier transform infrared analysis, suggesting a direct correlation of prostanoids in cellular homeostasis and survival.

  13. Laminin-Coated Poly(Methyl Methacrylate (PMMA Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population

    Directory of Open Access Journals (Sweden)

    Nor Kamalia Zahari

    2017-10-01

    Full Text Available Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate (PMMA nanofiber (PM scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h−1 and migration (0.26 ± 0.04 μm/min, while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h−1 and migration (0.23 ± 0.03 μm/min. Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.

  14. Laminin-Coated Poly(Methyl Methacrylate) (PMMA) Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population.

    Science.gov (United States)

    Zahari, Nor Kamalia; Idrus, Ruszymah Binti Haji; Chowdhury, Shiplu Roy

    2017-10-30

    Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h -1 ) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h -1 ) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.

  15. Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

    Science.gov (United States)

    Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

    1996-08-01

    To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

  16. NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats

    Energy Technology Data Exchange (ETDEWEB)

    Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Park, Ji-hoon [Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Kim, Soon Ha, E-mail: shakim@lgls.com [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of)

    2012-08-15

    Oxidative stress is one of the causes of cardiomyopathy. In the present study, NecroXs, novel class of mitochondrial ROS/RNS scavengers, were evaluated for cardioprotection in in vitro and in vivo model, and the putative mechanism of the cardioprotection of NecroX-7 was investigated by global gene expression profiling and subsequent biochemical analysis. NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced death of H9C2 rat cardiomyocytes at EC{sub 50} = 0.057 μM. In doxorubicin (DOX)-induced cardiomyopathy in rats, NecroX-7 significantly reduced the plasma levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) which were increased by DOX treatment (p < 0.05). Microarray analysis revealed that 21 genes differentially expressed in tBHP-treated H9C2 cells were involved in ‘Production of reactive oxygen species’ (p = 0.022), and they were resolved by concurrent NecroX-7 treatment. Gene-to-gene networking also identified that NecroX-7 relieved cell death through Ncf1/p47phox and Rac2 modulation. In subsequent biochemical analysis, NecroX-7 inhibited NADPH oxidase (NOX) activity by 53.3% (p < 0.001). These findings demonstrate that NecroX-7, in part, provides substantial protection of cardiomyopathy induced by tBHP or DOX via NOX-mediated cell death. -- Highlights: ► NecroX-7 prevented tert-butyl hydroperoxide-induced in vitro cardiac cell death. ► NecroX-7 ameliorated doxorubicin-induced in vivo cardiomyopathy. ► NecroX-7 prevented oxidative stress and necrosis-enriched transcriptional changes. ► NecroX-7 effectively inhibited NADPH oxidase activation. ► Cardioprotection of Necro-7 was brought on by modulation of NADPH oxidase activity.

  17. α-Syntrophin stabilizes catalase to reduce endogenous reactive oxygen species levels during myoblast differentiation.

    Science.gov (United States)

    Moon, Jae Yun; Choi, Su Jin; Heo, Cheol Ho; Kim, Hwan Myung; Kim, Hye Sun

    2017-07-01

    α-Syntrophin is a component of the dystrophin-glycoprotein complex that interacts with various intracellular signaling proteins in muscle cells. The α-syntrophin knock-down C2 cell line (SNKD), established by infecting lentivirus particles with α-syntrophin shRNA, is characterized by a defect in terminal differentiation and increase in cell death. Since myoblast differentiation is accompanied by intensive mitochondrial biogenesis, the generation of intracellular reactive oxygen species (ROS) is also increased during myogenesis. Two-photon microscopy imaging showed that excessive intracellular ROS accumulated during the differentiation of SNKD cells as compared with control cells. The formation of 4-hydroxynonenal adduct, a byproduct of lipid peroxidation during oxidative stress, significantly increased in differentiated SNKD myotubes and was dramatically reduced by epigallocatechin-3-gallate, a well-known ROS scavenger. Among antioxidant enzymes, catalase was significantly decreased during differentiation of SNKD cells without changes at the mRNA level. Of interest was the finding that the degradation of catalase was rescued by MG132, a proteasome inhibitor, in the SNKD cells. This study demonstrates a novel function of α-syntrophin. This protein plays an important role in the regulation of oxidative stress from endogenously generated ROS during myoblast differentiation by modulating the protein stability of catalase. © 2017 Federation of European Biochemical Societies.

  18. The MSX1 homeoprotein recruits G9a methyltransferase to repressed target genes in myoblast cells.

    Directory of Open Access Journals (Sweden)

    Jingqiang Wang

    Full Text Available Although the significance of lysine modifications of core histones for regulating gene expression is widely appreciated, the mechanisms by which these modifications are incorporated at specific regulatory elements during cellular differentiation remains largely unknown. In our previous studies, we have shown that in developing myoblasts the Msx1 homeoprotein represses gene expression by influencing the modification status of chromatin at its target genes. We now show that genomic binding by Msx1 promotes enrichment of the H3K9me2 mark on repressed target genes via recruitment of G9a histone methyltransferase, the enzyme responsible for catalyzing this histone mark. Interaction of Msx1 with G9a is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 mark in proximity to Msx1 binding sites on repressed target genes in myoblast cells as well as the developing limb. We propose that regulation of chromatin status by Msx1 recruitment of G9a and other histone modifying enzymes to regulatory regions of target genes represents an important means of regulating the gene expression during development.

  19. Rats

    Directory of Open Access Journals (Sweden)

    Alexey Kondrashov

    2012-01-01

    Full Text Available We aimed to perform a chemical analysis of both Alibernet red wine and an alcohol-free Alibernet red wine extract (AWE and to investigate the effects of AWE on nitric oxide and reactive oxygen species production as well as blood pressure development in normotensive Wistar Kyoto (WKY and spontaneously hypertensive rats (SHRs. Total antioxidant capacity together with total phenolic and selected mineral content was measured in wine and AWE. Young 6-week-old male WKY and SHR were treated with AWE (24,2 mg/kg/day for 3 weeks. Total NOS and SOD activities, eNOS and SOD1 protein expressions, and superoxide production were determined in the tissues. Both antioxidant capacity and phenolic content were significantly higher in AWE compared to wine. The AWE increased NOS activity in the left ventricle, aorta, and kidney of SHR, while it did not change NOS activity in WKY rats. Similarly, increased SOD activity in the plasma and left ventricle was observed in SHR only. There were no changes in eNOS and SOD1 expressions. In conclusion, phenolics and minerals included in AWE may contribute directly to increased NOS and SOD activities of SHR. Nevertheless, 3 weeks of AWE treatment failed to affect blood pressure of SHR.

  20. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  1. Adipose Tissue-Derived Stem Cell Secreted IGF-1 Protects Myoblasts from the Negative Effect of Myostatin

    Directory of Open Access Journals (Sweden)

    Sebastian Gehmert

    2014-01-01

    Full Text Available Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs, these cells (ASCs provide a therapeutic option for Duchenne Muscular Dystrophy (DMD. But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases.

  2. Biodistribution studies of {sup 99m}Tc-labeled myoblasts in a murine model of muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Colombo, F.R. E-mail: colombof@policlinico.mi.it; Torrente, Y.; Casati, R.; Benti, R.; Corti, S.; Salani, S.; D' Angelo, M.G.; DeLiso, A.; Scarlato, G.; Bresolin, N.; Gerundini, P

    2001-11-01

    The purpose of this study was twofold: first, to evaluate the myoblast labeling of various {sup 99m}Tc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding {sup 99m}Tc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt). One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [{sup 99m}Tc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin.

  3. Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle

    NARCIS (Netherlands)

    Vliet, A.K. van; Nègre-Arrariou, P.; Thiel, G.C.F. van; Bolhuis, P.A.; Cohen, L.H.

    1996-01-01

    Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC50 values of 19 ± 6 nM and 4.0 ± 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC50 value of 110 ± 38 nM). Through

  4. Biphasic regulation of intracellular calcium by gemfibrozil contributes to inhibiting L6 myoblast differentiation: implications for clinical myotoxicity.

    Science.gov (United States)

    Liu, Aiming; Yang, Julin; Gonzalez, Frank J; Cheng, Gary Q; Dai, Renke

    2011-02-18

    Gemfibrozil is the most myotoxic fibrate drug commonly used for dyslipidemia, but the mechanism is poorly understood. The current study revealed that gemfibrozil inhibits myoblast differentiation through the regulation of intracellular calcium ([Ca(2+)]i) as revealed in L6 myoblasts by use of laser scan confocal microscopy and flow cytometry using Fluo-4 AM as a probe. Gemfibrozil at 20-400 μM, could regulate [Ca(2+)]i in L6 cells in a biphasic manner, and sustained reduction was observed when the concentration reached 200 μM. Inhibition of L6 differentiation by gemfibrozil was concentration-dependent with maximal effect noted between 200 and 400 μM, as indicated by creatine kinase activities and the differentiation index, respectively. In differentiating L6 myoblasts, gemfibrozil at concentrations below 400 μM led to no significant signs of apoptosis or cytotoxicity, whereas differentiation, inhibited by 200 μM gemfibrozil, was only partially recovered. A good correlation was noted between gemfibrozil concentrations that regulate [Ca(2+)]i and inhibit L6 myoblasts differentiation, and both are within the range of total serum concentrations found in the clinic. These data suggest a potential pharmacodynamic effect of gemfibrozil on myogenesis as a warning sign, in addition to the complex pharmacokinetic interactions. It is also noteworthy that mobilization of [Ca(2+)]i by gemfibrozil may trigger complex biological responses besides myocyte differentiation. Information revealed in this study explores the mechanism of gemfibrozil-induced myotoxicity through the regulation of intracellular calcium.

  5. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    International Nuclear Information System (INIS)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven

    2014-01-01

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro

  6. Mechanical stimuli activation of calpain is required for myoblast differentiation and occurs via an ERK/MAP kinase signaling pathway

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    2006-01-01

    a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have shown that mechanical signals transmitted through the C2C12 cells interaction with laminin cause an increase in cellular differentiation. This signaling results in an increase in the number of myotubes formed in the cultures...

  7. Mesenchymal Stromal Cell Secreted Sphingosine 1-Phosphate (S1P) Exerts a Stimulatory Effect on Skeletal Myoblast Proliferation

    Science.gov (United States)

    Tani, Alessia; Anderloni, Giulia; Pierucci, Federica; Matteini, Francesca; Chellini, Flaminia; Zecchi Orlandini, Sandra; Meacci, Elisabetta

    2014-01-01

    Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration. PMID:25264785

  8. Myogenesis in the sea urchin embryo: the molecular fingerprint of the myoblast precursors

    Science.gov (United States)

    2013-01-01

    Background In sea urchin larvae the circumesophageal fibers form a prominent muscle system of mesodermal origin. Although the morphology and later development of this muscle system has been well-described, little is known about the molecular signature of these cells or their precise origin in the early embryo. As an invertebrate deuterostome that is more closely related to the vertebrates than other commonly used model systems in myogenesis, the sea urchin fills an important phylogenetic gap and provides a unique perspective on the evolution of muscle cell development. Results Here, we present a comprehensive description of the development of the sea urchin larval circumesophageal muscle lineage beginning with its mesodermal origin using high-resolution localization of the expression of several myogenic transcriptional regulators and differentiation genes. A few myoblasts are bilaterally distributed at the oral vegetal side of the tip of the archenteron and first appear at the late gastrula stage. The expression of the differentiation genes Myosin Heavy Chain, Tropomyosin I and II, as well as the regulatory genes MyoD2, FoxF, FoxC, FoxL1, Myocardin, Twist, and Tbx6 uniquely identify these cells. Interestingly, evolutionarily conserved myogenic factors such as Mef2, MyoR and Six1/2 are not expressed in sea urchin myoblasts but are found in other mesodermal domains of the tip of the archenteron. The regulatory states of these domains were characterized in detail. Moreover, using a combinatorial analysis of gene expression we followed the development of the FoxF/FoxC positive cells from the onset of expression to the end of gastrulation. Our data allowed us to build a complete map of the Non-Skeletogenic Mesoderm at the very early gastrula stage, in which specific molecular signatures identify the precursors of different cell types. Among them, a small group of cells within the FoxY domain, which also express FoxC and SoxE, have been identified as plausible myoblast

  9. Complex interactions between human myoblasts and the surrounding 3D fibrin-based matrix.

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    Stéphane Chiron

    Full Text Available Anchorage of muscle cells to the extracellular matrix is crucial for a range of fundamental biological processes including migration, survival and differentiation. Three-dimensional (3D culture has been proposed to provide a more physiological in vitro model of muscle growth and differentiation than routine 2D cultures. However, muscle cell adhesion and cell-matrix interplay of engineered muscle tissue remain to be determined. We have characterized cell-matrix interactions in 3D muscle culture and analyzed their consequences on cell differentiation. Human myoblasts were embedded in a fibrin matrix cast between two posts, cultured until confluence, and then induced to differentiate. Myoblasts in 3D aligned along the longitudinal axis of the gel. They displayed actin stress fibers evenly distributed around the nucleus and a cortical mesh of thin actin filaments. Adhesion sites in 3D were smaller in size than in rigid 2D culture but expression of adhesion site proteins, including α5 integrin and vinculin, was higher in 3D compared with 2D (p<0.05. Myoblasts and myotubes in 3D exhibited thicker and ellipsoid nuclei instead of the thin disk-like shape of the nuclei in 2D (p<0.001. Differentiation kinetics were faster in 3D as demonstrated by higher mRNA concentrations of α-actinin and myosin. More important, the elastic modulus of engineered muscle tissues increased significantly from 3.5 ± 0.8 to 7.4 ± 4.7 kPa during proliferation (p<0.05 and reached 12.2 ± 6.0 kPa during differentiation (p<0.05, thus attesting the increase of matrix stiffness during proliferation and differentiation of the myocytes. In conclusion, we reported modulations of the adhesion complexes, the actin cytoskeleton and nuclear shape in 3D compared with routine 2D muscle culture. These findings point to complex interactions between muscle cells and the surrounding matrix with dynamic regulation of the cell-matrix stiffness.

  10. Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

    International Nuclear Information System (INIS)

    Roura-Ferrer, Meritxell; Sole, Laura; Martinez-Marmol, Ramon; Villalonga, Nuria; Felipe, Antonio

    2008-01-01

    Voltage-dependent K + channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G 1 -phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletal muscle cell proliferation

  11. Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells.

    Science.gov (United States)

    Konigsberg, M; Pérez, V I; Ríos, C; Liu, Y; Lee, S; Shi, Y; Van Remmen, H

    2013-01-01

    In the majority of studies using primary cultures of myoblasts, the cells are maintained at ambient oxygen tension (21% O2), despite the fact that physiological O2 at the tissue level in vivo is much lower (~1-5% O2). We hypothesized that the cellular response in presence of high oxygen concentration might be particularly important in studies comparing energetic function or oxidative stress in cells isolated from young versus old animals. To test this, we asked whether oxygen tension plays a role in mitochondrial bioenergetics (oxygen consumption, glycolysis and fatty acid oxidation) or oxidative damage to proteins (protein disulfides, carbonyls and aggregates) in myoblast precursor cells (MPCs) isolated from young (3-4 m) and old (29-30 m) C57BL/6 mice. MPCs were grown under physiological (3%) or ambient (21%) O2 for two weeks prior to exposure to an acute oxidative insult (H2O2). Our results show significantly higher basal mitochondrial respiration in young versus old MPCs, an increase in basal respiration in young MPCs maintained at 3% O2 compared to cells maintained at 21% O2, and a shift toward glycolytic metabolism in old MPCs grown at 21% O2. H2O2 treatment significantly reduced respiration in old MPCs grown at 3% O2 but did not further repress respiration at 21% O2 in old MPCs. Oxidative damage to protein was higher in cells maintained at 21% O2 and increased in response to H2O2 in old MPCs. These data underscore the importance of understanding the effect of ambient oxygen tension in cell culture studies, in particular studies measuring oxidative damage and mitochondrial function.

  12. Phosphoinositide 3-kinase/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

    Science.gov (United States)

    Lim, Jeong A; Woo, Joo Hong; Kim, Hye Sun

    2008-09-01

    In this study, it was found that undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than differentiated myotubes. Cell death occurred with a relatively low concentration of menadione in myoblasts compared to myotubes. With the same concentration of menadione, the Bcl-2/Bax ratio decreased and nuclei containing condensed chromatin were observed in myoblasts to a greater extent than in myotubes. However, myotubes became increasingly susceptible to menadione when phosphoinositide 3-kinase (PI3-K) was blocked by pre-incubation with LY294002, a PI3-K inhibitor. Actually, PI3-K activity was reduced by menadione in myoblasts but not in myotubes. In addition, the phosphorylation of Akt, a downstream effector of PI3-K, was inhibited in myoblasts by menadione but increased in myotubes. Both LY294002 and API-2, an Akt inhibitor, decreased the Bcl-2/Bax ratio in menadione-exposed myotubes. These results suggest that the differential activity of PI3-K/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

  13. Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

    DEFF Research Database (Denmark)

    Sellathurai, Jeeva; Nielsen, Joachim; Hejbøl, Eva Kildall

    2016-01-01

    -PCR, immunocytochemistry and western blot. RESULTS AND CONCLUSIONS: We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET......, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch...... and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β...

  14. Myoblast sensitivity and fibroblast insensitivity to osteogenic conversion by BMP-2 correlates with the expression of Bmpr-1a

    Directory of Open Access Journals (Sweden)

    North Kathryn N

    2009-05-01

    Full Text Available Abstract Background Osteoblasts are considered to primarily arise from osseous progenitors within the periosteum or bone marrow. We have speculated that cells from local soft tissues may also take on an osteogenic phenotype. Myoblasts are known to adopt a bone gene program upon treatment with the osteogenic bone morphogenetic proteins (BMP-2,-4,-6,-7,-9, but their osteogenic capacity relative to other progenitor types is unclear. We further hypothesized that the sensitivity of cells to BMP-2 would correlate with BMP receptor expression. Methods We directly compared the BMP-2 sensitivity of myoblastic murine cell lines and primary cells with osteoprogenitors from osseous tissues and fibroblasts. Fibroblasts forced to undergo myogenic conversion by transduction with a MyoD-expressing lentiviral vector (LV-MyoD were also examined. Outcome measures included alkaline phosphatase expression, matrix mineralization, and expression of osteogenic genes (alkaline phosphatase, osteocalcin and bone morphogenetic protein receptor-1A as measured by quantitative PCR. Results BMP-2 induced a rapid and robust osteogenic response in myoblasts and osteoprogenitors, but not in fibroblasts. Myoblasts and osteoprogenitors grown in osteogenic media rapidly upregulated Bmpr-1a expression. Chronic BMP-2 treatment resulted in peak Bmpr-1a expression at day 6 before declining, suggestive of a negative feedback mechanism. In contrast, fibroblasts expressed low levels of Bmpr-1a that was only weakly up-regulated by BMP-2 treatment. Bioinformatics analysis confirmed the presence of myogenic responsive elements in the proximal promoter region of human and murine BMPR-1A/Bmpr-1a. Forced myogenic gene expression in fibroblasts was associated with a significant increase in Bmpr-1a expression and a synergistic increase in the osteogenic response to BMP-2. Conclusion These data demonstrate the osteogenic sensitivity of muscle progenitors and provide a mechanistic insight into the

  15. Improvement of maternal vitamin D status with 25-hydroxycholecalciferol positively impacts porcine fetal skeletal muscle development and myoblast activity.

    Science.gov (United States)

    Hines, E A; Coffey, J D; Starkey, C W; Chung, T K; Starkey, J D

    2013-09-01

    There is little information available regarding the influence of maternal vitamin D status on fetal skeletal muscle development. Therefore, we investigated the effect of improved vitamin D status resulting from 25-hydroxycholecalciferol (25OHD3) supplementation of dams on fetal skeletal muscle developmental characteristics and myoblast activity using Camborough 22 gilts (n = 40) randomly assigned to 1 of 2 corn-soybean meal-based diets. The control diet (CTL) contained 2,500 IU cholecalciferol (D3)/kg diet, whereas the experimental diet contained 500 IU D3/kg diet plus 50 µg 25OHD3/kg diet. Gilts were fed 2.7 kg of their assigned diet once daily beginning 43 d before breeding through d 90 of gestation. On gestational d 90 (± 1), fetal LM and semitendinosus muscle samples were collected for analysis of developmental characteristics and myoblast activity, respectively. No treatment difference was observed in fetal LM cross-sectional area (P = 0.25). Fetuses from 25OHD3-supplemented gilts had more LM fibers (P = 0.04) that tended to be smaller in cross-sectional area compared with CTL fetuses (P = 0.11). A numerical increase in the total number of Pax7+ myoblasts was also observed in fetuses from 25OHD3-supplemented gilts (P = 0.12). Myoblasts derived from the muscles of fetuses from 25OHD3-fed dams displayed an extended proliferative phase in culture compared with those from fetuses of dams fed only D3 (P importance of maternal vitamin D status on the development of fetal skeletal muscle.

  16. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

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    Haohan Wang

    2016-03-01

    Full Text Available Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

  17. Effects of creatine and its analog, β-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts.

    Science.gov (United States)

    Ohira, Yoshinobu; Matsuoka, Yoshikazu; Kawano, Fuminori; Ogura, Akihiko; Higo, Yoko; Ohira, Takashi; Terada, Masahiro; Oke, Yoshihiko; Nakai, Naoya

    2011-01-01

    The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.

  18. Possible role of TIEG1 as a feedback regulator of myostatin and TGF-{beta} in myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke; Chao, Guozheng; Takahashi, Hideyuki; Watanabe, Kouichi; Ohwada, Shyuichi; Aso, Hisashi [Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555 (Japan); Yamaguchi, Takahiro, E-mail: ty1010@bios.tohoku.ac.jp [Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555 (Japan)

    2010-03-19

    Myostatin and TGF-{beta} negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-{beta} signaling remains unclear. TGF-{beta} inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-{beta} signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-{beta} signaling using C2C12 myoblasts. Myostatin and TGF-{beta} induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage of differentiation. In contrast, TGF-{beta} enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-{beta} in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-{beta}. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-{beta} that prevents excess action in myoblasts.

  19. MicroRNA-128 targets myostatin at coding domain sequence to regulate myoblasts in skeletal muscle development.

    Science.gov (United States)

    Shi, Lei; Zhou, Bo; Li, Pinghua; Schinckel, Allan P; Liang, Tingting; Wang, Han; Li, Huizhi; Fu, Lingling; Chu, Qingpo; Huang, Ruihua

    2015-09-01

    MicroRNAs (miRNAs or miRs) play a critical role in skeletal muscle development. In a previous study we observed that miR-128 was highly expressed in skeletal muscle. However, its function in regulating skeletal muscle development is not clear. Our hypothesis was that miR-128 is involved in the regulation of the proliferation and differentiation of skeletal myoblasts. In this study, through bioinformatics analyses, we demonstrate that miR-128 specifically targeted mRNA of myostatin (MSTN), a critical inhibitor of skeletal myogenesis, at coding domain sequence (CDS) region, resulting in down-regulating of myostatin post-transcription. Overexpression of miR-128 inhibited proliferation of mouse C2C12 myoblast cells but promoted myotube formation; whereas knockdown of miR-128 had completely opposite effects. In addition, ectopic miR-128 regulated the expression of myogenic factor 5 (Myf5), myogenin (MyoG), paired box (Pax) 3 and 7. Furthermore, an inverse relationship was found between the expression of miR-128 and MSTN protein expression in vivo and in vitro. Taken together, these results reveal that there is a novel pathway in skeletal muscle development in which miR-128 regulates myostatin at CDS region to inhibit proliferation but promote differentiation of myoblast cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Possible role of TIEG1 as a feedback regulator of myostatin and TGF-β in myoblasts

    International Nuclear Information System (INIS)

    Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke; Chao, Guozheng; Takahashi, Hideyuki; Watanabe, Kouichi; Ohwada, Shyuichi; Aso, Hisashi; Yamaguchi, Takahiro

    2010-01-01

    Myostatin and TGF-β negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-β signaling remains unclear. TGF-β inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-β signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-β signaling using C2C12 myoblasts. Myostatin and TGF-β induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage of differentiation. In contrast, TGF-β enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-β in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-β. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-β that prevents excess action in myoblasts.

  1. Photobiomodulation Protects and Promotes Differentiation of C2C12 Myoblast Cells Exposed to Snake Venom.

    Directory of Open Access Journals (Sweden)

    Luciana Miato Gonçalves Silva

    Full Text Available Snakebites is a neglected disease and in Brazil is considered a serious health problem, with the majority of the snakebites caused by the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse local myonecrose which is the main sequel caused by the envenomation. Several studies have shown the effectiveness of low level laser (LLL therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown. In this in vitro study, we aimed to analyze the effect of LLL irradiation against cytotoxicity induced by Bothrops jararacussu venom on myoblast C2C12 cells.C2C12 were utilized as a model target and were incubated with B. jararacussu venom (12.5 μg/mL and immediately irradiated with LLL at wavelength of red 685 nm or infrared 830 nm with energy density of 2.0, 4.6 and 7.0 J/cm2. Effects of LLL on cellular responses of venom-induced cytotoxicity were examined, including cell viability, measurement of cell damage and intra and extracellular ATP levels, expression of myogenic regulatory factors, as well as cellular differentiation.In non-irradiated cells, the venom caused a decrease in cell viability and a massive release of LDH and CK levels indicating myonecrosis. Infrared and red laser at all energy densities were able to considerably decrease venom-induced cytotoxicity. Laser irradiation induced myoblasts to differentiate into myotubes and this effect was accompanied by up regulation of MyoD and specially myogenin. Moreover, LLL was able to reduce the extracellular while increased the intracellular ATP content after venom exposure. In addition, no difference in the intensity of cytotoxicity was shown by non-irradiated and irradiated venom.LLL irradiation caused a protective effect on C2C12 cells against the cytotoxicity caused by B. jararacussu venom and promotes differentiation of these cells by up regulation of myogenic factors. A modulatory effect of ATP synthesis may

  2. Sphingosine kinase/sphingosine 1-phosphate axis: a new player for insulin-like growth factor-1-induced myoblast differentiation

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    Bernacchioni Caterina

    2012-07-01

    Full Text Available Abstract Background Insulin-like growth factor-1 (IGF-1 is the most important physiological regulator of skeletal muscle progenitor cells, which are responsible for adult skeletal muscle regeneration. The ability of IGF-1 to affect multiple aspects of skeletal muscle cell biology such as proliferation, differentiation, survival and motility is well recognized, although the molecular mechanisms implicated in its complex biological action are not fully defined. Since sphingosine 1-phosphate (S1P has recently emerged as a key player in skeletal muscle regeneration, we investigated the possible involvement of the sphingosine kinase (SK/S1P receptor axis on the biological effects of IGF-1 in murine myoblasts. Methods RNA interference, chemical inhibition and immunofluorescence approaches were used to assess the role of the SK/S1P axis on the myogenic and mitogenic effects of IGF-1 in C2C12 myoblasts. Results We show that IGF-1 increases SK activity in mouse myoblasts. The effect of the growth factor does not involve transcriptional regulation of SK1 or SK2, since the protein content of both isoforms is not affected; rather, IGF-1 enhances the fraction of the active form of SK. Moreover, transactivation of the S1P2 receptor induced by IGF-1 via SK activation appears to be involved in the myogenic effect of the growth factor. Indeed, the pro-differentiating effect of IGF-1 in myoblasts is impaired when SK activity is pharmacologically inhibited, or SK1 or SK2 are specifically silenced, or the S1P2 receptor is downregulated. Furthermore, in this study we show that IGF-1 transactivates S1P1/S1P3 receptors via SK activation and that this molecular event negatively regulates the mitogenic effect elicited by the growth factor, since the specific silencing of S1P1 or S1P3 receptors increases cell proliferation induced by IGF-1. Conclusions We demonstrate a dual role of the SK/S1P axis in response to myoblast challenge with IGF-1, that likely is important to

  3. The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts.

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    Shy Cian Khor

    Full Text Available Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF and α-tocopherol (ATF in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal expression, myogenic differentiation and myogenic regulatory factors (MRFs expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts.

  4. Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis.

    Science.gov (United States)

    Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

    2013-09-06

    Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Effect of IR Laser on Myoblasts: Prospects of Application for Counteracting Microgravity-Induced Muscle Atrophy

    Science.gov (United States)

    Monici, Monica; Cialdai, Francesca; Romano, Giovanni; Corsetto, Paola Antonia; Rizzo, Angela Maria; Caselli, Anna; Ranaldi, Francesco

    2013-02-01

    Microgravity-induced muscle atrophy is a problem of utmost importance for the impact it may have on the health and performance of astronauts. Therefore, appropriate countermeasures are needed to prevent disuse atrophy and favour muscle recovery. Muscle atrophy is characterized by loss of muscle mass and strength, and a shift in substrate utilization from fat to glucose, that leads to a reduced metabolic efficiency and enhanced fatigability. Laser therapy is already used in physical medicine and rehabilitation to accelerate muscle recovery and in sports medicine to prevent damages produced by metabolic disturbances and inflammatory reactions after heavy exercise. The aim of the research we present was to get insights on possible benefits deriving from the application of an advanced infrared laser system to counteract deficits of muscle energy metabolism and stimulate the recovery of the hypotrophic tissue. The source used was a Multiwave Locked System (MLS) laser, which combines continuous and pulsed emissions at 808 nm and 905 nm, respectively. We studied the effect of MLS treatment on morphology and energy metabolism of C2C12 cells, a widely accepted myoblast model, previously exposed to microgravity conditions modelled by a Random Positioning Machine. The MLS laser treatment was able to restore basal levels of serine/threonine protein phosphatase activity and to counteract cytoskeletal alterations and increase in glycolytic enzymes activity that occurred following the exposure to modelled microgravity. In conclusion, the results provide interesting insights for the application of infrared laser in the treatment of muscle atrophy.

  6. Edible Scaffolds Based on Non-Mammalian Biopolymers for Myoblast Growth

    Directory of Open Access Journals (Sweden)

    Javier Enrione

    2017-12-01

    Full Text Available In vitro meat has recently emerged as a new concept in food biotechnology. Methods to produce in vitro meat generally involve the growth of muscle cells that are cultured on scaffolds using bioreactors. Suitable scaffold design and manufacture are critical to downstream culture and meat production. Most current scaffolds are based on mammalian-derived biomaterials, the use of which is counter to the desire to obviate mammal slaughter in artificial meat production. Consequently, most of the knowledge is related to the design and control of scaffold properties based on these mammalian-sourced materials. To address this, four different scaffold materials were formulated using non-mammalian sources, namely, salmon gelatin, alginate, and additives including gelling agents and plasticizers. The scaffolds were produced using a freeze-drying process, and the physical, mechanical, and biological properties of the scaffolds were evaluated. The most promising scaffolds were produced from salmon gelatin, alginate, agarose, and glycerol, which exhibited relatively large pore sizes (~200 μm diameter and biocompatibility, permitting myoblast cell adhesion (~40% and growth (~24 h duplication time. The biodegradation profiles of the scaffolds were followed, and were observed to be less than 25% after 4 weeks. The scaffolds enabled suitable myogenic response, with high cell proliferation, viability, and adequate cell distribution throughout. This system composed of non-mammalian edible scaffold material and muscle-cells is promising for the production of in vitro meat.

  7. First intron of nestin gene regulates its expression during C2C12 myoblast ifferentiation

    Institute of Scientific and Technical Information of China (English)

    Hua Zhong; Zhigang Jin; Yongfeng Chen; Ting Zhang; Wei Bian; Xing Cui; Naihe Jing

    2008-01-01

    Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Nestin is an intermediate filament protein expressed in neural progenitor cells and in developing skeletal muscle. Nestin has been widely used as a neural progenitor cell marker. It is well established that the specific expression of the nestin gene in neural progenitor cells is conferred by the neural-specific enhancer located in the second intron of the nestin gene. However, the transcriptional mechanism of nestin expression in developing muscle is still unclear. In this study, we identified a muscle cell-specific enhancer in the first intron of mouse nestin gene in mouse myoblast C2C12 cells.We localized the core enhancer activity to the 291-661 region of the first intron, and showed that the two E-boxes in the core enhancer region were important for enhancer activity in differentiating C2C12 cells. We also showed that MyoD protein was involved in the regulation of nestin expression in the myogenic differentiation of C2C12 cells.

  8. Disruption of myoblast alignment by highly motile rhabdomyosarcoma cell in tissue structure.

    Science.gov (United States)

    Li, Menglu; Nagamori, Eiji; Kino-Oka, Masahiro

    2017-02-01

    Rhabdomyosarcoma (RMS) is a highly malignant tumor type of skeletal muscle origin, hallmarked by local invasion. Interaction between invasive tumor cells and normal cells plays a major role in tumor invasion and metastasis. Culturing tumor cells in a three-dimensional (3D) model can translate tumor malignancy relevant cell-cell interaction. To mimic tumor heterogeneity in vitro, a co-culture system consisting of a malignant embryonal rhabdomyosarcoma (ERMS) cell line RD and a normal human skeletal muscle myoblast (HSMM) cell line was established by cell sheet technology. Various ratios of RDs to HSMMs were employed to understand the quantitative effect on intercellular interactions. Disruption of sheet structure was observed in heterogeneous cell sheets having a low ratio of RDs to HSMMs, whereas homogeneous HSMM or RD sheets maintained intact structure. Deeper exploration of dynamic tumor cell behavior inside HSMM sheets revealed that HSMM cell alignment was disrupted by highly motile RDs. This study demonstrated that RMS cells are capable of compromising their surrounding environment through induced decay of HSMMs alignment in a cell-based 3D system. This suggests that muscle disruption might be a major consequence of RMS cell invasion into muscles, which could be a promising target to preventing tumor invasion. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Human myotubes from myoblast cultures undergoing senescence exhibit defects in glucose and lipid metabolism

    DEFF Research Database (Denmark)

    Nehlin, Jan O; Just, Marlene; Rustan, Arild C

    2011-01-01

    Adult stem cells are known to have a finite replication potential. Muscle biopsy-derived human satellite cells (SCs) were grown at different passages and differentiated to human myotubes in culture to analyze the functional state of various carbohydrate and lipid metabolic pathways. As the prolif......Adult stem cells are known to have a finite replication potential. Muscle biopsy-derived human satellite cells (SCs) were grown at different passages and differentiated to human myotubes in culture to analyze the functional state of various carbohydrate and lipid metabolic pathways...... number and could be explained by reduced incorporation into diacyl- and triacylglycerols. The levels of long-chain acyl-CoA esters decreased with increased passage number. Late-passage, non-proliferating, myoblast cultures showed strong senescence-associated β-galactosidase activity indicating...... that the observed metabolic defects accompany the induction of a senescent state. The main function of SCs is regeneration and skeletal muscle-build up. Thus, the metabolic defects observed during aging of SC-derived myotubes could have a role in sarcopenia, the gradual age-related loss of muscle mass and strength....

  10. Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation

    Directory of Open Access Journals (Sweden)

    Elena Barbieri

    2011-01-01

    Full Text Available This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1 , NRF1 , and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD, a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1 involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

  11. Signals of Ezh2, Src, and Akt Involve in Myostatin-Pax7 Pathways Regulating the Myogenic Fate Determination during the Sheep Myoblast Proliferation and Differentiation

    Science.gov (United States)

    Li, Li; Liu, Ruizao; Zhang, Li; Zhao, Fuping; Lu, Jian; Zhang, Xiaoning; Du, Lixin

    2015-01-01

    Myostatin and Pax7 have been well documented individually, however, the mechanism by which Myostatin regulates Pax7 is seldom reported. Here, based on muscle transcriptome analysis in Texel (Myostatin mutant) and Ujumqin (wild type) sheep across the five fetal stages, we constructed and examined the Myostatin-Pax7 pathways in muscle. Then we validated the signals by RNAi in the proliferating and differentiating sheep myoblasts in vitro at mRNA, protein, and cell morphological levels. We reveal that Myostatin signals to Pax7 at least through Ezh2, Src, and Akt during the sheep myoblast proliferation and differentiation. Other signals such as p38MAPK, mTOR, Erk1/2, Wnt, Bmp2, Smad, Tgfb1, and p21 are most probably involved in the Myostatin-affected myogenic events. Myostatin knockdown significantly reduces the counts of nucleus and myotube, but not the fusion index of myoblasts during cell differentiation. In addition, findings also indicate that Myostatin is required for normal myogenic differentiation of the sheep myoblasts, which is different from the C2C12 myoblasts. We expand the regulatory network of Myostatin-Pax7 pathways and first illustrate that Myostatin as a global regulator participates in the epigenetic events involved in myogenesis, which contributes to understand the molecular mechanism of Myostatin in regulation of myogenesis. PMID:25811841

  12. Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Muratore, Massimo, E-mail: M.Muratore@ed.ac.uk [Institute of Integrated Micro and Nano System, School of Engineering, The University of Edinburgh, Edinburgh EH9 3JF (United Kingdom); Mitchell, Steve [Institute of Molecular Plant Science, School of Biological Science, The University of Edinburgh, Edinburgh EH9 3JF (United Kingdom); Waterfall, Martin [Institute of Immunology and Infection Research, School of Biological Science, The University of Edinburgh, Edinburgh EH9 3JT (United Kingdom)

    2013-09-06

    Highlights: •Dielectrophoretic separation/sorting of multipotent cells. •Plasma membrane microvilli structure of C2C12 and fibroblasts by SEM microscopy. •Cell cycle determination by Ki-67 in DEP-sorted cells. •Plasma membrane differences responsible for changes in membrane capacitance. -- Abstract: Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy.

  13. Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts

    International Nuclear Information System (INIS)

    Watanabe, Tomonobu M.; Higuchi, Sayaka; Kawauchi, Keiko; Tsukasaki, Yoshikazu; Ichimura, Taro; Fujita, Hideaki

    2012-01-01

    Highlights: ► Change in the epigenetic landscape during myogenesis was optically investigated. ► Mobility of nuclear proteins was used to state the epigenetic status of the cell. ► Mobility of nuclear proteins decreased as myogenesis progressed in C2C12. ► Differentiation state diagram was developed using parameters obtained. -- Abstract: Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extent of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage during myogenesis using the state diagram developed with the parameters obtained in this study.

  14. Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis

    International Nuclear Information System (INIS)

    Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

    2013-01-01

    Highlights: •Dielectrophoretic separation/sorting of multipotent cells. •Plasma membrane microvilli structure of C2C12 and fibroblasts by SEM microscopy. •Cell cycle determination by Ki-67 in DEP-sorted cells. •Plasma membrane differences responsible for changes in membrane capacitance. -- Abstract: Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy

  15. Anticancer activity of biologically synthesized silver and gold nanoparticles on mouse myoblast cancer cells and their toxicity against embryonic zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Ramachandran, Rajan [Centre for Advanced Studies in Botany, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu (India); Krishnaraj, Chandran [Department of Food Science & Technology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); M/s. Eureka Forbes Ltd, R & D Centre, Kudlu, Bangalore (India); Sivakumar, Allur Subramaniyan [Department of Animal Biotechnology, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Prasannakumar, Palaniappan [Advanced Biomedical Imaging Center, Department of Electronic Engineering, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Abhay Kumar, V.K. [M/s. Eureka Forbes Ltd, R & D Centre, Kudlu, Bangalore (India); Shim, Kwan Seob [Department of Animal Biotechnology, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Song, Chul-Gyu [Advanced Biomedical Imaging Center, Department of Electronic Engineering, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Yun, Soon-Il, E-mail: siyun@jbnu.ac.kr [Department of Food Science & Technology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

    2017-04-01

    The aim of this study was to evaluate the anticancer activity of bioinspired silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) against mouse myoblast cancer cells (C{sub 2}C{sub 12}). Both AgNPs and AuNPs were biologically synthesized using Spinacia oleracea Linn., aqueous leaves extract. UV–Vis. spectrophotometer, high resolution-transmission electron microscopy (HR-TEM), field emission-scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) studies supported the successful synthesis of AgNPs and AuNPs. Both these NPs have shown cytotoxicity against C{sub 2}C{sub 12} cells even at very low concentration (5 μg/mL). Acridine orange/Ethidium bromide (AO/EB) dual staining confirmed the apoptotic morphological features. The levels of caspase enzymes (caspase-3 and caspase-7) were significantly up-regulated in NPs treated myoblast cells than the plant extract. Furthermore, in zebrafish embryo toxicity study, AgNPs showed 100% mortality at 3 μg/mL concentration while AuNPs exhibited the same at much higher concentration (300 mg/mL). Taken together, these results provide a preliminary guidance for the development of biomaterials based drugs to fight against the fatal diseases for example cancer. - Highlights: • Anticancer activity was done for the first time against mouse myoblast cells. • AgNPs showed 100% growth inhibition against C{sub 2}C{sub 12} cells at 20 μg/mL concentration. • AO/EB dual staining and caspase assays confirmed the apoptotic features. • Nanoparticles treated embryos showed yolk sac edema and tail malformation. • AgNPs were found to be more toxic to embryonic zebrafishes than the AuNPs.

  16. Anticancer activity of biologically synthesized silver and gold nanoparticles on mouse myoblast cancer cells and their toxicity against embryonic zebrafish

    International Nuclear Information System (INIS)

    Ramachandran, Rajan; Krishnaraj, Chandran; Sivakumar, Allur Subramaniyan; Prasannakumar, Palaniappan; Abhay Kumar, V.K.; Shim, Kwan Seob; Song, Chul-Gyu; Yun, Soon-Il

    2017-01-01

    The aim of this study was to evaluate the anticancer activity of bioinspired silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) against mouse myoblast cancer cells (C 2 C 12 ). Both AgNPs and AuNPs were biologically synthesized using Spinacia oleracea Linn., aqueous leaves extract. UV–Vis. spectrophotometer, high resolution-transmission electron microscopy (HR-TEM), field emission-scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) studies supported the successful synthesis of AgNPs and AuNPs. Both these NPs have shown cytotoxicity against C 2 C 12 cells even at very low concentration (5 μg/mL). Acridine orange/Ethidium bromide (AO/EB) dual staining confirmed the apoptotic morphological features. The levels of caspase enzymes (caspase-3 and caspase-7) were significantly up-regulated in NPs treated myoblast cells than the plant extract. Furthermore, in zebrafish embryo toxicity study, AgNPs showed 100% mortality at 3 μg/mL concentration while AuNPs exhibited the same at much higher concentration (300 mg/mL). Taken together, these results provide a preliminary guidance for the development of biomaterials based drugs to fight against the fatal diseases for example cancer. - Highlights: • Anticancer activity was done for the first time against mouse myoblast cells. • AgNPs showed 100% growth inhibition against C 2 C 12 cells at 20 μg/mL concentration. • AO/EB dual staining and caspase assays confirmed the apoptotic features. • Nanoparticles treated embryos showed yolk sac edema and tail malformation. • AgNPs were found to be more toxic to embryonic zebrafishes than the AuNPs.

  17. A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

    Directory of Open Access Journals (Sweden)

    Knight James DR

    2012-03-01

    Full Text Available Abstract Background The p38α mitogen-activated protein kinase (MAPK is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear. Methods To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms. Results Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation. Conclusion Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.

  18. Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Siemionow, M; Cwykiel, J; Heydemann, A; Garcia-Martinez, J; Siemionow, K; Szilagyi, E

    2018-04-01

    Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin-deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 10 6 ) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

  19. The effect of low intensity shockwave treatment (Li-SWT) on human myoblasts and mouse skeletal muscle

    DEFF Research Database (Denmark)

    Hansen, Lise K; Schrøder, Henrik D; Lund, Lars

    2017-01-01

    , proliferation and differentiation were tested. Cardiotoxin induced injury was created in tibialis anterior muscles of 28 mice, and two days later, the lesions were treated with 500 impulses of Li-SWT on one of the legs. The treatment was repeated every third day of the period and ended on day 14 after...... incremental effect on expression of pro-angiogenic genes. However, we found no changes in the number of PAX7 positive cells or blood vessel density in Li-SWT treated and control muscle. Furthermore, Li-SWT in the selected doses did not decrease survival, proliferation or differentiation of myoblasts in vitro....

  20. Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken

    Science.gov (United States)

    Lin, Shumao; Luo, Wen; Ye, Yaqiong; Bekele, Endashaw J.; Nie, Qinghua; Li, Yugu; Zhang, Xiquan

    2017-01-01

    The sex-linked dwarf chicken is caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, lower body weight, smaller muscle fiber diameter and fewer muscle fiber number. However, the precise regulatory pathways that lead to the inhibition of skeletal muscle growth in dwarf chickens still remain unclear. Here we found a let-7b mediated pathway might play important role in the regulation of dwarf chicken skeletal muscle growth. Let-7b has higher expression in the skeletal muscle of dwarf chicken than in normal chicken, and the expression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which is a translational activator of IGF2, showed opposite expression trend to let-7b. In vitro cellular assays validated that let-7b directly inhibits IGF2BP3 expression through binding to its 3′UTR region, and the protein level but not mRNA level of IGF2 would be reduced in let-7b overexpressed chicken myoblast. Let-7b can inhibit cell proliferation and induce cell cycle arrest in chicken myoblast through let-7b-IGF2BP3-IGF2 signaling pathway. Additionally, let-7b can also regulate skeletal muscle growth through let-7b-GHR-GHR downstream genes pathway, but this pathway is non-existent in dwarf chicken because of the deletion mutation of GHR 3′UTR. Notably, as the loss binding site of GHR for let-7b, let-7b has enhanced its binding and inhibition on IGF2BP3 in dwarf myoblast, suggesting that the miRNA can balance its inhibiting effect through dynamic regulate its binding to target genes. Collectively, these results not only indicate that let-7b can inhibit skeletal muscle growth through let-7b-IGF2BP3-IGF2 signaling pathway, but also show that let-7b regulates myoblast proliferation by inhibiting IGF2BP3 expression in dwarf and normal chickens. PMID:28736533

  1. The Use of Platelet-Rich and Platelet-Poor Plasma to Enhance Differentiation of Skeletal Myoblasts: Implications for the Use of Autologous Blood Products for Muscle Regeneration.

    Science.gov (United States)

    Miroshnychenko, Olga; Chang, Wen-Teh; Dragoo, Jason L

    2017-03-01

    Platelet-rich plasma (PRP) has been used to augment tissue repair and regeneration after musculoskeletal injury. However, there is increasing clinical evidence that PRP does not show a consistent clinical effect. Purpose/Hypothesis: This study aimed to compare the effects of the following non-neutrophil-containing (leukocyte-poor) plasma fractions on human skeletal muscle myoblast (HSMM) differentiation: (1) PRP, (2) modified PRP (Mod-PRP), in which transforming growth factor β1 (TGF-β1) and myostatin (MSTN) were depleted, and (3) platelet-poor plasma (PPP). The hypothesis was that leukocyte-poor PRP would lead to myoblast proliferation (not differentiation), whereas certain modifications of PRP preparations would increase myoblast differentiation, which is necessary for skeletal muscle regeneration. Controlled laboratory study. Blood from 7 human donors was individually processed to simultaneously create leukocyte-poor fractions: PRP, Mod-PRP, PPP, and secondarily spun PRP and Mod-PRP (PRP ss and Mod-PRP ss , respectively). Mod-PRP was produced by removing TGF-β1 and MSTN from PRP using antibodies attached to sterile beads, while a second-stage centrifugal spin of PRP was performed to remove platelets. The biologics were individually added to cell culture groups. Analysis for induction into myoblast differentiation pathways included Western blot analysis, reverse-transcription polymerase chain reaction, and immunohistochemistry, as well as confocal microscopy to assess polynucleated myotubule formation. HSMMs cultured with PRP showed an increase in proliferation but no evidence of differentiation. Western blot analysis confirmed that MSTN and TGF-β1 could be decreased in Mod-PRP using antibody-coated beads, but this modification mildly improved myoblast differentiation. However, cell culture with PPP, PRP ss , and Mod-PRP ss led to a decreased proliferation rate but a significant induction of myoblast differentiation verified by increased multinucleated

  2. Activation of estrogen response elements is mediated both via estrogen and muscle contractions in rat skeletal muscle myotubes

    DEFF Research Database (Denmark)

    Wiik, A.; Hellsten, Ylva; Berthelson, P.

    2009-01-01

    is ER independent. The muscle contraction-induced transactivation of ERE and increase in ERbeta mRNA were instead found to be MAP kinase (MAPK) dependent. This study demonstrates for the first time that muscle contractions have a similar functional effect as estrogen in skeletal muscle myotubes, causing......The aim of the present study was to investigate the activation of estrogen response elements (EREs) by estrogen and muscle contractions in rat myotubes in culture and to assess whether the activation is dependent on the estrogen receptors (ERs). In addition, the effect of estrogen and contraction...... on the mRNA levels of ERalpha and ERbeta was studied to determine the functional consequence of the transactivation. Myoblasts were isolated from rat skeletal muscle and transfected with a vector consisting of sequences of EREs coupled to the gene for luciferase. The transfected myoblasts were...

  3. α-Syntrophin is involved in the survival signaling pathway in myoblasts under menadione-induced oxidative stress.

    Science.gov (United States)

    Lim, Jeong-A; Choi, Su Jin; Moon, Jae Yun; Kim, Hye Sun

    2016-05-15

    Dystrophin-deficient muscle is known to be more vulnerable to oxidative stress, but not much is known about the signaling pathway(s) responsible for this phenomenon. α-Syntrophin, a component of the dystrophin-glycoprotein complex, can function as a scaffold protein because of its multiple protein interaction domains. In this study, we investigated the role of α-syntrophin in C2 myoblasts under menadione-induced oxidative stress. We found that the protein level of α-syntrophin was elevated when cells were exposed to menadione. To investigate the function of α-syntrophin during oxidative stress, we established α-syntrophin-overexpressing and knockdown cell lines. The α-syntrophin-overexpressing cells were resistant to the menadione-induced oxidative stress. In addition, survival signalings such as protein kinase B (Akt) phosphorylation and the Bcl-2/BAX ratio were increased in these cells. On the other hand, apoptotic signals such as cleavage of caspase-3 and poly ADP ribose polymerase (PARP) were increased in the α-syntrophin knockdown cells. Furthermore, Ca(2+)influx, which is known to increase when cells are exposed to oxidative stress, decreased in the α-syntrophin-overexpressing cells, but increased in the knockdown cells. These results suggest that α-syntrophin plays a pivotal role in the survival pathway triggered by menadione-induced oxidative stress in cultured myoblasts. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Wnt/β-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

    International Nuclear Information System (INIS)

    Zhang, Long; Shi, Songting; Zhang, Juan; Zhou, Fangfang; Dijke, Peter ten

    2012-01-01

    Highlights: ► Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. ► Wnt3a induces Id3 expression via canonical Wnt/β-catenin pathway. ► Wnt3a-induced Id3 expression does not depend on BMP signaling activation. ► Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a β-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/β-catenin induced gene in myoblast cell fate determination.

  5. Degree of Suppression of Mouse Myoblast Cell Line C₂C12 Differentiation Varies According to Chondroitin Sulfate Subtype.

    Science.gov (United States)

    Warita, Katsuhiko; Oshima, Nana; Takeda-Okuda, Naoko; Tamura, Jun-Ichi; Hosaka, Yoshinao Z

    2016-10-21

    Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C₂C 12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion.

  6. Modulation of Protein Quality Control and Proteasome to Autophagy Switch in Immortalized Myoblasts from Duchenne Muscular Dystrophy Patients

    Directory of Open Access Journals (Sweden)

    Marion Wattin

    2018-01-01

    Full Text Available The maintenance of proteome integrity is of primary importance in post-mitotic tissues such as muscle cells; thus, protein quality control mechanisms must be carefully regulated to ensure their optimal efficiency, a failure of these processes being associated with various muscular disorders. Duchenne muscular dystrophy (DMD is one of the most common and severe forms of muscular dystrophies and is caused by mutations in the dystrophin gene. Protein quality control modulations have been diversely observed in degenerating muscles of patients suffering from DMD or in animal models of the disease. In this study, we investigated whether modulations of protein quality control mechanisms already pre-exist in undifferentiated myoblasts originating from DMD patients. We report for the first time that the absence of dystrophin in human myoblasts is associated with protein aggregation stress characterized by an increase of protein aggregates. This stress is combined with BAG1 to BAG3 switch, NFκB activation and up-regulation of BAG3/HSPB8 complexes that ensure preferential routing of misfolded/aggregated proteins to autophagy rather than to deficient 26S proteasome. In this context, restoration of pre-existing alterations of protein quality control processes might represent an alternative strategy for DMD therapies.

  7. Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model.

    Science.gov (United States)

    Choi, In Young; Lim, HoTae; Estrellas, Kenneth; Mula, Jyothi; Cohen, Tatiana V; Zhang, Yuanfan; Donnelly, Christopher J; Richard, Jean-Philippe; Kim, Yong Jun; Kim, Hyesoo; Kazuki, Yasuhiro; Oshimura, Mitsuo; Li, Hongmei Lisa; Hotta, Akitsu; Rothstein, Jeffrey; Maragakis, Nicholas; Wagner, Kathryn R; Lee, Gabsang

    2016-06-07

    Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model

    Directory of Open Access Journals (Sweden)

    In Young Choi

    2016-06-01

    Full Text Available Duchenne muscular dystrophy (DMD remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs. Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our “chemical-compound-based” strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological “dual-SMAD” inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form “rescued” multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human “DMD-in-a-dish” model using hiPSC-based disease modeling.

  9. MiRNA-199a-3p Regulates C2C12 Myoblast Differentiation through IGF-1/AKT/mTOR Signal Pathway

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    Long Jia

    2013-12-01

    Full Text Available MicroRNAs constitute a class of ~22-nucleotide non-coding RNAs. They modulate gene expression by associating with the 3' untranslated regions (3' UTRs of messenger RNAs (mRNAs. Although multiple miRNAs are known to be regulated during myoblast differentiation, their individual roles in muscle development are still not fully understood. In this study, we showed that miR-199a-3p was highly expressed in skeletal muscle and was induced during C2C12 myoblasts differentiation. We also identified and confirmed several genes of the IGF-1/AKT/mTOR signal pathway, including IGF-1, mTOR, and RPS6KA6, as important cellular targets of miR-199a-3p in myoblasts. Overexpression of miR-199a-3p partially blocked C2C12 myoblast differentiation and the activation of AKT/mTOR signal pathway, while interference of miR-199a-3p by antisense oligonucleotides promoted C2C12 differentiation and myotube hypertrophy. Thus, our studies have established miR-199a-3p as a potential regulator of myogenesis through the suppression of IGF-1/AKT/mTOR signal pathway.

  10. Gallic acid prevents isoproterenol-induced cardiac hypertrophy and fibrosis through regulation of JNK2 signaling and Smad3 binding activity

    Science.gov (United States)

    Ryu, Yuhee; Jin, Li; Kee, Hae Jin; Piao, Zhe Hao; Cho, Jae Yeong; Kim, Gwi Ran; Choi, Sin Young; Lin, Ming Quan; Jeong, Myung Ho

    2016-01-01

    Gallic acid, a type of phenolic acid, has been shown to have beneficial effects in inflammation, vascular calcification, and metabolic diseases. The present study was aimed at determining the effect and regulatory mechanism of gallic acid in cardiac hypertrophy and fibrosis. Cardiac hypertrophy was induced by isoproterenol (ISP) in mice and primary neonatal cardiomyocytes. Gallic acid pretreatment attenuated concentric cardiac hypertrophy. It downregulated the expression of atrial natriuretic peptide, brain natriuretic peptide, and beta-myosin heavy chain in vivo and in vitro. Moreover, it prevented interstitial collagen deposition and expression of fibrosis-associated genes. Upregulation of collagen type I by Smad3 overexpression was observed in cardiac myoblast H9c2 cells but not in cardiac fibroblasts. Gallic acid reduced the DNA binding activity of phosphorylated Smad3 in Smad binding sites of collagen type I promoter in rat cardiac fibroblasts. Furthermore, it decreased the ISP-induced phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) protein in mice. JNK2 overexpression reduced collagen type I and Smad3 expression as well as GATA4 expression in H9c2 cells and cardiac fibroblasts. Gallic acid might be a novel therapeutic agent for the prevention of cardiac hypertrophy and fibrosis by regulating the JNK2 and Smad3 signaling pathway. PMID:27703224

  11. Anticancer activity of synthetic bis(indolyl)methane-ortho-biaryls against human cervical cancer (HeLa) cells.

    Science.gov (United States)

    Jamsheena, Vellekkatt; Shilpa, Ganesan; Saranya, Jayaram; Harry, Nissy Ann; Lankalapalli, Ravi Shankar; Priya, Sulochana

    2016-03-05

    Bis(indolyl)methane appended biaryls were designed, synthesized and evaluated in human cervical cancer cell lines (HeLa) for their anticancer activities and compared against normal rat cardiac myoblasts (H9C2) cells. Compounds 1-12 were synthesized, with variations in one of the phenyl unit, in a single step by condensation of biaryl-2-carbaldehydes with indole in the presence of para-toluenesulfonic acid. Compound 1 exhibited a GI50 value of 11.00 ± 0.707 μM and the derivatives, compounds 4 and 11 showed a GI50 value of 8.33 ± 0.416 μM and 9.13 ± 0.177 μM respectively in HeLa cells and was found to be non-toxic to H9C2 cells up to 20 μM. Furthermore, compounds 1, 4 and 11 induced caspase dependent cellular apoptosis in a concentration-dependent manner, reduced mitochondrial membrane potential, inhibited the cell migration and downregulated the production of MMP-2 and MMP-9 in HeLa cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Patterning Muscles Using Organizers: Larval Muscle Templates and Adult Myoblasts Actively Interact to Pattern the Dorsal Longitudinal Flight Muscles of Drosophila

    Science.gov (United States)

    Roy, Sudipto; VijayRaghavan, K.

    1998-01-01

    Pattern formation in muscle development is often mediated by special cells called muscle organizers. During metamorphosis in Drosophila, a set of larval muscles function as organizers and provide scaffolding for the development of the dorsal longitudinal flight muscles. These organizers undergo defined morphological changes and dramatically split into templates as adult fibers differentiate during pupation. We have investigated the cellular mechanisms involved in the use of larval fibers as templates. Using molecular markers that label myoblasts and the larval muscles themselves, we show that splitting of the larval muscles is concomitant with invasion by imaginal myoblasts and the onset of differentiation. We show that the Erect wing protein, an early marker of muscle differentiation, is not only expressed in myoblasts just before and after fusion, but also in remnant larval nuclei during muscle differentiation. We also show that interaction between imaginal myoblasts and larval muscles is necessary for transformation of the larval fibers. In the absence of imaginal myoblasts, the earliest steps in metamorphosis, such as the escape of larval muscles from histolysis and changes in their innervation, are normal. However, subsequent events, such as the splitting of these muscles, fail to progress. Finally, we show that in a mutant combination, null for Erect wing function in the mesoderm, the splitting of the larval muscles is aborted. These studies provide a genetic and molecular handle for the understanding of mechanisms underlying the use of muscle organizers in muscle patterning. Since the use of such organizers is a common theme in myogenesis in several organisms, it is likely that many of the processes that we describe are conserved. PMID:9606206

  13. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    International Nuclear Information System (INIS)

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    2013-01-01

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP 3 /calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation

  14. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    Energy Technology Data Exchange (ETDEWEB)

    Zuloaga, R. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Fuentes, E.N.; Molina, A. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile); Valdés, J.A., E-mail: jvaldes@unab.cl [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile)

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  15. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo.

    Directory of Open Access Journals (Sweden)

    Sebastian Frese

    Full Text Available Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human

  16. Perturbations of NAD+ salvage systems impact mitochondrial function and energy homeostasis in mouse myoblasts and intact skeletal muscle

    DEFF Research Database (Denmark)

    Andersen, Marianne Agerholm; Dall, Morten; Jensen, Benjamin Anderschou Holbech

    2018-01-01

    Nicotinamide adenine dinucleotide (NAD+) can be synthesized by nicotinamide phosphoribosyltransferase (NAMPT). We aimed to determine the role of NAMPT for maintaining NAD+ levels, mitochondrial function, and metabolic homeostasis in skeletal muscle cells. We generated stable Nampt knockdown (sh......Nampt KD) C2C12 cells using a shRNA lentiviral approach. Moreover, we applied gene electrotransfer to express cre recombinase in tibialis anterior muscle of floxed Nampt mice. In shNampt KD C2C12 myoblasts, Nampt and NAD+ levels were reduced by 70% and 50%, respectively, and maximal respiratory capacity...... was reduced by 25%. Moreover, anaerobic glycolytic flux increased by 55% and 2-deoxyglucose uptake increased by 25% in shNampt KD cells. Treatment with the NAD+ precursor nicotinamide riboside restored NAD+ levels in shNampt cells and increased maximal respiratory capacity by 18% and 32% in control and sh...

  17. Skeletal Myoblast Cell Sheet Implantation Ameliorates Both Systolic and Diastolic Cardiac Performance in Canine Dilated Cardiomyopathy Model.

    Science.gov (United States)

    Shirasaka, Tomonori; Miyagawa, Shigeru; Fukushima, Satsuki; Kawaguchi, Naomasa; Nakatani, Satoshi; Daimon, Takashi; Okita, Yutaka; Sawa, Yoshiki

    2016-02-01

    Improving both systolic and diastolic function may be the most important factor in treating heart failure. In this study, we hypothesized that cell-sheet transplantation could improve these function in the damaged heart. We generated a dilated cardiomyopathy model in beagles by continuous ventricle pacing at 240 beats per minute. After 4 weeks, the beagles underwent skeletal myoblast cell sheet transplantation (SMCST) or a sham operation, and rapid ventricle pacing continued for an additional 4 weeks. Six of the e8 beagles treated by SMCST were still alive 4 weeks after the procedure. We evaluated SMCST's cardiotherapeutic effects by comparing beagles treated by SMCST with beagles that underwent a sham operation (control, n = 5). Diastolic function, as well as systolic function improved significantly in the SMCST group as compared with the sham group (control vs SMCST group, median [interquartile range]: E/E', 16 [0.9] vs 11 [1.0]; P dilated cardiomyopathy heart.

  18. Enhanced contractile force generation by artificial skeletal muscle tissues using IGF-I gene-engineered myoblast cells.

    Science.gov (United States)

    Sato, Masanori; Ito, Akira; Kawabe, Yoshinori; Nagamori, Eiji; Kamihira, Masamichi

    2011-09-01

    The aim of this study was to investigate whether insulin-like growth factor (IGF)-I gene delivery to myoblast cells promotes the contractile force generated by hydrogel-based tissue-engineered skeletal muscles in vitro. Two retroviral vectors allowing doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into mouse myoblast C2C12 cells to evaluate the effects of IGF-I gene expression on these cells. IGF-I gene expression stimulated the proliferation of C2C12 cells, and a significant increase in the growth rate was observed for IGF-I-transduced C2C12 cells with Dox addition, designated C2C12/IGF (Dox+) cells. Quantitative morphometric analyses showed that the myotubes induced from C2C12/IGF (Dox+) cells had a larger area and a greater width than control myotubes induced from normal C2C12 cells. Artificial skeletal muscle tissues were prepared from the respective cells using hydrogels composed of type I collagen and Matrigel. Western blot analyses revealed that the C2C12/IGF (Dox+) tissue constructs showed activation of a skeletal muscle hypertrophy marker (Akt) and enhanced expression of muscle-specific markers (myogenin, myosin heavy chain and tropomyosin). Moreover, the creatine kinase activity was increased in the C2C12/IGF (Dox+) tissue constructs. The C2C12/IGF (Dox+) tissue constructs contracted in response to electrical pulses, and generated a significantly higher physical force than the control C2C12 tissue constructs. These findings indicate that IGF-I gene transfer has the potential to yield functional skeletal muscle substitutes that are capable of in vivo restoration of the load-bearing function of injured muscle or acting as in vitro electrically-controlled bio-actuators. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Functional analysis of SH3 domain containing ring finger 2 during the myogenic differentiation of quail myoblast cells

    Directory of Open Access Journals (Sweden)

    Si Won Kim

    2017-08-01

    Full Text Available Objective Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. Methods Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2 gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7 cells during muscle differentiation. Results Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Conclusion We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth.

  20. Detection of Pancreatic Cancer-Induced Cachexia Using a Fluorescent Myoblast Reporter System and Analysis of Metabolite Abundance.

    Science.gov (United States)

    Winnard, Paul T; Bharti, Santosh K; Penet, Marie-France; Marik, Radharani; Mironchik, Yelena; Wildes, Flonne; Maitra, Anirban; Bhujwalla, Zaver M

    2016-03-15

    The dire effects of cancer-induced cachexia undermine treatment and contribute to decreased survival rates. Therapeutic options for this syndrome are limited, and therefore efforts to identify signs of precachexia in cancer patients are necessary for early intervention. The applications of molecular and functional imaging that would enable a whole-body "holistic" approach to this problem may lead to new insights and advances for diagnosis and treatment of this syndrome. Here we have developed a myoblast optical reporter system with the purpose of identifying early cachectic events. We generated a myoblast cell line expressing a dual tdTomato:GFP construct that was grafted onto the muscle of mice-bearing human pancreatic cancer xenografts to provide noninvasive live imaging of events associated with cancer-induced cachexia (i.e., weight loss). Real-time optical imaging detected a strong tdTomato fluorescent signal from skeletal muscle grafts in mice with weight losses of only 1.2% to 2.7% and tumor burdens of only approximately 79 to 170 mm(3). Weight loss in cachectic animals was also associated with a depletion of lipid, cholesterol, valine, and alanine levels, which may provide informative biomarkers of cachexia. Taken together, our findings demonstrate the utility of a reporter system that is capable of tracking tumor-induced weight loss, an early marker of cachexia. Future studies incorporating resected tissue from human pancreatic ductal adenocarcinoma into a reporter-carrying mouse may be able to provide a risk assessment of cachexia, with possible implications for therapeutic development. ©2015 American Association for Cancer Research.

  1. NEU3 sialidase strictly modulates GM3 levels in skeletal myoblasts C2C12 thus favoring their differentiation and protecting them from apoptosis.

    Science.gov (United States)

    Anastasia, Luigi; Papini, Nadia; Colazzo, Francesca; Palazzolo, Giacomo; Tringali, Cristina; Dileo, Loredana; Piccoli, Marco; Conforti, Erika; Sitzia, Clementina; Monti, Eugenio; Sampaolesi, Maurilio; Tettamanti, Guido; Venerando, Bruno

    2008-12-26

    Membrane-bound sialidase NEU3, often referred to as the "ganglioside sialidase," has a critical regulatory function on the sialoglycosphingolipid pattern of the cell membrane, with an anti-apoptotic function, especially in cancer cells. Although other sialidases have been shown to be involved in skeletal muscle differentiation, the role of NEU3 had yet to be disclosed. Herein we report that NEU3 plays a key role in skeletal muscle differentiation by strictly modulating the ganglioside content of adjacent cells, with special regard to GM3. Induced down-regulation of NEU3 in murine C2C12 myoblasts, even when partial, totally inhibits their capability to differentiate by increasing the GM3 level above a critical point, which causes epidermal growth factor receptor inhibition (and ultimately its down-regulation) and an higher responsiveness of myoblasts to the apoptotic stimuli.

  2. Lipopolysaccharide inhibits myogenic differentiation of C2C12 myoblasts through the Toll-like receptor 4-nuclear factor-κB signaling pathway and myoblast-derived tumor necrosis factor-α.

    Directory of Open Access Journals (Sweden)

    Yuko Ono

    Full Text Available Circulating lipopolysaccharide (LPS concentrations are often elevated in patients with sepsis or with various endogenous diseases that are associated with metabolic endotoxemia. Involuntary loss of skeletal muscle, termed muscle wasting, is commonly observed in these conditions, suggesting that circulating LPS might play an essential role in its development. Although impairment of muscle regeneration is an important determinant of skeletal muscle wasting, it is unclear whether LPS affects this process and, if so, by what mechanism. Here, we used the C2C12 myoblast cell line to investigate the effects of LPS on myogenesis.C2C12 myoblasts were grown to 80% confluence and induced to differentiate in the absence or presence of LPS (0.1 or 1 μg/mL; TAK-242 (1 μM, a specific inhibitor of Toll-like receptor 4 (TLR4 signaling; and a tumor necrosis factor (TNF-α neutralizing antibody (5 μg/mL. Expression of a skeletal muscle differentiation marker (myosin heavy chain II, two essential myogenic regulatory factors (myogenin and MyoD, and a muscle negative regulatory factor (myostatin was analyzed by western blotting. Nuclear factor-κB (NF-κB DNA-binding activity was measured using an enzyme-linked immunosorbent assay.LPS dose-dependently and significantly decreased the formation of multinucleated myotubes and the expression of myosin heavy chain II, myogenin, and MyoD, and increased NF-κB DNA-binding activity and myostatin expression. The inhibitory effect of LPS on myogenic differentiation was reversible, suggesting that it was not caused by nonspecific toxicity. Both TAK-242 and anti-TNF-α reduced the LPS-induced increase in NF-κB DNA-binding activity, downregulation of myogenic regulatory factors, and upregulation of myostatin, thereby partially rescuing the impairment of myogenesis.Our data suggest that LPS inhibits myogenic differentiation via a TLR4-NF-κB-dependent pathway and an autocrine/paracrine TNF-α-induced pathway. These pathways

  3. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    Energy Technology Data Exchange (ETDEWEB)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany); Renkawitz-Pohl, Renate, E-mail: renkawit@biologie.uni-marburg.de [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany)

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  4. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    International Nuclear Information System (INIS)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev; Renkawitz-Pohl, Renate

    2013-01-01

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity

  5. DNA replication timing is maintained genome-wide in primary human myoblasts independent of D4Z4 contraction in FSH muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Benjamin D Pope

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is linked to contraction of an array of tandem 3.3-kb repeats (D4Z4 at 4q35.2 from 11-100 copies to 1-10 copies. The extent to which D4Z4 contraction at 4q35.2 affects overall 4q35.2 chromatin organization remains unclear. Because DNA replication timing is highly predictive of long-range chromatin interactions, we generated genome-wide replication-timing profiles for FSHD and control myogenic precursor cells. We compared non-immortalized myoblasts from four FSHD patients and three control individuals to each other and to a variety of other human cell types. This study also represents the first genome-wide comparison of replication timing profiles in non-immortalized human cell cultures. Myoblasts from both control and FSHD individuals all shared a myoblast-specific replication profile. In contrast, male and female individuals were readily distinguished by monoallelic differences in replication timing at DXZ4 and other regions across the X chromosome affected by X inactivation. We conclude that replication timing is a robust cell-type specific feature that is unaffected by FSHD-related D4Z4 contraction.

  6. L6E9 Myoblasts Are Deficient of Myostatin and Additional TGF- Members Are Candidates to Developmentally Control Their Fiber Formation

    Directory of Open Access Journals (Sweden)

    Stefania Rossi

    2010-01-01

    Full Text Available This work provides evidence that the robust myoblast differentiation observed in L6E9 cells is causally linked to deficiency of myostatin, which, conversely, has been found to be expressed in C2C12 cells. However, despite the absence of endogenous myostatin, L6E9 myoblasts expressed functional Activin receptors type II (ActRIIs and follistatin as well as the highly related TGF- members Activins and GDF11, suggesting that in this cell line the regulation of fiber size might be under the control of multiple regulators regardless of myostatin. In line with this hypothesis, delivery of a dominant-negative ActRIIb form or the increase of follistatin, as obtained via Trichostatin treatment or stable transfection of a short human follistatin form, enhanced the L6E9 cell differentiation and further increased the size of myotubes, suggesting that L6E9 myoblasts provide a spontaneous myostatin knock-out in vitro model to study TGF- ligands involved in developmental regulation of fiber size.

  7. 4,4'-Dichlorodiphenyltrichloroethane (DDT) and 4,4'-dichlorodiphenyldichloroethylene (DDE) inhibit myogenesis in C2C12 myoblasts.

    Science.gov (United States)

    Kim, Jonggun; Park, Min Young; Kim, Yoo; Yoon, Kyong Sup; Clark, John Marshall; Park, Yeonhwa; Whang, Kwang-Youn

    2017-12-01

    Most countries have banned the use of 4,4'-dichlorodiphenyltrichloroethane (DDT). However, owing to its extremely high lipophilic characteristics, DDT and its metabolite 4,4'-dichlorodiphenyldichloroethylene (DDE) are ubiquitous in the environment and in many types of food. The positive correlation between exposure to insecticides, including DDT and DDE, and weight gain, resulting in impaired energy metabolism in offspring following perinatal DDT and DDE exposure, was previously reported. Therefore the influence of DDT and DDE on myogenesis using C2C12 myoblasts was investigated in this study. DDT and DDE decreased myotube formation dose- and time-dependently. Among myogenic regulatory factors, DDT and DDE mainly decreased MyoD1 and Myf5 expression. DDT and DDE treatment also altered Myostatin expression, phosphorylation of protein kinase B, p70 ribosomal protein S6 kinase, forkhead box O protein 3 and mammalian target of rapamycin, resulting in attenuation of myotube formation. These results may have significant implications for understanding the effects of developmental exposure of DDT and DDE on myogenesis and development of obesity and type 2 diabetes later in life. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  8. Liposome-based vascular endothelial growth factor-165 transfection with skeletal myoblast for treatment of ischaemic limb disease.

    Science.gov (United States)

    Ye, Lei; Haider, Husnain Kh; Esa, Wahidah Bte; Su, Liping; Law, Peter K; Zhang, Wei; Lim, Yeanteng; Poh, Kian Keong; Sim, Eugene K W

    2010-01-01

    The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (VEGF(165)) gene transfer into skeletal myoblasts (SkMs) for treatment of acute hind limb ischaemia in a rabbit model. The feasibility and efficacy of CD liposome mediated gene transfer with rabbit SkMs were characterized using plasmid carrying enhanced green fluorescent protein (pEGFP) and assessed by flow cytometry. After optimization, SkMs were transfected with CD lipoplexes carrying plasmid-VEGF(165) (CD-pVEGF(165)) and transplanted into rabbit ischaemic limb. Animals were randomized to receive intramuscular injection of Medium199 (M199; group 1), non-transfected SkM (group 2) or CD-pVEGF(165) transfected SkM (group 3). Flow cytometry revealed that up to 16% rabbit SkMs were successfully transfected with pEGFP. Based on the optimized transfection condition, transfected rabbit SkM expressed VEGF(165) up to day 18 with peak at day 2. SkMs were observed in all cell-transplanted groups, as visualized with 6-diamidino-2-phenylindole and bromodeoxyuridine. Angiographic blood vessel score revealed increased collateral vessel development in group 3 (39.7 +/- 2.0) compared with group 2 (21.6 +/- 1.1%, P limb and may serve as a safe and new therapeutic modality for the repair of acute ischaemic limb disease.

  9. Acute insulin resistance mediated by advanced glycation endproducts in severely burned rats.

    Science.gov (United States)

    Zhang, Xing; Xu, Jie; Cai, Xiaoqing; Ji, Lele; Li, Jia; Cao, Bing; Li, Jun; Hu, Dahai; Li, Yan; Wang, Haichang; Xiong, Lize; Xiao, Ruiping; Gao, Feng

    2014-06-01

    Hyperglycemia often occurs in severe burns; however, the underlying mechanisms and importance of managing postburn hyperglycemia are not well recognized. This study was designed to investigate the dynamic changes of postburn hyperglycemia and the underlying mechanisms and to evaluate whether early glycemic control is beneficial in severe burns. Prospective, randomized experimental study. Animal research laboratory. Sprague-Dawley rats. Anesthetized rats were subjected to a full-thickness burn injury comprising 40% of the total body surface area and were randomized to receive vehicle, insulin, and a soluble form of receptor for advanced glycation endproducts treatments. An in vitro study was performed on cultured H9C2 cells subjected to vehicle or carboxymethyllysine treatment. We found that blood glucose change presented a distinct pattern with two occurrences of hyperglycemia at 0.5- and 3-hour postburn, respectively. Acute insulin resistance evidenced by impaired insulin signaling and glucose uptake occurred at 3-hour postburn, which was associated with the second hyperglycemia and positively correlated with mortality. Mechanistically, we found that serum carboxymethyllysine, a dominant species of advanced glycation endproducts, increased within 1-hour postburn, preceding the occurrence of insulin resistance. More importantly, treatment of animals with soluble form of receptor for advanced glycation endproducts, blockade of advanced glycation endproducts signaling, alleviated severe burn-induced insulin resistance. In addition, early hyperglycemic control with insulin not only reduced serum carboxymethyllysine but also blunted postburn insulin resistance and reduced mortality. These findings suggest that severe burn-induced insulin resistance is partly at least mediated by serum advanced glycation endproducts and positively correlated with mortality. Early glycemic control with insulin or inhibition of advanced glycation endproducts with soluble form of receptor

  10. Establishment of bipotent progenitor cell clone from rat skeletal muscle.

    Science.gov (United States)

    Murakami, Yousuke; Yada, Erica; Nakano, Shin-ichi; Miyagoe-Suzuki, Yuko; Hosoyama, Tohru; Matsuwaki, Takashi; Yamanouchi, Keitaro; Nishihara, Masugi

    2011-12-01

    The present study describes the isolation, cloning and characterization of adipogenic progenitor cells from rat skeletal muscle. Among the obtained 10 clones, the most highly adipogenic progenitor, 2G11 cells, were further characterized. In addition to their adipogenicity, 2G11 cells retain myogenic potential as revealed by formation of multinucleated myotubes when co-cultured with myoblasts. 2G11 cells were resistant to an inhibitory effect of basic fibroblast growth factor on adipogenesis, while adipogenesis of widely used preadipogenic cell line, 3T3-L1 cells, was suppressed almost completely by the same treatment. In vivo transplantation experiments revealed that 2G11 cells are able to possess both adipogenicity and myogenicity in vivo. These results indicate the presence of bipotent progenitor cells in rat skeletal muscle, and suggest that such cells may contribute to ectopic fat formation in skeletal muscle. © 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

  11. Analysis of microRNA Expression Profiles Induced by Yiqifumai Injection in Rats with Chronic Heart Failure

    Directory of Open Access Journals (Sweden)

    Yu Zhao

    2018-02-01

    Full Text Available Background: Yiqifumai Injection (YQFM is clinically used to treat various cardiovascular diseases including chronic heart failure (CHF. The efficacy of YQFM for treating heart failure has been suggested, but the mechanism of action for pharmacological effects of YQFM is unclear.Methods: Echocardiography detection, left ventricular intubation evaluation, histopathology and immunohistochemical examination were performed in CHF rats to evaluate the cardioprotective effect of YQFM. Rat miRNA microarray and bioinformatics analysis were employed to investigate the differentially expressed microRNAs. In vitro models of AngII-induced hypertrophy and t-BHP induced oxidative stress in H9c2 myocardial cells were used to validate the anti-hypertrophy and anti-apoptosis effects of YQFM. Measurement of cell surface area, ATP content and cell viability, Real-time PCR and Western blot were performed.Results: YQFM significantly improved the cardiac function of CHF rats by increasing left ventricular ejection fraction and fractional shortening, decreasing left ventricular internal diameter and enhancing cardiac output. Seven microRNAs which have a reversible regulation by YQFM treatment were found. Among them, miR-21-3p and miR-542-3p are related to myocardial hypertrophy and cell proliferation, respectively and were further verified by RT-PCR. Target gene network was established and potential related signaling pathways were predicted. YQFM could significantly alleviate AngII induced hypertrophy in cellular model. It also significantly increased cell viabilities and ATP content in t-BHP induced apoptotic cell model. Western blot analysis showed that YQFM could increase the phosphorylation of Akt.Conclusion: Our findings provided scientific evidence to uncover the mechanism of action of YQFM on miRNAs regulation against CHF by miRNA expression profile technology. The results indicated that YQFM has a potential effect on alleviate cardiac hypertrophy and apoptosis

  12. Extracellular creatine regulates creatine transport in rat and human muscle cells.

    OpenAIRE

    Loike, J D; Zalutsky, D L; Kaback, E; Miranda, A F; Silverstein, S C

    1988-01-01

    Muscle cells do not synthesize creatine; they take up exogenous creatine by specific Na+-dependent plasma membrane transporters. We found that extracellular creatine regulates the level of expression of these creatine transporters in L6 rat muscle cells. L6 myoblasts maintained for 24 hr in medium containing 1 mM creatine exhibited 1/3rd of the creatine transport activity of cells maintained for 24 hr in medium without creatine. Down-regulation of creatine transport was partially reversed whe...

  13. Efficient non-viral reprogramming of myoblasts to stemness with a single small molecule to generate cardiac progenitor cells.

    Directory of Open Access Journals (Sweden)

    Zeeshan Pasha

    Full Text Available The current protocols for generation of induced pluripotent stem (iPS cells involve genome integrating viral vectors which may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulation for reprogramming of skeletal myoblasts (SMs using small molecules.SMs from young male Oct3/4-GFP(+ transgenic mouse were treated with DNA methyltransferase (DNMT inhibitor, RG108. Two weeks later, GFP(+ colonies of SM derived iPS cells (SiPS expressing GFP and with morphological similarity of mouse embryonic stem (ESCs were formed and propagated in vitro. SiPS were positive for alkaline phosphatase activity, expressed SSEA1, displayed ES cell specific pluripotency markers and formed teratoma in nude mice. Optimization of culture conditions for embryoid body (EBs formation yielded spontaneously contracting EBs having morphological, molecular, and ultra-structural similarities with cardiomyocytes and expressed early and late cardiac markers. miR profiling showed abrogation of let-7 family and upregulation of ESCs specific miR-290-295 cluster thus indicating that SiPS were similar to ESCs in miR profile. Four weeks after transplantation into the immunocompetent mice model of acute myocardial infarction (n = 12 per group, extensive myogenesis was observed in SiPS transplanted hearts as compared to DMEM controls (n = 6 per group. A significant reduction in fibrosis and improvement in global heart function in the hearts transplanted with SiPS derived cardiac progenitor cells were observed.Reprogramming of SMs by DNMT inhibitor is a simple, reproducible and efficient technique more likely to generate transgene integration-free iPS cells. Cardiac progenitors derived from iPS cells propagated extensively in the infarcted myocardium without tumorgenesis and improved cardiac function.

  14. Serotonin and Histamine Therapy Increases Tetanic Forces of Myoblasts, Reduces Muscle Injury, and Improves Grip Strength Performance of Dmdmdx Mice

    Directory of Open Access Journals (Sweden)

    Volkan Gurel

    2015-11-01

    Full Text Available Duchenne muscular dystrophy (DMD is a recessive X-linked fatal disorder caused by a mutation in the dystrophin gene. Although several therapeutic approaches have been studied, none has led to substantial long-term effects in patients. The aim of this study was to test a serotonin and histamine (S&H combination on human skeletal myoblasts and Dmdmdx mice for its effects on muscle strength and injury. Normal human bioartificial muscles (BAMs were treated, and muscle tetanic forces and muscle injury tests were performed using the MyoForce Analysis System. Dmdmdx mice, the murine model of DMD, were administered serotonin, histamine, or S&H combination twice daily for 6 weeks, and functional performance tests were conducted once a week. The S&H combination treatment caused significant increases in tetanic forces at all time points and concentrations tested as compared to the saline controls. Dose response of the BAMs to the treatment demonstrated a significant increase in force generation at all concentrations compared to the controls after 3 to 4 days of drug treatment. The highest 3 concentrations had a significant effect on lowering contractile-induced injury as measured by a reduction in the release of adenylate kinase. Histamine-only and S&H treatments improved grip strength of Dmdmdx mice, whereas serotonin-only treatment resulted in no significant improvement in muscle strength. The results of this study indicate that S&H therapy might be a promising new strategy for muscular dystrophies and that the mechanism should be further investigated.

  15. Thimerosal-induced apoptosis in mouse C2C12 myoblast cells occurs through suppression of the PI3K/Akt/survivin pathway.

    Directory of Open Access Journals (Sweden)

    Wen-Xue Li

    Full Text Available BACKGROUND: Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells. METHODOLOGY/PRINCIPAL FINDINGS: The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125-500 nM for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Akt(ser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic

  16. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Young Woo [Cell Therapy and Tissue Engineering Center, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Biomedical Research Institute, Lifeliver Co., Ltd., Suwon (Korea, Republic of); Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin [Biomedical Research Institute, Lifeliver Co., Ltd., Suwon (Korea, Republic of); Park, Won Jin [Dr. Park' s Aesthetic Clinic, Seoul (Korea, Republic of); Kong, Jee Hyun; Shim, Kwang Yong [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Lee, Jong In, E-mail: oncochem@yonsei.ac.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Kim, Hyun Soo, E-mail: khsmd@unitel.co.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of)

    2011-04-29

    Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  17. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    International Nuclear Information System (INIS)

    Eom, Young Woo; Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin; Park, Won Jin; Kong, Jee Hyun; Shim, Kwang Yong; Lee, Jong In; Kim, Hyun Soo

    2011-01-01

    Highlights: → hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. → Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. → hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  18. YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Song, Young Joon [Department of Biological Sciences, College of Natural Science, Inha University, 253 Yonghyun-dong, Nam-Gu, Incheon, Korea, 402-751 (Korea, Republic of); Lee, Hansol, E-mail: hlee@inha.ac.kr [Department of Biological Sciences, College of Natural Science, Inha University, 253 Yonghyun-dong, Nam-Gu, Incheon, Korea, 402-751 (Korea, Republic of)

    2010-02-15

    Precisely controlled cellular differentiation is essential for the proper development of vertebrate embryo and deregulated differentiation is a major cause of many human congenital diseases as well as cancer. Msx1 is a member of the homeoprotein family implicated in these processes, which inhibits the differentiation of skeletal muscle and other cell types, presumably by regulating transcription of target genes through interaction with other cellular factors. We presently show that YB1/p32, a nuclear Y-box binding protein 1, interacts with Msx1 homeoprotein and functions as a regulator of C2C12 myoblast differentiation. We demonstrate that YB1/p32 functionally interacts with Msx1 through its N-terminal region and colocalizes with Msx1 at the nuclear periphery. Moreover, we find that YB1/p32 is competent for inhibition of C2C12 myoblast differentiation, which is correlated with its activity as a negative regulator of MyoD gene expression and binding to the MyoD core enhancer region (CER). Furthermore, YB1/p32 cooperates with Msx1 in transcriptional repression and knocking down the expression of endogenous YB1 attenuates the effects of Msx1. Taken together, our study has uncovered a new function of YB1/p32, a regulator of skeletal muscle differentiation.

  19. YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein

    International Nuclear Information System (INIS)

    Song, Young Joon; Lee, Hansol

    2010-01-01

    Precisely controlled cellular differentiation is essential for the proper development of vertebrate embryo and deregulated differentiation is a major cause of many human congenital diseases as well as cancer. Msx1 is a member of the homeoprotein family implicated in these processes, which inhibits the differentiation of skeletal muscle and other cell types, presumably by regulating transcription of target genes through interaction with other cellular factors. We presently show that YB1/p32, a nuclear Y-box binding protein 1, interacts with Msx1 homeoprotein and functions as a regulator of C2C12 myoblast differentiation. We demonstrate that YB1/p32 functionally interacts with Msx1 through its N-terminal region and colocalizes with Msx1 at the nuclear periphery. Moreover, we find that YB1/p32 is competent for inhibition of C2C12 myoblast differentiation, which is correlated with its activity as a negative regulator of MyoD gene expression and binding to the MyoD core enhancer region (CER). Furthermore, YB1/p32 cooperates with Msx1 in transcriptional repression and knocking down the expression of endogenous YB1 attenuates the effects of Msx1. Taken together, our study has uncovered a new function of YB1/p32, a regulator of skeletal muscle differentiation.

  20. α-linolenic acid reduces TNF-induced apoptosis in C2C12 myoblasts by regulating expression of apoptotic proteins

    Directory of Open Access Journals (Sweden)

    Felicia Carotenuto

    2016-11-01

    Full Text Available Impaired regeneration and consequent muscle wasting is a major feature of muscle degenerative diseases. Nutritional interventions as adjuvant strategy for preventing such conditions are recently gaining increasing attention. Ingestion of n3-polyunsaturated fatty acids has been suggested to have a positive impact on muscle diseases. We recently demonstrated that the dietary n3-fatty acid, alpha-linolenic acid (ALA, exerts potent beneficial effects in preserving skeletal muscle regeneration in models of muscle dystrophy. To better elucidate the underlying mechanism we investigate here on the expression level of the anti- and pro-apototic proteins, as well as caspase-3 activity, in C2C12 myoblasts challenged with pathological levels of TNF. The results demonstrated that ALA protective effect on C2C12 myoblasts was associated to an increased Bcl-2/Bax ratio. Indeed, the effect of ALA was directed to rescue Bcl-2 expression and decrease Bax expression both affected in an opposite way by TNF treatment. This effect was associated with a decrease in caspase-3 activity by ALA. TNF is a major pro-inflammatory cytokine that is expressed in damaged skeletal muscle, therefore, counteract inflammatory signals in the muscle microenvironment represents a critical strategy to ameliorate skeletal muscle pathologies

  1. Gallic acid attenuates calcium calmodulin-dependent kinase II-induced apoptosis in spontaneously hypertensive rats.

    Science.gov (United States)

    Jin, Li; Piao, Zhe Hao; Liu, Chun Ping; Sun, Simei; Liu, Bin; Kim, Gwi Ran; Choi, Sin Young; Ryu, Yuhee; Kee, Hae Jin; Jeong, Myung Ho

    2018-03-01

    Hypertension causes cardiac hypertrophy and leads to heart failure. Apoptotic cells are common in hypertensive hearts. Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is associated with apoptosis. We recently demonstrated that gallic acid reduces nitric oxide synthase inhibition-induced hypertension. Gallic acid is a trihydroxybenzoic acid and has been shown to have beneficial effects, such as anti-cancer, anti-calcification and anti-oxidant activity. The purpose of this study was to determine whether gallic acid regulates cardiac hypertrophy and apoptosis in essential hypertension. Gallic acid significantly lowered systolic and diastolic blood pressure in spontaneously hypertensive rats (SHRs). Wheat germ agglutinin (WGA) and H&E staining revealed that gallic acid reduced cardiac enlargement in SHRs. Gallic acid treatment decreased cardiac hypertrophy marker genes, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), in SHRs. The four isoforms, α, β, δ and γ, of CaMKII were increased in SHRs and were significantly reduced by gallic acid administration. Gallic acid reduced cleaved caspase-3 protein as well as bax, p53 and p300 mRNA levels in SHRs. CaMKII δ overexpression induced bax and p53 expression, which was attenuated by gallic acid treatment in H9c2 cells. Gallic acid treatment reduced DNA fragmentation and the TUNEL positive cells induced by angiotensin II. Taken together, gallic acid could be a novel therapeutic for the treatment of hypertension through suppression of CaMKII δ-induced apoptosis. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  2. Subcellular localization of anthracyclines in cultured rat cardiomyoblasts as possible predictors of cardiotoxicity.

    Science.gov (United States)

    Studzian, Kazimierz; Kik, Krzysztof; Lukawska, Malgorzata; Oszczapowicz, Irena; Strek, Malgorzata; Szmigiero, Leszek

    2015-10-01

    In this study, we compared the cellular uptake, intracellular localization and cytotoxicity of two groups of anthracycline derivatives in cultured H9c2(2-1) rat cardiomyoblasts. The first group consisted of doxorubicin (DOX) and two of its derivatives containing a formamidino group (-N = CH-N<) at the C-3' position with a morpholine (DOXM) or a hexamethyleneimine (DOXH) ring. The second group consisted of daunorubicin (DRB) and its derivatives containing a morpholine (DRBM) or a hexamethyleneimine (DRBH) ring. DOXH and DRBH were taken up by cardiomyoblasts more efficiently than estimated for other tested anthracyclines. The cellular uptakes of DOXM and DRBM were reduced compared to those of the parent compounds. Applied structural modifications of DOX and DRB influenced the subcellular localization of the tested derivatives. DOX and DOXH were localized primarily in nuclei, whereas the other anthracyclines were found in the nuclei and cytoplasm. The percentages of the compounds that accumulated in the nuclei were 80.2 and 54.2 % for DOX and DOXH, respectively. The lowest nuclear accumulation values were observed for DRBM (19.9 %), DRBH (21.9 %) and DOXM (23.7 %). The ability of anthracyclines to accumulate in the nuclei correlated with their DNA binding constants (r = 0.858, P = 0.029). A correlation was found between the accumulation of the tested anthracyclines in the nuclei of cardiomyoblasts and their cardiotoxicity in vivo, which was observed in our previous study. We suggest that cytotoxicity and the anthracycline accumulation level in the nuclei of cultured cardiomyoblasts could be used for early prediction of their cardiotoxicity.

  3. The CDM Superfamily Protein MBC Directs Myoblast Fusion through a Mechanism That Requires Phosphatidylinositol 3,4,5-Triphosphate Binding but Is Independent of Direct Interaction with DCrk▿§

    Science.gov (United States)

    Balagopalan, Lakshmi; Chen, Mei-Hui; Geisbrecht, Erika R.; Abmayr, Susan M.

    2006-01-01

    myoblast city (mbc), a member of the CDM superfamily, is essential in the Drosophila melanogaster embryo for fusion of myoblasts into multinucleate fibers. Using germ line clones in which both maternal and zygotic contributions were eliminated and rescue of the zygotic loss-of-function phenotype, we established that mbc is required in the fusion-competent subset of myoblasts. Along with its close orthologs Dock180 and CED-5, MBC has an SH3 domain at its N terminus, conserved internal domains termed DHR1 and DHR2 (or “Docker”), and C-terminal proline-rich domains that associate with the adapter protein DCrk. The importance of these domains has been evaluated by the ability of MBC mutations and deletions to rescue the mbc loss-of-function muscle phenotype. We demonstrate that the SH3 and Docker domains are essential. Moreover, ethyl methanesulfonate-induced mutations that change amino acids within the MBC Docker domain to residues that are conserved in other CDM family members nevertheless eliminate MBC function in the embryo, which suggests that these sites may mediate interactions specific to Drosophila MBC. A functional requirement for the conserved DHR1 domain, which binds to phosphatidylinositol 3,4,5-triphosphate, implicates phosphoinositide signaling in myoblast fusion. Finally, the proline-rich C-terminal sites mediate strong interactions with DCrk, as expected. These sites are not required for MBC to rescue the muscle loss-of-function phenotype, however, which suggests that MBC's role in myoblast fusion can be carried out independently of direct DCrk binding. PMID:17030600

  4. MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Zhiyuan; Sun, Xiaorui; Xu, Dequan; Xiong, Yuanzhu; Zuo, Bo, E-mail: zuobo@mail.hzau.edu.cn

    2015-11-27

    Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MyoD, MyoG, Myf5 and Myf6 (also known as MRF4). MicroRNA is a kind of ∼22 nt-long non-coding small RNAs, and act as key transcriptional or post-transcriptional regulators of gene expression. Identification of miRNAs involved in the regulation of muscle genes could improve our understanding of myogenesis process. In this study, we investigated the regulation of Myf6 gene by miRNAs. We showed that miR-374b specifically bound to the 3'untranslated region (UTR) of Myf6 and down-regulated the expression of Myf6 gene at both mRNA and protein level. Furthermore, miR-374b is ubiquitously expressed in the tissues of adult C57BL6 mouse, and the mRNA abundance increases first and then decreases during C2C12 myoblasts differentiation. Over-expression of miR-374b impaired C2C12 cell differentiation, while inhibiting miR-374b expression by 2′-O-methyl antisense oligonucleotides promoted C2C12 cell differentiation. Taken together, our findings identified miR-374b directly targets Myf6 and negatively regulates myogenesis. - Highlights: • MiR-374b directly targets 3′UTR of Myf6. • MiR-374b negatively regulates Myf6 in C2C12 cells. • MiR-374b abundance significiently changes during C2C12 cells differentiation. • MiR-374b negatively regulates C2C12 cells differentiation.

  5. Assessment of the antioxidant activity of an olive oil total polyphenolic fraction and hydroxytyrosol from a Greek Olea europea variety in endothelial cells and myoblasts.

    Science.gov (United States)

    Kouka, Paraskevi; Priftis, Alexandros; Stagos, Dimitrios; Angelis, Apostolis; Stathopoulos, Panagiotis; Xinos, Nikos; Skaltsounis, Alexios-Léandros; Mamoulakis, Charalampos; Tsatsakis, Aristides M; Spandidos, Demetrios A; Kouretas, Demetrios

    2017-09-01

    Olive oil (OO) constitutes the basis of the Mediterranean diet, and it seems that its biophenols, such as hydroxytyrosol (HT) may scavenge free radicals, attracting distinct attention due to their beneficial effects in many pathological conditions, such as cancer. To the best of our knowedge, this is the first study in which the functional properties of an OO total polyphenolic fraction (TPF) and pure HT were examined in order to determine their antioxidant effects at a cellular level in endothelial cells and myoblasts. The test compounds were isolated using a green gradient‑elution centrifugal partition chromatography‑based method that allows the isolation of large volumes of OO in a continuous extraction procedure and with extremely low solvent consumption. For the isolation of HT, a combination of two chromatographic techniques was used, which is effective for the recovery of pure compounds from complex natural extracts. Moreover, TPF and HT exhibited potent free radical scavenging activity in vitro. The cells were treated with non‑cytotoxic concentrations and their redox status [in terms of glutathione (GSH) and reactive oxygen species (ROS) levels] was assessed. TPF extract was less cytotoxic than HT, and the observed differences between the two cell lines used suggest a tissue‑specific activity. Finally, flow cytometric analysis revealed that both TPF and HT improved the redox status by increasing the levels of GSH, one of the most important antioxidant molecules, in both endothelial cells and myoblasts, while the ROS levels were not significantly affected.

  6. Combined use of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and platelet rich plasma (PRP) stimulates proliferation and differentiation of myoblasts in vitro: new therapeutic perspectives for skeletal muscle repair/regeneration.

    Science.gov (United States)

    Sassoli, Chiara; Vallone, Larissa; Tani, Alessia; Chellini, Flaminia; Nosi, Daniele; Zecchi-Orlandini, Sandra

    2018-02-05

    Satellite cell-mediated skeletal muscle repair/regeneration is compromised in cases of extended damage. Bone marrow mesenchymal stromal cells (BM-MSCs) hold promise for muscle healing but some criticisms hamper their clinical application, including the need to avoid animal serum contamination for expansion and the scarce survival after transplant. In this context, platelet-rich plasma (PRP) could offer advantages. Here, we compare the effects of PRP or standard culture media on C2C12 myoblast, satellite cell and BM-MSC viability, survival, proliferation and myogenic differentiation and evaluate PRP/BM-MSC combination effects in promoting myogenic differentiation. PRP induced an increase of mitochondrial activity and Ki67 expression comparable or even greater than that elicited by standard media and promoted AKT signaling activation in myoblasts and BM-MSCs and Notch-1 pathway activation in BM-MSCs. It stimulated MyoD, myogenin, α-sarcomeric actin and MMP-2 expression in myoblasts and satellite cell activation. Notably, PRP/BM-MSC combination was more effective than PRP alone. We found that BM-MSCs influenced myoblast responses through a paracrine activation of AKT signaling, contributing to shed light on BM-MSC action mechanisms. Our results suggest that PRP represents a good serum substitute for BM-MSC manipulation in vitro and could be beneficial towards transplanted cells in vivo. Moreover, it might influence muscle resident progenitors' fate, thus favoring the endogenous repair/regeneration mechanisms. Finally, within the limitations of an in vitro experimentation, this study provides an experimental background for considering the PRP/BM-MSC combination as a potential therapeutic tool for skeletal muscle damage, combining the beneficial effects of BM-MSCs and PRP on muscle tissue, while potentiating BM-MSC functionality.

  7. Bupivacaine can enhance lysosomal activity in mouse muscle myoblasts%布比卡因增强小鼠成肌细胞溶酶体的活性

    Institute of Scientific and Technical Information of China (English)

    熊静薇; 毛雨; 李荣荣; 丁正年

    2015-01-01

    Objective To investigate the effects of bupivacaine on lysosomal abundance and activity in mouse muscle myoblasts.Methods Mouse myoblasts C2C12 was randomly divided into control group (without any treatment) and bupivacaine group (treated with bupivacaine 600 μ mol/L for 6 h).After then,the changes of lysosomal pH was assessed by LysoSensor pH indicator.The content of lysosomes was detected by LysoTracker probe.The expression of lysosomal-associated membrane protein-1 (LAMP-1) and Cathepsin B was detected by Western blot analysis.The activity of lysosomal proteolytic enzymes Cathepsin B was determined by MagicRed assay kit.Results Bupivacaine did not affect lysosomal pH.However,compared with the controls,lysosomal abundance was significantly increased 15.15% following bupivacaine treatment(P<0.01).Moreover,protein expression levels of LAMP-1 and Cathepsin B were significantly upregulated 36.41% and 35.29% respetctively by bupivacaine (P<0.01).Furthermore,the activity of Cathepsin B was significantly increased 23.74% by bupivacaine(P<0.01).Conclusions Bupivacaine increased lysosomal content and enhance lysosomal activity in mouse muscle myoblasts.%目的 探讨局部麻醉药布比卡因对小鼠成肌细胞溶酶体的影响. 方法 将体外培养的小鼠成肌细胞C2C12分为2组.对照组:不加任何药物;布比卡因组:以600μmol/L布比卡因刺激细胞6h.实验结束后,用LysoSensor探针评价溶酶体腔pH,用LysoTrackor探针检测溶酶体含量,用蛋白免疫印迹法检测溶酶体相关膜蛋白-1(LAMP-1)和溶酶体蛋白水解酶Cathepsin B的表达水平,并以MagicRed染色法测定Cathepsin B的活性.结果 布比卡因对溶酶体腔pH没有影响.但是,与对照组相比,布比卡因组溶酶体含量增加15.15% (P<0.01),LAMP-1与Cathepsin B表达量分别增加36.41%、35.29% (P<0.01),Cathepsin B活性增加23.74%(P<0.01).结论 布比卡因能增加小鼠成肌细胞溶酶体含量,增强溶酶体活性.

  8. Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1

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    Hong, Gia-Ming [Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Present address: The University of Chicago, Section of Hematology/Oncology, 900 E. 57th Street, Room 7134, Chicago, IL 60637 (United States); Bain, Lisa J., E-mail: lbain@clemson.edu [Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States)

    2012-05-01

    Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (∼ 2-fold) to the myogenin promoter at the transcription start site (− 40 to + 42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (− 40 to + 42). -- Highlights: ► Igf-1 expression is decreased in C2C12 cells after 20 nM arsenite exposure. ► Arsenic exposure alters histone remodeling on the myogenin promoter. ► Glp expression, a H3–K9 methyltransferase, was increased in arsenic-exposed cells. ► Ezh2

  9. Notch1 Mediates Preconditioning Protection Induced by GPER in Normotensive and Hypertensive Female Rat Hearts

    Directory of Open Access Journals (Sweden)

    Carmine Rocca

    2018-05-01

    Full Text Available G protein-coupled estrogen receptor (GPER is an estrogen receptor expressed in the cardiovascular system. G1, a selective GPER ligand, exerts cardiovascular effects through activation of the PI3K-Akt pathway and Notch signaling in normotensive animals. Here, we investigated whether the G1/GPER interaction is involved in the limitation of infarct size, and improvement of post-ischemic contractile function in female spontaneous hypertensive rat (SHR hearts. In this model, we also studied Notch signaling and key components of survival pathway, namely PI3K-Akt, nitric oxide synthase (NOS and mitochondrial K+-ATP (MitoKATP channels. Rat hearts isolated from female SHR underwent 30 min of global, normothermic ischemia and 120 min of reperfusion. G1 (10 nM alone or specific inhibitors of GPER, PI3K/NOS and MitoKATP channels co-infused with G1, just before I/R, were studied. The involvement of Notch1 was studied by Western blotting. Infarct size and left ventricular pressure were measured. To confirm endothelial-independent G1-induced protection by Notch signaling, H9c2 cells were studied with specific inhibitor, N-[N-(3,5 difluorophenacetyl-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, 5 μM, of this signaling. Using DAPT, we confirmed the involvement of G1/Notch signaling in limiting infarct size in heart of normotensive animals. In the hypertensive model, G1-induced reduction in infarct size and improvement of cardiac function were prevented by the inhibition of GPER, PI3K/NOS, and MitoKATP channels. The involvement of Notch was confirmed by western blot in the hypertensive model and by the specific inhibitor in the normotensive model and cardiac cell line. Our results suggest that GPERs play a pivotal role in mediating preconditioning cardioprotection in normotensive and hypertensive conditions. The G1-induced protection involves Notch1 and is able to activate the survival pathway in the presence of comorbidity. Several pathological conditions

  10. 3,5-Diiodo-l-Thyronine Increases Glucose Consumption in Cardiomyoblasts Without Affecting the Contractile Performance in Rat Heart

    Directory of Open Access Journals (Sweden)

    Ginevra Sacripanti

    2018-05-01

    Full Text Available 3,5-diiodo-l-thyronine (T2 is an endogenous derivative of thyroid hormone that has been suggested to regulate energy expenditure, resting metabolic rate and oxygen consumption with a mechanism that involves the activation of mitochondrial function. In this study, we focused on the cardiac effects of T2, which have been poorly investigated so far, by using both in vitro and ex vivo models. As a comparison, the response to T3 and T4 was also determined. Rat cardiomyoblasts (H9c2 cells were used to determine T2, T3, and T4 uptake by high-performance liquid chromatography–tandem mass spectrometry. In the same experimental model, MTT test, crystal violet staining, and glucose consumption were investigated, using T2 concentrations ranging from 0.1 to 10 µM. To assess cardiac functional effects, isolated working rat hearts were perfused with T2, T3, or T4 in Krebs-Ringer buffer, and the hemodynamic variables were recorded. T2 was taken up by cardiomyoblasts, and in cell lysate T2 levels increased slowly over time, reaching higher concentrations than in the incubation medium. T2 significantly decreased MTT staining at 0.5–10 µM concentration (P < 0.05. Crystal violet staining confirmed a reduction of cell viability only upon treatment with 10 µM T2, while equimolar T3 and T4 did not share this effect. Glucose consumption was also significantly affected as indicated by glucose uptake being increased by 24 or 35% in cells exposed to 0.1 or 1.0 µM T2 (P < 0.05 in both cases. On the contrary, T3 did not affect glucose consumption which, in turn, was significantly reduced by 1 and 10 µM T4 (−24 and −41% vs control, respectively, P < 0.05 and P < 0.01. In the isolated perfused rat heart, 10 µM T2 produced a slight and transient reduction in cardiac output, while T3 and T4 did not produce any hemodynamic effect. Our findings indicate that T2 is taken up by cardiomyoblasts, and at 0.1–1.0 µM concentration it can

  11. Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

    Science.gov (United States)

    Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D

    2005-01-01

    Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes.

  12. Microfluidic-enhanced 3D bioprinting of aligned myoblast-laden hydrogels leads to functionally organized myofibers in vitro and in vivo.

    Science.gov (United States)

    Costantini, Marco; Testa, Stefano; Mozetic, Pamela; Barbetta, Andrea; Fuoco, Claudia; Fornetti, Ersilia; Tamiro, Francesco; Bernardini, Sergio; Jaroszewicz, Jakub; Święszkowski, Wojciech; Trombetta, Marcella; Castagnoli, Luisa; Seliktar, Dror; Garstecki, Piotr; Cesareni, Gianni; Cannata, Stefano; Rainer, Alberto; Gargioli, Cesare

    2017-07-01

    We present a new strategy for the fabrication of artificial skeletal muscle tissue with functional morphologies based on an innovative 3D bioprinting approach. The methodology is based on a microfluidic printing head coupled to a co-axial needle extruder for high-resolution 3D bioprinting of hydrogel fibers laden with muscle precursor cells (C2C12). To promote myogenic differentiation, we formulated a tailored bioink with a photocurable semi-synthetic biopolymer (PEG-Fibrinogen) encapsulating cells into 3D constructs composed of aligned hydrogel fibers. After 3-5 days of culture, the encapsulated myoblasts started migrating and fusing, forming multinucleated myotubes within the 3D bioprinted fibers. The obtained myotubes showed high degree of alignment along the direction of hydrogel fiber deposition, further revealing maturation, sarcomerogenesis, and functionality. Following subcutaneous implantation in the back of immunocompromised mice, bioprinted constructs generated organized artificial muscle tissue in vivo. Finally, we demonstrate that our microfluidic printing head allows to design three dimensional multi-cellular assemblies with an exquisite compartmentalization of the encapsulated cells. Our results demonstrate an enhanced myogenic differentiation with the formation of parallel aligned long-range myotubes. The approach that we report here represents a robust and valid candidate for the fabrication of macroscopic artificial muscle to scale up skeletal muscle tissue engineering for human clinical application. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  13. Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells

    Directory of Open Access Journals (Sweden)

    Kumiko Terada

    2013-01-01

    Full Text Available Background. Wnt signaling is involved in muscle formation through β-catenin-dependent or -independent pathways, but interactions with other signaling pathways including transforming growth factor β/Smad have not been precisely elucidated. Results. As Wnt4 stimulates myogenic differentiation by antagonizing myostatin (GDF8 activity, we examined the role of Wnt4 signaling during muscle differentiation in the C2C12 myoblast cell line. Among several extrinsic signaling molecules examined in a microarray analysis of C2C12 cells during the transition from cell proliferation to differentiation after mitogen deprivation, bone morphogenetic protein 4 (BMP4 expression was prominently increased. Wnt4 overexpression had similar effects on BMP4 expression. BMP4 was able to inhibit muscle differentiation when added to the culture medium. BMP4 and noggin had no effects on the cellular localization of β-catenin induced by Wnt3a; however, the BMP4-induced phosphorylation of Smad1/5/8 was enhanced by Wnt4, but not by Wnt3a. The BMP antagonist noggin effectively stimulated muscle differentiation through binding to endogenous BMPs, and the effect of noggin was enhanced by the presence of Wnt3a and Wnt4. Conclusion. These results suggest that BMP/Smad pathways are modified through Wnt signaling during the transition from progenitor cell proliferation to myogenic differentiation, although Wnt/β-catenin signaling is not modified with BMP/Smad signaling.

  14. Temperature-responsive poly(ε-caprolactone) cell culture platform with dynamically tunable nano-roughness and elasticity for control of myoblast morphology.

    Science.gov (United States)

    Uto, Koichiro; Ebara, Mitsuhiro; Aoyagi, Takao

    2014-01-21

    We developed a dynamic cell culture platform with dynamically tunable nano-roughness and elasticity. Temperature-responsive poly(ε-caprolactone) (PCL) films were successfully prepared by crosslinking linear and tetra-branched PCL macromonomers. By optimizing the mixing ratios, the crystal-amorphous transition temperature (Tm) of the crosslinked film was adjusted to the biological relevant temperature (~33 °C). While the crosslinked films are relatively stiff (50 MPa) below the Tm, they suddenly become soft (1 MPa) above the Tm. Correspondingly, roughness of the surface was decreased from 63.4-12.4 nm. It is noted that the surface wettability was independent of temperature. To investigate the role of dynamic surface roughness and elasticity on cell adhesion, cells were seeded on PCL films at 32 °C. Interestingly, spread myoblasts on the film became rounded when temperature was suddenly increased to 37 °C, while significant changes in cell morphology were not observed for fibroblasts. These results indicate that cells can sense dynamic changes in the surrounding environment but the sensitivity depends on cell types.

  15. Retinoid acid-induced microRNA-27b-3p impairs C2C12 myoblast proliferation and differentiation by suppressing α-dystrobrevin

    Energy Technology Data Exchange (ETDEWEB)

    Li, Nan; Tang, Yi; Liu, Bo; Cong, Wei; Liu, Chao, E-mail: liuchao_19760711@yahoo.com; Xiao, Jing, E-mail: xiaoj@dmu.edu.cn

    2017-01-15

    We previously reported that excess retinoic acid (RA) resulted in hypoplastic and derangement of myofilaments in embryonic tongue by inhibiting myogenic proliferation and differentiation through CamKIID pathway. Our further studies revealed that the expression of a series of miRNAs was altered by RA administration in embryonic tongue as well as in C2C12 cells. Thus, if excess RA impairs myogenic proliferation and differentiation through miRNAs is taken into account. In present study, miR-27b-3p was found up-regulated in RA-treated C2C12 cells as in embryonic tongue, and predicted to target the 3′UTR of α-dystrobrevin (DTNA). Luciferase reporter assays confirmed the direct interaction between miR-27b-3p and the 3′UTR of DTNA. MiR-27b-3p mimics recapitulated the RA repression on DTNA expression, C2C12 proliferation and differentiation, while the miR-27b-3p inhibitor circumvented these defects resulting from excess RA. As expected, the effects of siDTNA on C2C12 were coincided with those by RA treatment or miR-27b-3p mimics. Therefore, these findings indicated that excess RA inhibited the myoblast proliferation and differentiation by up-regulating miR-27b-3p to target DTNA, which implied a new mechanism in myogenic hypoplasia. - Highlights: • A mechanism that RA results in tongue deformity by disrupting the myogenesis. • A non-muscle specific miR mediating the RA suppression on tongue myogenesis. • A target gene of non-muscle specific miR involved in RA induced tongue deformity.

  16. G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

    Science.gov (United States)

    Wu, Melissa P; Doyle, Jamie R; Barry, Brenda; Beauvais, Ariane; Rozkalne, Anete; Piao, Xianhua; Lawlor, Michael W; Kopin, Alan S; Walsh, Christopher A; Gussoni, Emanuela

    2013-12-01

    Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities. © 2013 FEBS.

  17. Differential expression of FGF receptors and of myogenic regulatory factors in primary cultures of satellite cells originating from fast (EDL) and slow (Soleus) twitch rat muscles.

    Science.gov (United States)

    Martelly, I; Soulet, L; Bonnavaud, S; Cebrian, J; Gautron, J; Barritault, D

    2000-11-01

    In the rat, the fast and slow twitch muscles respectively Extensor digitorum longus (EDL) and Soleus present differential characteristics during regeneration. This suggests that their satellite cells responsible for muscle growth and repair represent distinct cellular populations. We have previously shown that satellite cells dissociated from Soleus and grown in vitro proliferate more readily than those isolated from EDL muscle. Fibroblast growth factors (FGFs) are known as regulators of myoblast proliferation and several studies have revealed a relationship between the response of myoblasts to FGF and the expression of myogenic regulatory factors (MRF) of the MyoD family by myoblasts. Therefore, we presently examined the possibility that the satellite cells isolated from EDL and Soleus muscles differ in the expression of FGF receptors (FGF-R) and of MRF expression. FGF-R1 and -R4 were strongly expressed in proliferating cultures whereas FGF-R2 and R3 were not detected in these cultures. In differentiating cultures, only -R1 was present in EDL satellite cells while FGF-R4 was also still expressed in Soleus cells. Interestingly, the unconventional receptor for FGF called cystein rich FGF receptor (CFR), of yet unknown function, was mainly detected in EDL satellite cell cultures. Soleus and EDL satellite cell cultures also differed in the expression MRFs. These results are consistent with the notion that satellite cells from fast and slow twitch muscles belong to different types of myogenic cells and suggest that satellite cells might play distinct roles in the formation and diversification of fast and slow fibres.

  18. The role of aryl hydrocarbon receptor signaling pathway in cardiotoxicity of acute lead intoxication in vivo and in vitro rat model.

    Science.gov (United States)

    Ansari, Mushtaq A; Maayah, Zaid H; Bakheet, Saleh A; El-Kadi, Ayman O; Korashy, Hesham M

    2013-04-05

    Lead (Pb(2+)) is a naturally occurring systemic toxicant heavy metal that affects several organs in the body including the kidneys, liver, and central nervous system. However, Pb(2+)-induced cardiotoxicity has never been investigated yet and the exact mechanism of Pb(2+) associated cardiotoxicity has not been studied. The current study was designed to investigate the potential effect of Pb(2+) to induce cardiotoxicity in vivo and in vitro rat model and to explore the molecular mechanisms and the role of aryl hydrocarbon receptor (AhR) and regulated gene, cytochrome P4501A1 (CYP1A1), in Pb(2+)-mediated cardiotoxicity. For these purposes, Wistar albino rats were treated with Pb(2+) (25, 50 and 100mg/kg, i.p.) for three days and the effects on physiological and histopathological parameters of cardiotoxicity were determined. At the in vitro level, rat cardiomyocyte H9c2 cell lines were incubated with increasing concentration of Pb(2+) (25, 50, and 100 μM) and the expression of hypertrophic genes, α- and β-myosin heavy chain (α-MHC and β-MHC), brain Natriuretic Peptide (BNP), and CYP1A1 were determined at the mRNA and protein levels using real-time PCR and Western blot analysis, respectively. The results showed that Pb(2+) significantly induced cardiotoxicity and heart failure as evidenced by increase cardiac enzymes, lactate dehydrogenase and creatine kinase and changes in histopathology in vivo. In addition, Pb(2+) treatment induced β-MHC and BNP whereas inhibited α-MHC mRNA and protein levels in vivo in a dose-dependent manner. In contrast, at the in vitro level, Pb(2+) treatment induced both β-MHC and α-MHC mRNA levels in time- and dose-dependent manner. Importantly, these changes were accompanied with a proportional increase in the expression of CYP1A1 mRNA and protein expression levels, suggesting a role for the CYP1A1 in cardiotoxicity. The direct evidence for the involvement of CYP1A1 in the induction of cardiotoxicity by Pb(2+) was evidenced by the

  19. IGF-II-mediated downregulation of peroxisome proliferator-activated receptor-γ coactivator-1α in myoblast cells involves PI3K/Akt/FoxO1 signaling pathway.

    Science.gov (United States)

    Mu, Xiaoyu; Qi, Weihong; Liu, Yunzhang; Zhou, Jianfeng; Li, Yun; Rong, Xiaozhi; Lu, Ling

    2017-08-01

    Insulin-like growth factor II (IGF-II) can stimulate myogenesis and is critically involved in skeletal muscle differentiation. The presence of negative regulators of this process, however, is not well explored. Here, we showed that in myoblast cells, IGF-II negatively regulated peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA expression, while constitutive expression of PGC-1α induced myoblast differentiation. These results suggest that the negative regulation of PGC-1α by IGF-II may act as a negative feedback mechanism in IGF-II-induced myogenic differentiation. Reporter assays demonstrated that IGF-II suppresses the basal PGC-1α promoter activity. Blocking the IGF-II signaling pathway increased the endogenous PGC-1α levels. In addition, pharmacological inhibition of PI3 kinase activity prevented the downregulation of PGC-1α but the activation of mTOR was not required for this process. Importantly, further analysis showed that forkhead transcription factor FoxO1 contributes to mediating the effects of IGF-II on PGC-1 promoter activity. These findings indicate that IGF-II reduces PGC-1α expression in skeletal muscle cells through a mechanism involving PI3K-Akt-FoxO1 but not p38 MAPK or Erk1/2 MAPK pathways.

  20. Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery-Dreifuss muscular dystrophy is improved by inhibition of the MEK-ERK pathway and stimulation of the PI3-kinase pathway

    International Nuclear Information System (INIS)

    Favreau, Catherine; Delbarre, Erwan; Courvalin, Jean-Claude; Buendia, Brigitte

    2008-01-01

    Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process

  1. ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin-Hua [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Bauman, William A. [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Cardozo, Christopher, E-mail: chris.cardozo@va.gov [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States)

    2015-08-14

    Transcription factors of the nuclear factor-kappa B (NF-κB) family play a pivotal role in inflammation, immunity and cell survival responses. Recent studies revealed that NF-κB also regulates the processes of muscle atrophy. NF-κB activity is regulated by various factors, including ankyrin repeat domain 2 (AnkrD2), which belongs to the muscle ankyrin repeat protein family. Another member of this family, AnkrD1 is also a transcriptional effector. The expression levels of AnkrD1 are highly upregulated in denervated skeletal muscle, suggesting an involvement of AnkrD1 in NF-κB mediated cellular responses to paralysis. However, the molecular mechanism underlying the interactive role of AnkrD1 in NF-κB mediated cellular responses is not well understood. In the current study, we examined the effect of AnkrD1 on NF-κB activity and determined the interactions between AnkrD1 expression and NF-κB signaling induced by TNFα in differentiating C2C12 myoblasts. TNFα upregulated AnkrD1 mRNA and protein levels. AnkrD1-siRNA significantly increased TNFα-induced transcriptional activation of NF-κB, whereas overexpression of AnkrD1 inhibited TNFα-induced NF-κB activity. Co-immunoprecipitation studies demonstrated that AnkrD1 was able to bind p50 subunit of NF-κB and vice versa. Finally, CHIP assays revealed that AnkrD1 bound chromatin at a NF-κB binding site in the AnrkD2 promoter and required NF-κB to do so. These results provide evidence of signaling integration between AnkrD1 and NF-κB pathways, and suggest a novel anti-inflammatory role of AnkrD1 through feedback inhibition of NF-κB transcriptional activity by which AnkrD1 modulates the balance between physiological and pathological inflammatory responses in skeletal muscle. - Highlights: • AnkrD1 is upregulated by TNFα and represses NF-κB-induced transcriptional activity. • AnkrD1 binds to p50 subunit of NF-κB and is recruited to NF-κB bound to chromatin. • AnkrD1 mediates a feed-back inhibitory loop

  2. Cartap-induced cytotoxicity in mouse C2C12 myoblast cell line and the roles of calcium ion and oxidative stress on the toxic effects.

    Science.gov (United States)

    Liao, Jiunn-Wang; Kang, Jaw-Jou; Jeng, Chian-Ren; Chang, Shao-Kuang; Kuo, Ming-Jang; Wang, Shun-Cheng; Liu, Michael R S; Pang, Victor Fei

    2006-02-15

    Our previous study has demonstrated that instead of neuromuscular blockage cartap, an organonitrogen insecticide, could cause a marked irreversible Ca2+-dependent contracture in both isolated mouse and rabbit phrenic nerve-diaphragms. We further examined the potential of direct myocytotoxicity of cartap and the possible roles of calcium ion and oxidative stress on cartap-induced muscle cell injury using the mouse myoblast cell line, C2C12. Cartap exerted a dose- and time-dependent cytotoxic effect in C2C12 cells measured by MTT colorimetric assay and trypan blue dye exclusion. The extracellular activities of both creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated in the cartap-treated groups at or greater than 100 microM. The isoenzymatic profiles showed that the elevations were mainly due to CK-3, LDH-3, and LDH-4. Following the addition of 0.5-2.5mM EGTA, a Ca2+ chelator, or 30-100 microM verapamil, an L-type Ca2+ channel blocker, the cartap-induced reduction in MTT metabolic rate of C2C12 cells was significantly restored in a dose-dependent manner in both EGTA and verapamil-treated cells. Furthermore, EGTA could significantly reduce the cartap-induced elevation in the levels of total extracellular CK and LDH activities. Additionally, cartap significantly increased the level of endogenous reactive oxygen species (ROS) in C2C12 cells in a dose- and time-dependent manner. The cartap-induced ROS generation could be significantly inhibited by antioxidants, including Vitamins C and E, catalase, and superoxide dismutase, with catalase the most effective. EGTA could significantly inhibit cartap-induced ROS generation in a dose-dependent manner. The results suggested that cartap could induce ROS generation in C2C12 cells via a Ca2+-dependent mechanism resulting in subsequent cytotoxicity, at least partially, to C2C12 cells. It is speculated that both Ca2+ and Ca2+-induced ROS may also play the central role on the myogenic contracture and myofiber injury

  3. Bone morphogenetic protein-2 functions as a negative regulator in the differentiation of myoblasts, but not as an inducer for the formations of cartilage and bone in mouse embryonic tongue

    Directory of Open Access Journals (Sweden)

    Suzuki Erika

    2011-07-01

    Full Text Available Abstract Background In vitro studies using the myogenic cell line C2C12 demonstrate that bone morphogenetic protein-2 (BMP-2 converts the developmental pathway of C2C12 from a myogenic cell lineage to an osteoblastic cell lineage. Further, in vivo studies using null mutation mice demonstrate that BMPs inhibit the specification of the developmental fate of myogenic progenitor cells. However, the roles of BMPs in the phases of differentiation and maturation in skeletal muscles have yet to be determined. The present study attempts to define the function of BMP-2 in the final stage of differentiation of mouse tongue myoblast. Results Recombinant BMP-2 inhibited the expressions of markers for the differentiation of skeletal muscle cells, such as myogenin, muscle creatine kinase (MCK, and fast myosin heavy chain (fMyHC, whereas BMP-2 siRNA stimulated such markers. Neither the recombinant BMP-2 nor BMP-2 siRNA altered the expressions of markers for the formation of cartilage and bone, such as osteocalcin, alkaline phosphatase (ALP, collagen II, and collagen X. Further, no formation of cartilage and bone was observed in the recombinant BMP-2-treated tongues based on Alizarin red and Alcian blue stainings. Neither recombinant BMP-2 nor BMP-2 siRNA affected the expression of inhibitor of DNA binding/differentiation 1 (Id1. The ratios of chondrogenic and osteogenic markers relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a house keeping gene were approximately 1000-fold lower than those of myogenic markers in the cultured tongue. Conclusions BMP-2 functions as a negative regulator for the final differentiation of tongue myoblasts, but not as an inducer for the formation of cartilage and bone in cultured tongue, probably because the genes related to myogenesis are in an activation mode, while the genes related to chondrogenesis and osteogenesis are in a silencing mode.

  4. treated rats

    African Journals Online (AJOL)

    aghomotsegin

    2014-01-08

    Jan 8, 2014 ... nucleus, bizarre segmentation; (I) shows hypersegmentation, bizarre segmentation of neutrophils in the shape of ring nucleus with polychromatophilic RBCs. 1998; Muller and Tobin, 1980). The current study shows that rats administered C. edulis hydro-ethanol extract, orally for 28 days, developed anemia, ...

  5. Small activating RNA induces myogenic differentiation of rat adipose-derived stem cells by upregulating MyoD

    Directory of Open Access Journals (Sweden)

    Chenghe Wang

    2015-08-01

    Full Text Available ABSTRACTPurpose:RNA activation (RNAa is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs, also known as small activating RNAs (saRNAs. Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI–a condition primarily resulted from urethral sphincter deficiency.

  6. RatMap--rat genome tools and data.

    Science.gov (United States)

    Petersen, Greta; Johnson, Per; Andersson, Lars; Klinga-Levan, Karin; Gómez-Fabre, Pedro M; Ståhl, Fredrik

    2005-01-01

    The rat genome database RatMap (http://ratmap.org or http://ratmap.gen.gu.se) has been one of the main resources for rat genome information since 1994. The database is maintained by CMB-Genetics at Goteborg University in Sweden and provides information on rat genes, polymorphic rat DNA-markers and rat quantitative trait loci (QTLs), all curated at RatMap. The database is under the supervision of the Rat Gene and Nomenclature Committee (RGNC); thus much attention is paid to rat gene nomenclature. RatMap presents information on rat idiograms, karyotypes and provides a unified presentation of the rat genome sequence and integrated rat linkage maps. A set of tools is also available to facilitate the identification and characterization of rat QTLs, as well as the estimation of exon/intron number and sizes in individual rat genes. Furthermore, comparative gene maps of rat in regard to mouse and human are provided.

  7. Exercise training and return to a well-balanced diet activate the neuregulin 1/ErbB pathway in skeletal muscle of obese rats

    Science.gov (United States)

    Ennequin, Gaël; Boisseau, Nathalie; Caillaud, Kevin; Chavanelle, Vivien; Gerbaix, Maude; Metz, Lore; Etienne, Monique; Walrand, Stéphane; Masgrau, Aurélie; Guillet, Christelle; Courteix, Daniel; Niu, Airu; Li, Yi-Ping; Capel, Fréderic; Sirvent, Pascal

    2015-01-01

    Some studies suggest that the signalling pathway of neuregulin 1 (NRG1), a protein involved in the regulation of skeletal muscle metabolism, could be altered by nutritional and exercise interventions. We hypothesized that diet-induced obesity could lead to alterations of the NRG1 signalling pathway and that chronic exercise could improve NRG1 signalling in rat skeletal muscle. To test this hypothesis, male Wistar rats received a high fat/high sucrose (HF/HS) diet for 16 weeks. At the end of this period, NRG1 and ErbB expression/activity in skeletal muscle was assessed. The obese rats then continued the HF/HS diet or were switched to a well-balanced diet. Moreover, in both groups, half of the animals also performed low intensity treadmill exercise training. After another 8 weeks, NRG1 and ErbB expression/activity in skeletal muscle were tested again. The 16 week HF/HS diet induced obesity, but did not significantly affect the NRG1/ErbB signalling pathway in rat skeletal muscle. Conversely, after the switch to a well-balanced diet, NRG1 cleavage ratio and ErbB4 amount were increased. Chronic exercise training also promoted NRG1 cleavage, resulting in increased ErbB4 phosphorylation. This result was associated with increased protein expression and phosphorylation ratio of the metalloprotease ADAM17, which is involved in NRG1 shedding. Similarly, in vitro stretch-induced activation of ADAM17 in rat myoblasts induced NRG1 cleavage and ErbB4 activation. These results show that low intensity endurance training and well-balanced diet activate the NRG1-ErbB4 pathway, possibly via the metalloprotease ADAM17, in skeletal muscle of diet-induced obese rats. PMID:25820551

  8. Rat Genome Database (RGD)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Rat Genome Database (RGD) is a collaborative effort between leading research institutions involved in rat genetic and genomic research to collect, consolidate,...

  9. A novel fission-independent role of dynamin-related protein 1 in cardiac mitochondrial respiration.

    Science.gov (United States)

    Zhang, Huiliang; Wang, Pei; Bisetto, Sara; Yoon, Yisang; Chen, Quan; Sheu, Shey-Shing; Wang, Wang

    2017-02-01

    Mitochondria in adult cardiomyocytes exhibit static morphology and infrequent dynamic changes, despite the high abundance of fission and fusion regulatory proteins in the heart. Previous reports have indicated that fusion proteins may bear functions beyond morphology regulation. Here, we investigated the role of fission protein, dynamin-related protein 1 (DRP1), on mitochondrial respiration regulation in adult cardiomyocytes. By using genetic or pharmacological approaches, we manipulated the activity or protein level of fission and fusion proteins and found they mildly influenced mitochondrial morphology in adult rodent cardiomyocytes, which is in contrast to their significant effect in H9C2 cardiac myoblasts. Intriguingly, inhibiting endogenous DRP1 by dominant-negative DRP1 mutation (K38A), shRNA, or Mdivi-1 suppressed maximal respiration and respiratory control ratio in isolated mitochondria from adult mouse heart or in adult cardiomyocytes from rat. Meanwhile, basal respiration was increased due to increased proton leak. Facilitating mitofusin-mediated fusion by S3 compound, however, failed to inhibit mitochondrial respiration in adult cardiomyocytes. Mechanistically, DRP1 inhibition did not affect the maximal activity of individual respiratory chain complexes or the assembly of supercomplexes. Knocking out cyclophilin D, a regulator of mitochondrial permeability transition pore (mPTP), abolished the effect of DRP1 inhibition on respiration. Finally, DRP1 inhibition decreased transient mPTP-mediated mitochondrial flashes, delayed laser-induced mPTP opening and suppressed mitochondrial reactive oxygen species (ROS). These results uncover a novel non-canonical function of the fission protein, DRP1 in maintaining or positively stimulating mitochondrial respiration, bioenergetics and ROS signalling in adult cardiomyocyte, which is likely independent of morphological changes. Published on behalf of the European Society of Cardiology. All rights reserved. © The

  10. High glucose-induced Ca2+ overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways.

    Science.gov (United States)

    Kumar, Sandeep; Kain, Vasundhara; Sitasawad, Sandhya L

    2012-07-01

    Cardiac cell apoptosis is the initiating factor of cardiac complications especially diabetic cardiomyopathy. Mitochondria are susceptible to the damaging effects of elevated glucose condition. Calcium overload and oxidative insult are the two mutually non-exclusive phenomena suggested to cause cardiac dysfunction. Here, we examined the effect of high-glucose induced calcium overload in calpain-1 mediated cardiac apoptosis in an in vitro setting. H9c2, rat ventricular myoblast cell line was treated with elevated glucose condition and the cellular consequences were studied. Intracellular calcium trafficking, ROS generation, calpain-1 activation and caspase-12 and caspase-9 pathway were studied using flow cytometry, confocal microscopy and Western blot analysis. High-glucose treatment resulted in increased intracellular calcium ([Ca2+]i) which was mobilized to the mitochondria. Concomitant intra-mitochondrial calcium ([Ca2+]m) increase resulted in enhanced reactive oxygen and nitrogen species generation. These events led to mitochondrial dysfunction and apoptosis. Cardiomyocyte death exhibited several classical markers of apoptosis, including activation of caspases, appearance of annexin V on the outer plasma membrane, increased population of cells with sub-G0/G1 DNA content and nuclear condensation. Key findings include elucidation of cell signaling mechanism of high-glucose induced calcium-dependent cysteine protease calpain-1 activation, which triggers non-conventional caspases as alternate mode of cell death. This information increases the understanding of cardiac cell death under hyperglycemic condition and can possibly be extended for designing new therapeutic strategies for diabetic cardiomyopathy. The novel findings of the study reveal that high glucose induces apoptosis by both mitochondria-dependent and independent pathways via concomitant rise in intracellular calcium. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Exercise training and return to a well-balanced diet activate the neuregulin 1/ErbB pathway in skeletal muscle of obese rats.

    Science.gov (United States)

    Ennequin, Gaël; Boisseau, Nathalie; Caillaud, Kevin; Chavanelle, Vivien; Gerbaix, Maude; Metz, Lore; Etienne, Monique; Walrand, Stéphane; Masgrau, Aurélie; Guillet, Christelle; Courteix, Daniel; Niu, Airu; Li, Yi-Ping; Capel, Fréderic; Sirvent, Pascal

    2015-06-15

    associated with increased protein expression and phosphorylation ratio of the metalloprotease ADAM17, which is involved in NRG1 shedding. Similarly, in vitro stretch-induced activation of ADAM17 in rat myoblasts induced NRG1 cleavage and ErbB4 activation. These results show that low intensity endurance training and well-balanced diet activate the NRG1-ErbB4 pathway, possibly via the metalloprotease ADAM17, in skeletal muscle of diet-induced obese rats. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  12. Effects of resistance training on fast- and slow-twitch muscles in rats

    Directory of Open Access Journals (Sweden)

    M Umnova

    2010-09-01

    Full Text Available The purpose of this study was to investigate the effect of resistance training (RT on muscle strength, the dependence of that on the fast-twitch (FT and slow-twitch (ST fibers hypertrophy, nuclear domain size, synthesis and degradation rate of contractile proteins and on the expression of myosin isoforms’. 16 weeks old Wistar rats were trained on a vertical treadmill for six days a week during six weeks. The power of exercise increased 4.9% per session. In RT group the mass of studied muscles increased about 10%, hindlimb grip strength increased from 5.20±0.27 N/100g bw to the 6.05±0.29 N/100g bw (p<0.05. Cross-sectional area and number of myonuclei of FT and ST fibers in plantaris (Pla and soleus (Sol muscles increased, myonuclear domain size did not change significantly. RT increased the MyHC IId isoforms relative content and decreased that of IIb and IIa isoforms in Pla muscle, in Sol muscle increased only IIa isoform. In Pla muscle the relative content of myosin light chain (MyLC 1slow and 2slow isoforms decreased and that of MyLC 2fast isoforms increased during RT. MyLC 3 and MyLC 2 ratio did not change significantly in Pla but increased in Sol muscle by 14.3±3.4�0(p<0.01. The rat RT programme caused hypertrophy of FT and ST muscle fibers, increase of myonuclear number via fusion of satellite cells with damaged fibers or formation of new muscle fibers as a result of myoblast fusion and myotubes formation, maintaining myonuclear domain size.

  13. Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats.

    Science.gov (United States)

    Zargar, Sana; Moreira, Tracy S; Samimi-Seisan, Helena; Jeganathan, Senthure; Kakade, Dhanshri; Islam, Nushaba; Campbell, Jonathan; Adegoke, Olasunkanmi A J

    2011-06-01

    Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.

  14. Immunology taught by rats

    OpenAIRE

    Klenerman, P; Barnes, EJ

    2017-01-01

    Immunology may be best taught by viruses, and possibly by humans, but the rats of New York City surprisingly also have plenty to offer. A survey published in 2014 of the pathogens carried by rats trapped in houses and parks in Manhattan identified a huge burden of infectious agents in these animals, including several novel viruses. Among these are Norway rat hepaciviruses (NrHVs), which belong to the same family as hepatitis C virus (HCV). NrHVs were found in rat livers, raising the possibili...

  15. Role of androgen receptor on cyclic mechanical stretch-regulated proliferation of C2C12 myoblasts and its upstream signals: IGF-1-mediated PI3K/Akt and MAPKs pathways.

    Science.gov (United States)

    Ma, Yiming; Fu, Shaoting; Lu, Lin; Wang, Xiaohui

    2017-07-15

    To detect the effects of androgen receptor (AR) on cyclic mechanical stretch-modulated proliferation of C2C12 myoblasts and its pathways: roles of IGF-1, PI3K and MAPK. C2C12 were randomly divided into five groups: un-stretched control, six or 8 h of fifteen percent stretch, and six or 8 h of twenty percent stretch. Cyclic mechanical stretch of C2C12 were completed using a computer-controlled FlexCell Strain Unit. Cell proliferation and IGF-1 concentration in medium were detected by CCK8 and ELISA, respectively. Expressions of AR and IGF-1R, and expressions and activities of PI3K, p38 and ERK1/2 in stretched C2C12 cells were determined by Western blot. ①The proliferation of C2C12 cells, IGF-1 concentration in medium, expressions of AR and IGF-1R, and activities of PI3K, p38 and ERK1/2 were increased by 6 h of fifteen percent stretch, while decreased by twenty percent stretch for six or 8 h ②The fifteen percent stretch-increased proliferation of C2C12 cells was reversed by AR inhibitor, Flutamide. ③The increases of AR expression, activities of PI3K, p38 and ERK1/2 resulted from fifteen percent stretch were attenuated by IGF-1 neutralizing antibody, while twenty percent stretch-induced decreases of the above indicators were enhanced by recombinant IGF-1. ④Specific inhibitors of p38, ERK1/2 and PI3K all decreased the expression of AR in fifteen percent and twenty percent of stretched C2C12 cells. Cyclic mechanical stretch modulated the proliferation of C2C12 cells, which may be attributed to the alterations of AR via IGF-1-PI3K/Akt and IGF-1-MAPK (p38, ERK1/2) pathways in C2C12 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation.

    Science.gov (United States)

    Nisr, Raid B; Affourtit, Charles

    2014-02-01

    Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. © 2013.

  17. Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation☆

    Science.gov (United States)

    Nisr, Raid B.; Affourtit, Charles

    2014-01-01

    Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. PMID:24212054

  18. RatMap—rat genome tools and data

    Science.gov (United States)

    Petersen, Greta; Johnson, Per; Andersson, Lars; Klinga-Levan, Karin; Gómez-Fabre, Pedro M.; Ståhl, Fredrik

    2005-01-01

    The rat genome database RatMap (http://ratmap.org or http://ratmap.gen.gu.se) has been one of the main resources for rat genome information since 1994. The database is maintained by CMB–Genetics at Göteborg University in Sweden and provides information on rat genes, polymorphic rat DNA-markers and rat quantitative trait loci (QTLs), all curated at RatMap. The database is under the supervision of the Rat Gene and Nomenclature Committee (RGNC); thus much attention is paid to rat gene nomenclature. RatMap presents information on rat idiograms, karyotypes and provides a unified presentation of the rat genome sequence and integrated rat linkage maps. A set of tools is also available to facilitate the identification and characterization of rat QTLs, as well as the estimation of exon/intron number and sizes in individual rat genes. Furthermore, comparative gene maps of rat in regard to mouse and human are provided. PMID:15608244

  19. SWEEP Project RAT

    DEFF Research Database (Denmark)

    Ibsen, Lars Bo; Madsen, Søren; Petersen, L. B.

    This report presents the results from the design analyses made for the clustered suction caisson used as foundation for a Riser Access Tower (RAT). The RAT is intended built next to the K15-FA-1 Platform in the Dutch Sector of the North Sea....

  20. Colon of the rat

    International Nuclear Information System (INIS)

    Lindstroem, C.G.; Rosengren, J.-E.; Fork, F.-T.

    1979-01-01

    The anatomy and radiologic appearance of the colon in rats are described on the basis of 300 animals treated with carcinogenic agents and 40 normal rats. The macroscopic and microscopic appearance of the mucosa varies in the different parts of the colon. Lymphoid plaques are normal structures. The results justify a new anatomic nomenclature. (Auth.)

  1. Shikonin increases glucose uptake in skeletal muscle cells and improves plasma glucose levels in diabetic Goto-Kakizaki rats.

    Directory of Open Access Journals (Sweden)

    Anette I Öberg

    Full Text Available BACKGROUND: There is considerable interest in identifying compounds that can improve glucose homeostasis. Skeletal muscle, due to its large mass, is the principal organ for glucose disposal in the body and we have investigated here if shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increases glucose uptake in skeletal muscle cells. METHODOLOGY/PRINCIPAL FINDINGS: Shikonin increases glucose uptake in L6 skeletal muscle myotubes, but does not phosphorylate Akt, indicating that in skeletal muscle cells its effect is medaited via a pathway distinct from that used for insulin-stimulated uptake. Furthermore we find no evidence for the involvement of AMP-activated protein kinase in shikonin induced glucose uptake. Shikonin increases the intracellular levels of calcium in these cells and this increase is necessary for shikonin-mediated glucose uptake. Furthermore, we found that shikonin stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myoblasts. The beneficial effect of shikonin on glucose uptake was investigated in vivo by measuring plasma glucose levels and insulin sensitivity in spontaneously diabetic Goto-Kakizaki rats. Treatment with shikonin (10 mg/kg intraperitoneally once daily for 4 days significantly decreased plasma glucose levels. In an insulin sensitivity test (s.c. injection of 0.5 U/kg insulin, plasma glucose levels were significantly lower in the shikonin-treated rats. In conclusion, shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium. CONCLUSIONS/SIGNIFICANCE: Shikonin increases glucose uptake in skeletal muscle cells via an insulin-independent pathway dependent on calcium. The beneficial effects of shikonin on glucose metabolism, both in vitro and in vivo, show that the compound possesses properties that make it of considerable interest for developing novel treatment of type 2 diabetes.

  2. Individually reared rats

    International Nuclear Information System (INIS)

    Kraeuchi, K.; Gentsch, C.; Feer, H.

    1981-01-01

    The influence of social isolation in rats on postsynaptic alpha 1 - and beta-adrenergic receptors, on the cAMP generating system and on the presynaptic uptake mechanism in the central noradrenergic system was examined in different brain regions. Rearing rats in isolation from the 19th day of life for 12 weeks leads in all regions to a general tendency for a reduction in 3 H-DHA binding, to an enhanced 3 H-WB4101 binding and to a decreased responsiveness of the noradrenaline sensitive cAMP generating system. These changes reach significance only in the pons-medulla-thallamusregion. Isolated rats showed an increased synaptosomal uptake of noradrenaline, most pronounced and significant in the hypothalamus. Our data provide further support for a disturbance in central noradrenergic function in isolated rats. (author)

  3. Detection of satellite cells during skeletal muscle wound healing in rats: time-dependent expressions of Pax7 and MyoD in relation to wound age.

    Science.gov (United States)

    Tian, Zhi-Ling; Jiang, Shu-Kun; Zhang, Miao; Wang, Meng; Li, Jiao-Yong; Zhao, Rui; Wang, Lin-Lin; Li, Shan-Shan; Liu, Min; Zhang, Meng-Zhou; Guan, Da-Wei

    2016-01-01

    The study was focused on time-dependent expressions of paired-box transcription factor 7 (Pax7) and myoblast determination protein (MyoD) during skeletal muscle wound healing. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male rats. Samples were taken at 1, 3, 5, 7, 9, 13, 17, and 21 days after injury, respectively (five rats in each posttraumatic interval). Five rats were employed as control. By morphometric analysis, the data based on the number of Pax7(+)/MyoD(-), Pax7(+)/MyoD(+), and Pax7(-)/MyoD(+) cells were highly correlated with the wound age. Pax7 and MyoD expressions were upregulated after injury by Western blot and quantitative real-time PCR assays. The relative quantity of Pax7 protein peaked at 5 days after injury, which was >1.13, and decreased thereafter. Similarly, the relative quantity of MyoD mRNA expression peaked at 3 days after injury, which was >2.59. The relative quantity of Pax7 protein >0.73 or mRNA expression >2.38 or the relative quantity of MyoD protein >1.33 suggested a wound age of 3 to 7 days. The relative quantity of MyoD mRNA expression >2.02 suggested a wound age of 1 to 7 days post-injury. In conclusion, the expressions of Pax7 and MyoD are upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting that Pax7 and MyoD may be potential markers for wound age estimation in skeletal muscle.

  4. Calpain 3 Expression Pattern during Gastrocnemius Muscle Atrophy and Regeneration Following Sciatic Nerve Injury in Rats

    Directory of Open Access Journals (Sweden)

    Ronghua Wu

    2015-11-01

    Full Text Available Calpain 3 (CAPN3, also known as p94, is a skeletal muscle-specific member of the calpain family that is involved in muscular dystrophy; however, the roles of CAPN3 in muscular atrophy and regeneration are yet to be understood. In the present study, we attempted to explain the effect of CAPN3 in muscle atrophy by evaluating CAPN3 expression in rat gastrocnemius muscle following reversible sciatic nerve injury. After nerve injury, the wet weight ratio and cross sectional area (CSA of gastrocnemius muscle were decreased gradually from 1–14 days and then recovery from 14–28 days. The active form of CAPN3 (~62 kDa protein decreased slightly on day 3 and then increased from day 7 to 14 before a decrease from day 14 to 28. The result of linear correlation analysis showed that expression of the active CAPN3 protein level was negatively correlated with muscle wet weight ratio. CAPN3 knockdown by short interfering RNA (siRNA injection improved muscle recovery on days 7 and 14 after injury as compared to that observed with control siRNA treatment. Depletion of CAPN3 gene expression could promote myoblast differentiation in L6 cells. Based on these findings, we conclude that the expression pattern of the active CAPN3 protein is linked to muscle atrophy and regeneration following denervation: its upregulation during early stages may promote satellite cell renewal by inhibiting differentiation, whereas in later stages, CAPN3 expression may be downregulated to stimulate myogenic differentiation and enhance recovery. These results provide a novel mechanistic insight into the role of CAPN3 protein in muscle regeneration after peripheral nerve injury.

  5. Fine-structural distribution of MMP-2 and MMP-9 activities in the rat skeletal muscle upon training: a study by high-resolution in situ zymography.

    Science.gov (United States)

    Yeghiazaryan, Marine; Żybura-Broda, Katarzyna; Cabaj, Anna; Włodarczyk, Jakub; Sławińska, Urszula; Rylski, Marcin; Wilczyński, Grzegorz M

    2012-07-01

    Matrix metalloproteinases (MMPs) are key regulators of extracellular matrix remodeling, but have also important intracellular targets. The purpose of this study was to examine the activity and subcellular localization of the gelatinases MMP-2 and MMP-9 in skeletal muscle of control and physically trained rats. In control hind limb muscle, the activity of the gelatinases was barely detectable. In contrast, after 5 days of intense exercise, in Soleus (Sol), but not Extensor digitorum longus (EDL) muscle, significant upregulation of gelatinolytic activity in myofibers was observed mainly in the nuclei, as assessed by high resolution in situ zymography. The nuclei of quiescent satellite cells did not contain the activity. Within the myonuclei, the gelatinolytic activity colocalized with an activated RNA Polymerase II. Also in Sol, but not in EDL, there were few foci of mononuclear cells with strongly positive cytoplasm, associated with apparent necrotic myofibers. These cells were identified as activated satellite cells/myoblasts. No extracellular gelatinase activity was observed. Gel zymography combined with subcellular fractionation revealed training-related upregulation of active MMP-2 in the nuclear fraction, and increase of active MMP-9 in the cytoplasmic fraction of Sol. Using RT-PCR, selective increase in MMP-9 mRNA was observed. We conclude that training activates nuclear MMP-2, and increases expression and activity of cytoplasmic MMP-9 in Sol, but not in EDL. Our results suggest that the gelatinases are involved in muscle adaptation to training, and that MMP-2 may play a novel role in myonuclear functions.

  6. Structure of the vitreoretinal border region in spontaneously hypertensive rats (SHR rats)

    DEFF Research Database (Denmark)

    Heegaard, Steffen

    1993-01-01

    Øjenpatologi, vitreoretinal border region, inner limiting membrane of the retina, spontaneously hypertensive rats, SHR rats, ultrastructure......Øjenpatologi, vitreoretinal border region, inner limiting membrane of the retina, spontaneously hypertensive rats, SHR rats, ultrastructure...

  7. Rat bite fever.

    NARCIS (Netherlands)

    Gaastra, W.; Boot, R.G.A.; Ho, H.; Lipman, L.J.A.

    2009-01-01

    Rat bite fever (RBF) is a bacterial zoonosis for which two causal bacterial species have been identified: Streptobacillis moniliformis and Spirillum minus. Haverhill fever (HF) is a form of S. moniliformis infection believed to develop after ingestion of contaminated food or water. Here the

  8. Novel Sulfur Metabolites of Garlic Attenuate Cardiac Hypertrophy and Remodeling through Induction of Na+/K+-ATPase Expression.

    Science.gov (United States)

    Khatua, Tarak N; Borkar, Roshan M; Mohammed, Soheb A; Dinda, Amit K; Srinivas, R; Banerjee, Sanjay K

    2017-01-01

    Epidemiologic studies show an inverse correlation between garlic consumption and progression of cardiovascular disease. However, the molecular basis for the beneficial effect of garlic on the heart is not known. Therefore, the objective of the present study was to (1) investigate the effect of raw garlic on isoproterenol (Iso) induced cardiac hypertrophy (2) find the active metabolites of garlic responsible for the beneficial effect. Cardiac hypertrophy was induced in rats by subcutaneous single injection of Iso 5 mg kg -1 day -1 for 15 days and the effect of garlic (250 mg/kg/day orally) was evaluated. Garlic metabolites in in vivo were identified by LC/MS study. The effect of garlic and its metabolites were evaluated against hypertrophy in H9C2 cells. Garlic normalized cardiac oxidative stress after Iso administration. Cardiac pathology and mitochondrial enzyme activities were improved in hypertrophy heart after garlic administration. Decreased Na + /K + -ATPase protein level that observed in hypertrophy heart was increased after garlic administration. We identified three garlic metabolites in rat serum. To confirm the role of garlic metabolites on cardiac hypertrophy, Na + /K + -ATPase expression and intracellular calcium levels were measured after treating H9C2 cells with raw garlic and two of its active metabolites, allyl methyl sulfide and allyl methyl sulfoxide. Raw garlic and both metabolites increased Na + /K + -ATPase protein level and decreased intracellular calcium levels and cell size in Iso treated H9C2 cells. This antihypertrophic effect of garlic and its sulfur metabolites were lost in H9C2 cells in presence of Na + /K + -ATPase inhibitor. In conclusion, garlic and its active metabolites increased Na + /K + -ATPase in rat heart, and attenuated cardiac hypertrophy and associated remodeling. Our data suggest that identified new garlic metabolites may be useful for therapeutic intervention against cardiac hypertrophy.

  9. Imidazoquinolinone, imidazopyridine, and isoquinolindione derivatives as novel and potent inhibitors of the poly(ADP-ribose) polymerase (PARP): a comparison with standard PARP inhibitors.

    Science.gov (United States)

    Eltze, Tobias; Boer, Rainer; Wagner, Thomas; Weinbrenner, Steffen; McDonald, Michelle C; Thiemermann, Christoph; Bürkle, Alexander; Klein, Thomas

    2008-12-01

    We have identified three novel structures for inhibitors of the poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA and implicated in DNA repair, apoptosis, organ dysfunction or necrosis. 2-[4-(5-Methyl-1H-imidazol-4-yl)-piperidin-1-yl]-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK49187), 2-(4-pyridin-2-yl-phenyl)-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK236864), 6-chloro-8-hydroxy-2,3-dimethyl-imidazo-[1,2-alpha]-pyridine (BYK20370), and 4-(1-methyl-1H-pyrrol-2-ylmethylene)-4H-isoquinolin-1,3-dione (BYK204165) inhibited cell-free recombinant human PARP-1 with pIC(50) values of 8.36, 7.81, 6.40, and 7.35 (pK(i) 7.97, 7.43, 5.90, and 7.05), and murine PARP-2 with pIC(50) values of 7.50, 7.55, 5.71, and 5.38, respectively. BYK49187, BYK236864, and BYK20370 displayed no selectivity for PARP-1/2, whereas BYK204165 displayed 100-fold selectivity for PARP-1. The IC(50) values for inhibition of poly(ADP-ribose) synthesis in human lung epithelial A549 and cervical carcinoma C4I cells as well in rat cardiac myoblast H9c2 cells after PARP activation by H(2)O(2) were highly significantly correlated with those at cell-free PARP-1 (r(2) = 0.89-0.96, P < 0.001) but less with those at PARP-2 (r(2) = 0.78-0.84, P < 0.01). The infarct size caused by coronary artery occlusion and reperfusion in the anesthetized rat was reduced by 22% (P < 0.05) by treatment with BYK49187 (3 mg/kg i.v. bolus and 3 mg/kg/h i.v. during 2-h reperfusion), whereas the weaker PARP inhibitors, BYK236864 and BYK20370, were not cardioprotective. In conclusion, the imidazoquinolinone BYK49187 is a potent inhibitor of human PARP-1 activity in cell-free and cellular assays in vitro and reduces myocardial infarct size in vivo. The isoquinolindione BYK204165 was found to be 100-fold more selective for PARP-1. Thus, both compounds might be novel and valuable tools for investigating PARP-1-mediated effects.

  10. Laughing rats are optimistic.

    Directory of Open Access Journals (Sweden)

    Rafal Rygula

    Full Text Available Emotions can bias human decisions- for example depressed or anxious people tend to make pessimistic judgements while those in positive affective states are often more optimistic. Several studies have reported that affect contingent judgement biases can also be produced in animals. The animals, however, cannot self-report; therefore, the valence of their emotions, to date, could only be assumed. Here we present the results of an experiment where the affect-contingent judgement bias has been produced by objectively measured positive emotions. We trained rats in operant Skinner boxes to press one lever in response to one tone to receive a food reward and to press another lever in response to a different tone to avoid punishment by electric foot shock. After attaining a stable level of discrimination performance, the animals were subjected to either handling or playful, experimenter-administered manual stimulation - tickling. This procedure has been confirmed to induce a positive affective state in rats, and the 50-kHz ultrasonic vocalisations (rat laughter emitted by animals in response to tickling have been postulated to index positive emotions akin to human joy. During the tickling and handling sessions, the numbers of emitted high-frequency 50-kHz calls were scored. Immediately after tickling or handling, the animals were tested for their responses to a tone of intermediate frequency, and the pattern of their responses to this ambiguous cue was taken as an indicator of the animals' optimism. Our findings indicate that tickling induced positive emotions which are directly indexed in rats by laughter, can make animals more optimistic. We demonstrate for the first time a link between the directly measured positive affective state and decision making under uncertainty in an animal model. We also introduce innovative tandem-approach for studying emotional-cognitive interplay in animals, which may be of great value for understanding the emotional

  11. Tubuloglomerular feedback in Dahl rats

    DEFF Research Database (Denmark)

    Karlsen, F M; Leyssac, P P; Holstein-Rathlou, N H

    1998-01-01

    in both Dahl-S and salt-resistant Dahl rats on high- and low-salt diets. TGF was investigated in the closed-loop mode with a videometric technique, in which the response in late proximal flow rate to perturbations in Henle flow rate was measured. All Dahl rats showed a similar compensatory response...

  12. Neutron radiography of osteopetrotic rat

    International Nuclear Information System (INIS)

    Graf, B.; Renard, G.; Le Gall, J.; Laporte, A.

    1983-01-01

    The osteopetrotic disease is characterized by bone and cartilage tissue coexistence in the medullary space of long bones. The authors have studied ''congenital osteopetrosis'' of ''op'' rats. Comparing radiography, neutrography and histology, the evolution of the ''osteopetrotic disease'' and the healing of the ill rats by a single injection of bone marrow from normal animals is shown. (Auth.)

  13. The three-kidney rat

    International Nuclear Information System (INIS)

    Provoost, A.P.; Van Aken, M.

    1984-01-01

    In contrast to the numerous research into the adaption of renal function when nephons are lost, much less attention has been paid to the effects of an extra kidney. Through the availability of inbred rat strains, techniques to transplant rat kidneys, and methods to measure total and individual kidney function repeatedly in the same animal, it became possible to study the renal function in rats with three kidneys. Adult male rats of a highly inbred Wistar strain were used. Nine recipients of a third kidney (3-K) were compared with 5 sham operated control (2-K) rats. The total GFR, as measured by the plasma clearance of Cr-5l EDTA, was taken 1,3,6,9, and 15 weeks after operation. The contribution of each kidney to the total renal function was determined by a Tc-99m DTPA scan performed at weeks 10 and 16. After transplantation the total GFR of 3-K rats was, in general, not different from the value before transplantation or from that of 2-K rats. The lack of increase of the GFR of 3-K rats was not the result of a non-functioning graft

  14. Do rats have orgasms?

    Science.gov (United States)

    Pfaus, James G.; Scardochio, Tina; Parada, Mayte; Gerson, Christine; Quintana, Gonzalo R.; Coria-Avila, Genaro A.

    2016-01-01

    Background Although humans experience orgasms with a degree of statistical regularity, they remain among the most enigmatic of sexual responses; difficult to define and even more difficult to study empirically. The question of whether animals experience orgasms is hampered by similar lack of definition and the additional necessity of making inferences from behavioral responses. Method Here we define three behavioral criteria, based on dimensions of the subjective experience of human orgasms described by Mah and Binik, to infer orgasm-like responses (OLRs) in other species: 1) physiological criteria that include pelvic floor and anal muscle contractions that stimulate seminal emission and/or ejaculation in the male, or that stimulate uterine and cervical contractions in the female; 2) short-term behavioral changes that reflect immediate awareness of a pleasurable hedonic reward state during copulation; and 3) long-term behavioral changes that depend on the reward state induced by the OLR, including sexual satiety, the strengthening of patterns of sexual arousal and desire in subsequent copulations, and the generation of conditioned place and partner preferences for contextual and partner-related cues associated with the reward state. We then examine whether physiological and behavioral data from observations of male and female rats during copulation, and in sexually-conditioned place- and partner-preference paradigms, are consistent with these criteria. Results Both male and female rats display behavioral patterns consistent with OLRs. Conclusions The ability to infer OLRs in rats offers new possibilities to study the phenomenon in neurobiological and molecular detail, and to provide both comparative and translational perspectives that would be useful for both basic and clinical research. PMID:27799081

  15. Do rats have orgasms?

    Directory of Open Access Journals (Sweden)

    James G. Pfaus

    2016-10-01

    Full Text Available Background: Although humans experience orgasms with a degree of statistical regularity, they remain among the most enigmatic of sexual responses; difficult to define and even more difficult to study empirically. The question of whether animals experience orgasms is hampered by similar lack of definition and the additional necessity of making inferences from behavioral responses. Method: Here we define three behavioral criteria, based on dimensions of the subjective experience of human orgasms described by Mah and Binik, to infer orgasm-like responses (OLRs in other species: 1 physiological criteria that include pelvic floor and anal muscle contractions that stimulate seminal emission and/or ejaculation in the male, or that stimulate uterine and cervical contractions in the female; 2 short-term behavioral changes that reflect immediate awareness of a pleasurable hedonic reward state during copulation; and 3 long-term behavioral changes that depend on the reward state induced by the OLR, including sexual satiety, the strengthening of patterns of sexual arousal and desire in subsequent copulations, and the generation of conditioned place and partner preferences for contextual and partner-related cues associated with the reward state. We then examine whether physiological and behavioral data from observations of male and female rats during copulation, and in sexually-conditioned place- and partner-preference paradigms, are consistent with these criteria. Results: Both male and female rats display behavioral patterns consistent with OLRs. Conclusions: The ability to infer OLRs in rats offers new possibilities to study the phenomenon in neurobiological and molecular detail, and to provide both comparative and translational perspectives that would be useful for both basic and clinical research.

  16. The academic rat race

    DEFF Research Database (Denmark)

    Landes, Xavier; Andersen, Martin Marchman; Nielsen, Morten Ebbe Juul

    2012-01-01

    : an increased pressure to produce articles (in peer-reviewed journals) has created an unbalanced emphasis on the research criterion at the expense of the latter two. More fatally, this pressure has turned academia into a rat race, leading to a deep change in the fundamental structure of academic behaviour......, and entailing a self-defeating and hence counter-productive pattern, where more publications is always better and where it becomes increasingly difficult for researchers to keep up with the new research in their field. The article identifies the pressure to publish as a problem of collective action. It ends up...

  17. The Rat Race

    CERN Multimedia

    Stephen Haywood

    Dear Muriel, Being an animal, you are probably more familiar with rats than most of us. Yet it seems to me that our Community (not just ATLAS) is stuck in a huge "rat race". I am somewhat mystified as to how we have got ourselves into this and I wonder whether you or your readers could explain this - I give my own observations below. In HEP and ATLAS specifically, we are all working long hours and we are all becoming exhausted. There are people at Point 1 who are working day and night, every day of the week; there are people writing software who send emails round the clock, including weekends. It is one thing to have bursts of activity which require us to put in some longer hours, but in ATLAS, the bursts last months or years. I have been on ATLAS 14 years and it has felt like one endless rush. Why do we do this? We are all highly motivated, we love our work and want to succeed individually and collectively. We are parts of various teams, and we do not want to let the side down. We worked hard at school an...

  18. Serotonin metabolism in rat brain

    International Nuclear Information System (INIS)

    Schutte, H.H.

    1976-01-01

    The metabolism of serotonin in rat brain was studied by measuring specific activities of tryptophan in plasma and of serotonin, 5-hydroxyindole acetic acid and tryptophan in the brain after intravenous injection of tritiated tryptophan. For a detailed analysis of the specific activities, a computer simulation technique was used. It was found that only a minor part of serotonin in rat brain is synthesized from tryptophan rapidly transported from the blood. It is suggested that the brain tryptophan originates from brain proteins. It was also found that the serotonin in rat brain is divided into more than one metabolic compartment

  19. Cardioprotection against experimental myocardial ischemic injury using cornin

    Directory of Open Access Journals (Sweden)

    Y. Xu

    2016-01-01

    Full Text Available Phosphorylated-cyclic adenosine monophosphate response element-binding protein (Phospho-CREB has an important role in the pathogenesis of myocardial ischemia. We isolated the iridoid glycoside cornin from the fruit of Verbena officinalis L, investigated its effects against myocardial ischemia and reperfusion (I/R injury in vivo, and elucidated its potential mechanism in vitro. Effects of cornin on cell viability, as well as expression of phospho-CREB and phospho-Akt in hypoxic H9c2 cells in vitro, and myocardial I/R injury in vivo, were investigated. Cornin attenuated hypoxia-induced cytotoxicity significantly in H9c2 cells in a concentration-dependent manner. Treatment of H9c2 cells with cornin (10 µM blocked the reduction of expression of phospho-CREB and phospho-Akt in a hypoxic condition. Treatment of rats with cornin (30 mg/kg, iv protected them from myocardial I/R injury as indicated by a decrease in infarct volume, improvement in hemodynamics, and reduction of severity of myocardial damage. Cornin treatment also attenuated the reduction of expression of phospho-CREB and phospho-Akt in ischemic myocardial tissue. These data suggest that cornin exerts protective effects due to an increase in expression of phospho-CREB and phospho-Akt.

  20. Influence of interleukin-1 beta gene polymorphisms on the risk of myocardial infarction and ischemic stroke at young age in vivo and in vitro.

    Science.gov (United States)

    Yang, Bo; Zhao, Hua; X, Bin; Wang, Ya-Bin; Zhang, Jian; Cao, Yu-Kang; Wu, Qing; Cao, Feng

    2015-01-01

    In this study, by using vivo and vitro model, we assessed whether interleukin (IL)-1beta gene polymorphisms influence on the risk of myocardial infarction and ischemic stroke at young age. 147 patients (age stroke were deeded as control group and greed to give blood samples for DNA analysis and biochemical measurements by written informed consent. IL-1β-511 wild type (WT, CC) and SNP (TT) were established and transfected into Rat myocardial H9c2 cell and Mouse brain endothelial bEND.3 cells. In Young Age MI or stroke patients, the IL-1β levels of patients with 511CC are higher than that of patients with 511TT. In our study, NF-κB miRNA, iNOS activity, NF-κB, iNOS and Bax protein expressions of MI-induced H9c2 cell or stroke-induced bEND.3 cells in IL-1β-511TT group were lower than those of IL-1β-511CC. Additionally, the protein expression of MMP-2 of MI-induced H9c2 cell or stroke-induced bEND.3 cells in IL-1β-511TT group were higher than that of IL-1β 511CC group. In conclusion, our data indicate that IL-1β-511TT/CC influence on the risk of myocardial infarction and ischemic stroke at young age through NF-κB, iNOS, MMP-2 and Bax.

  1. Beta-endorphin in genetically hypoprolactinemic rat: IPL nude rat

    International Nuclear Information System (INIS)

    Cohen, H.; Sabbagh, I.; Abou-Samra, A.B.; Bertrand, J.

    1986-01-01

    Beta-endorphin has been reported to regulate not only stress- and suckling-induced but also basal prolactin secretion. In the aim to better evaluate the endogenous beta-endorphin-prolactin interrelation, the authors measured beta-endorphin levels in a new rat strain, genetically hypoprolactinemic and characterized by a total lack of lactation: IPL nude rat. Beta-endorphin was measured using a specific anti-h-β endorphin in plasma and extracts of anterior and neurointermediate lobes of the pituitary, hypothalamus and brain. Pituitary extracts were also chromatographed on Sephadex G50 column. Results obtained showed that in IPL nude females on diestrus and males, the beta-endorphin contents of the neurointermediate lobe was significantly lower than in normal rats, while the values found in the other organs and plasma were similar. However, elution pattern of the anterior pituitary extracts from male rats showed greater immunoactivity eluting as I 125 h-beta-endorphin than in normal rat; this was not the case for the female rat. These results are consistent with a differential regulation of beta-endorphin levels of anterior and neurointermediate lobe by catecholamines. Moreover they suggest that PRL secretion was more related to neurointermediate beta-endorphin. 40 references, 2 figures, 4 tables

  2. Beta-endorphin in genetically hypoprolactinemic rat: IPL nude rat

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, H.; Sabbagh, I.; Abou-Samra, A.B.; Bertrand, J.

    1986-01-20

    Beta-endorphin has been reported to regulate not only stress- and suckling-induced but also basal prolactin secretion. In the aim to better evaluate the endogenous beta-endorphin-prolactin interrelation, the authors measured beta-endorphin levels in a new rat strain, genetically hypoprolactinemic and characterized by a total lack of lactation: IPL nude rat. Beta-endorphin was measured using a specific anti-h-..beta.. endorphin in plasma and extracts of anterior and neurointermediate lobes of the pituitary, hypothalamus and brain. Pituitary extracts were also chromatographed on Sephadex G50 column. Results obtained showed that in IPL nude females on diestrus and males, the beta-endorphin contents of the neurointermediate lobe was significantly lower than in normal rats, while the values found in the other organs and plasma were similar. However, elution pattern of the anterior pituitary extracts from male rats showed greater immunoactivity eluting as I/sup 125/ h-beta-endorphin than in normal rat; this was not the case for the female rat. These results are consistent with a differential regulation of beta-endorphin levels of anterior and neurointermediate lobe by catecholamines. Moreover they suggest that PRL secretion was more related to neurointermediate beta-endorphin. 40 references, 2 figures, 4 tables.

  3. Intramitochondrial autonomy in rat tissues

    International Nuclear Information System (INIS)

    Subramanian, M.; Rajwade, M.S.; Satav, J.G.; Katyare, S.S.; Fatterpaker, P.; Sreenivasan, A.

    1974-01-01

    The biogenesis of mitochondria in rat liver and their protein turnover has been investigated using 1- 14 C leucine. The results indicate that intramitochondrial autonomy exists both with respect to their genesis and turnover. (M.G.B.)

  4. Autoshaping in micrencephalic rats

    International Nuclear Information System (INIS)

    Goldstein, L.H.; Oakley, D.A.

    1989-01-01

    An autoshaping procedure in which the illumination of a lever was predictive of food reinforcement was used to compare learning in rats with micrencephaly induced by irradiation on the 16th day of gestation and in sham-irradiated controls. Both groups showed equivalent levels of lever-directed activity, and the micrencephalic animals differentiated as well as the control animals between the predictive lever and a nonpredictive lever. The micrencephalic animals were able to redistribute their lever-directed activity when the significance of the levers was reversed and did so more readily than the control animals. Results support the claim that association learning survives either traumatic or developmental neocortical damage and have implications for remedial procedures following both head injury and developmental cerebral pathology in humans

  5. Autoshaping in micrencephalic rats.

    Science.gov (United States)

    Goldstein, L H; Oakley, D A

    1989-06-01

    An autoshaping procedure in which the illumination of a lever was predictive of food reinforcement was used to compare learning in rats with micrencephaly induced by irradiation on the 16th day of gestation and in sham-irradiated controls. Both groups showed equivalent levels of lever-directed activity, and the micrencephalic animals differentiated as well as the control animals between the predictive lever and a nonpredictive lever. The micrencephalic animals were able to redistribute their lever-directed activity when the significance of the levers was reversed and did so more readily than the control animals. Results support the claim that association learning survives either traumatic or developmental neocortical damage and have implications for remedial procedures following both head injury and developmental cerebral pathology in humans.

  6. Biokinetics of radiotellurium in rats

    International Nuclear Information System (INIS)

    Nishimura, Y.; Sahoo, S.K.; Kim, S.; Homma-Takeda, S.; Watanabe, Y.; Inaba, J.

    2003-01-01

    Radiotellurium is present in the environment primarily due to its release during nuclear reactor accidents. Little is known of tellurium metabolism in juveniles, although the element is relatively abundant and has a number of industrial uses. A biokinetic study of radiotellurium in rats was done using gamma-ray counting. Wistar strain rats were used to determine the uptake of H 2 123 Te m O 3 by the whole-body retention of juvenile rats and the conceptus in relation to its gestational stages, by measurements in the placenta, fetal membranes, fetal fluid, and fetus. The whole-body retention of 123 Te m in juvenile rats was higher than that of adult rats. The relative concentration in the placenta and fetal membranes was higher than in the fetus. No activity was observed in the fetal fluid. These results indicate that the placenta and fetal membranes play significant roles as barriers to the transfer of 123 Te m into the fetus. The ratio, relative concentration in fetus/relative concentration in mother (C F /C M ), was calculated. The C F /C M ratio was dependent on the stage of gestation and ranged from 0.2 to 0.5. A little 123 Te m was transferred to the suckling rats through the mother's milk when the isotope was administered intravenously to the mother. (author)

  7. Tryptophan depletion affects compulsive behaviour in rats

    DEFF Research Database (Denmark)

    Merchán, A; Navarro, S V; Klein, A B

    2017-01-01

    investigated whether 5-HT manipulation, through a tryptophan (TRP) depletion by diet in Wistar and Lister Hooded rats, modulates compulsive drinking in schedule-induced polydipsia (SIP) and locomotor activity in the open-field test. The levels of dopamine, noradrenaline, serotonin and its metabolite were......-depleted HD Wistar rats, while the LD Wistar and the Lister Hooded rats did not exhibit differences in SIP. In contrast, the TRP-depleted Lister Hooded rats increased locomotor activity compared to the non-depleted rats, while no differences were found in the Wistar rats. Serotonin 2A receptor binding...

  8. Population Structure of Rat-Derived Pneumocystis carinii in Danish Wild Rats

    DEFF Research Database (Denmark)

    Palmer, Robert J.; Settnes, Osvald P.; Lodal, Jens

    2000-01-01

    The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. cari...

  9. DOPA, norepinephrine, and dopamine in rat tissues

    DEFF Research Database (Denmark)

    Eldrup, E; Richter, Erik; Christensen, N J

    1989-01-01

    We studied the effect of unilateral sympathectomy on rat quadriceps and gastrocnemius muscle concentrations of endogenous dihydroxyphenylalanine (DOPA), dopamine (DA), and norepinephrine (NE) and assessed the relationships between these catecholamines in several rat tissues. Catecholamines were...

  10. False Context Fear Memory in Rats

    Science.gov (United States)

    Bae, Sarah; Holmes, Nathan M.; Westbrook, R. Frederick

    2015-01-01

    Four experiments used rats to study false context fear memories. In Experiment 1, rats were pre-exposed to a distinctive chamber (context A) or to a control environment (context C), shocked after a delay in a second chamber (context B) and tested either in B or A. Rats pre-exposed to A froze just as much as control rats in B but more than control…

  11. EFFECT OF ETHANOL ON HEPATOBILIARY TRANSPORT OF CATIONIC DRUGS - A STUDY IN THE ISOLATED-PERFUSED RAT-LIVER, RAT HEPATOCYTES AND RAT MITOCHONDRIA

    NARCIS (Netherlands)

    STEEN, H; MEIJER, DKF; Merema, M.T.

    The effect of ethanol on the hepatic uptake of various cationic drugs was studied in isolated perfused rat livers, isolated rat hepatocytes and isolated rat liver mitochondria. In isolated rat hepatocytes and in isolated perfused rat livers, the uptake of the model organic cation

  12. Apigenin reduce lipoteichoic acid-induced inflammatory response in rat cardiomyoblast cells.

    Science.gov (United States)

    Gutiérrez-Venegas, Gloria; González-Rosas, Zeltzin

    2017-02-01

    Infective endocarditis is caused by Streptococcus sanguinis present in dental plaque, which can induce inflammatory responses in the endocardium. The present study depicts research on the properties of apigenin in embryonic mouse heart cells (H9c2) treated with lipoteichoic acid (LTA) obtained from S. sanguinis. Interleukin-1β and cyclooxygenase (COX)-2 expression were detected by reverse transcriptase polymerase chain reaction. In addition, western blot assays and immuno-fluorescence staining were used to assess translocation of nuclear factor kappa beta (NF-κB), degradation of IκB, as well as activity of the mitogen activated protein kinases: extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). Effect of apigenin on cell viability was equally assessed in other experimental series. Our results showed that apigenin blocked activation of ERK, JNK, and p38 in cardiomyocytes treated with LTA in a dose-dependent fashion. Moreover, apigenin showed no cytotoxic effects; it blocked NF-κB translocation and IκB degradation. Our findings suggested that apigenin possessed potential value in the treatment of infectious endocarditis.

  13. The serotonin transporter knockout rat : A review

    NARCIS (Netherlands)

    Olivier, Jocelien; Cools, Alexander; Ellenbroek, Bart A.; Cuppen, E.; Homberg, Judith; Kalueff, Allan V.; LaPorte, Justin L.

    2010-01-01

    This chapter dicusses the most recent data on the serotonin transporter knock-out rat, a unique rat model that has been generated by target-selected N-ethyl-N-nitrosourea (ENU) driven mutagenesis. The knock-out rat is the result of a premature stopcodon in the serotonin transporter gene, and the

  14. Tuberculosis Detection by Giant African Pouched Rats

    Science.gov (United States)

    Poling, Alan; Weetjens, Bart; Cox, Christophe; Beyene, Negussie; Durgin, Amy; Mahoney, Amanda

    2011-01-01

    In recent years, operant discrimination training procedures have been used to teach giant African pouched rats to detect tuberculosis (TB) in human sputum samples. This article summarizes how the rats are trained and used operationally, as well as their performance in studies published to date. Available data suggest that pouched rats, which can…

  15. Teratology studies in the rat.

    Science.gov (United States)

    Leroy, Mariline; Allais, Linda

    2013-01-01

    The rat is the rodent species of choice for the regulatory safety testing of xenobiotics, such as medicinal products, food additives, and other chemicals. Many decades of experience and extensive data have accumulated for both general and developmental toxicology investigations in this species. The high fertility and large litter size of the rat are advantages for teratogenicity testing. The study designs are well defined in the regulatory guidelines and are relatively standardized between testing laboratories across the world. Teratology studies address maternal- and embryo-toxicity following exposure during the period of organogenesis. This chapter describes the design and conduct of a teratology study in the rat in compliance with the regulatory guidelines. The procedures for the handling and housing of the pregnant animals, the caesarean examinations and the sampling of fetuses for morphological examinations are described. The utility and design of preliminary studies and the inclusion of satellite animals in the main study for toxicokinetic sampling are discussed.

  16. Voluntary Sleep Loss in Rats

    Science.gov (United States)

    Oonk, Marcella; Krueger, James M.; Davis, Christopher J.

    2016-01-01

    Study Objectives: Animal sleep deprivation (SDEP), in contrast to human SDEP, is involuntary and involves repeated exposure to aversive stimuli including the inability of the animal to control the waking stimulus. Therefore, we explored intracranial self-stimulation (ICSS), an operant behavior, as a method for voluntary SDEP in rodents. Methods: Male Sprague-Dawley rats were implanted with electroencephalography/electromyography (EEG/EMG) recording electrodes and a unilateral bipolar electrode into the lateral hypothalamus. Rats were allowed to self-stimulate, or underwent gentle handling-induced SDEP (GH-SDEP), during the first 6 h of the light phase, after which they were allowed to sleep. Other rats performed the 6 h ICSS and 1 w later were subjected to 6 h of noncontingent stimulation (NCS). During NCS the individual stimulation patterns recorded during ICSS were replayed. Results: After GH-SDEP, ICSS, or NCS, time in nonrapid eye movement (NREM) sleep and rapid eye movement (REM) sleep increased. Further, in the 24 h after SDEP, rats recovered all of the REM sleep lost during SDEP, but only 75% to 80% of the NREM sleep lost, regardless of the SDEP method. The magnitude of EEG slow wave responses occurring during NREM sleep also increased after SDEP treatments. However, NREM sleep EEG slow wave activity (SWA) responses were attenuated following ICSS, compared to GH-SDEP and NCS. Conclusions: We conclude that ICSS and NCS can be used to sleep deprive rats. Changes in rebound NREM sleep EEG SWA occurring after ICSS, NCS, and GH-SDEP suggest that nonspecific effects of the SDEP procedure differentially affect recovery sleep phenotypes. Citation: Oonk M, Krueger JM, Davis CJ. Voluntary sleep loss in rats. SLEEP 2016;39(7):1467–1479. PMID:27166236

  17. Rat bite fever without fever.

    Science.gov (United States)

    Stehle, P; Dubuis, O; So, A; Dudler, J

    2003-09-01

    Rat bite fever is a rarely reported acute febrile bacterial illness caused by Streptobacillus moniliformis or Spirillum minus following a rat bite. It is classically characterised by abrupt onset of fever with rigors, myalgias, headache, and the appearance of a generalised maculopapular petechial skin rash. Polyarthritis complicates the course of the disease in up to 50% of infected patients, and numerous hurdles can make the diagnosis particularly difficult in the absence of fever or rash, as in the present case. A high degree of awareness is necessary to make the correct diagnosis in such cases. Diagnosis has important prognostic implications as the disease is potentially lethal, but easily treatable.

  18. Treatment of diabetic rats with encapsulated islets.

    Science.gov (United States)

    Sweet, Ian R; Yanay, Ofer; Waldron, Lanaya; Gilbert, Merle; Fuller, Jessica M; Tupling, Terry; Lernmark, Ake; Osborne, William R A

    2008-12-01

    Immunoprotection of islets using bioisolator systems permits introduction of allogeneic cells to diabetic patients without the need for immunosuppression. Using TheraCyte immunoisolation devices, we investigated two rat models of type 1 diabetes mellitus (T1DM), BB rats and rats made diabetic by streptozotocin (STZ) treatment. We chose to implant islets after the onset of diabetes to mimic the probable treatment of children with T1DM as they are usually diagnosed after disease onset. We encapsulated 1000 rat islets and implanted them subcutaneously (SQ) into diabetic biobreeding (BB) rats and STZ-induced diabetic rats, defined as two or more consecutive days of blood glucose>350 mg/dl. Rats were monitored for weight and blood glucose. Untreated BB rats rapidly lost weight and were euthanized at >20% weight loss that occurred between 4 and 10 days from implantation. For period of 30-40 days following islet implantation weights of treated rats remained steady or increased. Rapid weight loss occurred after surgical removal of devices that contained insulin positive islets. STZ-treated rats that received encapsulated islets showed steady weight gain for up to 130 days, whereas untreated control rats showed steady weight loss that achieved >20% at around 55 days. Although islet implants did not normalize blood glucose, treated rats were apparently healthy and groomed normally. Autologous or allogeneic islets were equally effective in providing treatment. TheraCyte devices can sustain islets, protect allogeneic cells from immune attack and provide treatment for diabetic-mediated weight loss in both BB rats and STZ-induced diabetic rats.

  19. Induced Chronic Prostatitis in Rats

    African Journals Online (AJOL)

    Similarly, compared with reference group (176.1 ± 12.1 pg/ml), IL-1β level of prostate tissues of high-dose PCS ..... Fig 1: Effect of PCS extract on the histomorphology of prostate tissues in rats. ... involved with cellular recruitment, fever, acute.

  20. Isolating Lysosomes from Rat Liver.

    Science.gov (United States)

    Pryor, Paul R

    2016-04-01

    This protocol describes the generation of a fraction enriched in lysosomes from rat liver. The lysosomes are rapidly isolated using density-gradient centrifugation with gradient media that retain the osmolarity of the lysosomes such that they are functional and can be used in in vitro assays. © 2016 Cold Spring Harbor Laboratory Press.

  1. CCl4 cirrhosis in rats

    DEFF Research Database (Denmark)

    Fischer-Nielsen, A; Poulsen, H E; Hansen, B A

    1991-01-01

    Cirrhosis of the rat liver was induced by a 12 week individualized CCl4/phenobarbital treatment. After treatment, all surviving animals (81%) showed cirrhosis of the liver. The cirrhosis induced was irreversible when evaluated 24 weeks after cessation of treatment. Quantitative liver function...

  2. Whiskers aid anemotaxis in rats.

    Science.gov (United States)

    Yu, Yan S W; Graff, Matthew M; Bresee, Chris S; Man, Yan B; Hartmann, Mitra J Z

    2016-08-01

    Observation of terrestrial mammals suggests that they can follow the wind (anemotaxis), but the sensory cues underlying this ability have not been studied. We identify a significant contribution to anemotaxis mediated by whiskers (vibrissae), a modality previously studied only in the context of direct tactile contact. Five rats trained on a five-alternative forced-choice airflow localization task exhibited significant performance decrements after vibrissal removal. In contrast, vibrissal removal did not disrupt the performance of control animals trained to localize a light source. The performance decrement of individual rats was related to their airspeed threshold for successful localization: animals that found the task more challenging relied more on the vibrissae for localization cues. Following vibrissal removal, the rats deviated more from the straight-line path to the air source, choosing sources farther from the correct location. Our results indicate that rats can perform anemotaxis and that whiskers greatly facilitate this ability. Because air currents carry information about both odor content and location, these findings are discussed in terms of the adaptive significance of the interaction between sniffing and whisking in rodents.

  3. Acute renal failure in rats

    International Nuclear Information System (INIS)

    Cederholm, C.; Almen, T.; Bergquist, D.; Golman, K.; Takolander, R.; Malmoe Allmaenna Sjukhus

    1989-01-01

    It was demonstrated in rats that renal injury which follows transient renal hypoxia is potentiated by the contrast media metrizoate, ioxaglate, iopamidol and iohexol. Intravenous injection of 1 g I/kg of all four media alone to 82 rats caused no significant increase in serum urea 1, 3 and 7 days later. The percentage increase of serum urea is given in median values and interquartile range (in parentheses). Bilateral renal arterial occlusion alone for 40 minutes in 42 rats increased serum urea one day later by 40% (20-130). Intravenous injection of the media followed in one hour by bilateral renal arterial occlusion for 40 minutes in 104 rats caused serum urea to increase one day later by 130% (70-350) after metrizoate, by 220% (50-380) after ioxaglate, by 290 % (60-420) after iopamidol and by 160% (50-330) after iohexol. There were no significant differences between the potentiating effects of the various media on ischemic renal failure. (orig.)

  4. August rats are more resistant to arrhythmogenic effect of myocardial ischemia and reperfusion than Wistar rats.

    Science.gov (United States)

    Belkina, L M; Kirillina, T N; Pshennikova, M G; Arkhipenko, Yu V

    2002-06-01

    As differentiated from Wistar rats, myocardial ischemia and reperfusion produce no ventricular fibrillation in August rats. Pretreatment with nitric oxide synthase inhibitor Nw-nitro-L-arginine increased mortality rate in August rats with acute myocardial infarction from 20 to 40%. Under these conditions mortality rate in Wistar rats increased from 50 to 71%. Interstrain differences in the resistance of these animals to the arrhythmogenic effect of ischemia are probably associated with higher activity of the nitric oxide system in August rats compared to Wistar rats.

  5. Diminished hormonal responses to exercise in trained rats

    DEFF Research Database (Denmark)

    Galbo, H; Richter, Erik; Holst, J J

    1977-01-01

    Male rats (120 g) either were subjected to a 12-wk physical training program (T rats) or were sedentary controls (C rats). Subsequently the rats were killed at rest or after a 45- or 90-min forced swim. At rest, T rats had higher liver and muscle glycogen concentrations but lower plasma insulin...

  6. Dithiobiuret toxicity in the rat

    International Nuclear Information System (INIS)

    Williams, K.D.

    1985-01-01

    Raising the daily dose of dithiobiuret (DTB) in male rats from 0.5 to 1 to 5 mg/kg shortened the latency to the onset of flaccid muscle tone and associated diminished performance in a treadmill test from 7 to 5 to 3 days, respectively. Concomitant with the development of flaccid muscle tone gastrocnemius muscle contractions elicited by high frequency motor nerve stimulation were lower in peak tension and tended to fade more rapidly in DTB-treated rats than in control rats. Remarkably, rats treated with highly daily doses (10-16 mg/kg) of DTB were resistant to the expected development of DTB-induced flaccid muscle tone, and tetanic contractile abnormalities but a corresponding refractoriness to body weight loss, decreased fed and water intake, diuresis, and depression in water balance was not present. This nonselectivity of the refractory responses supported the results of a histopathological study indicating that DTB-induced neuromuscular toxicity was unlikely to be secondary to effect on other organ systems. It is not known whether the ultimate neurotoxin is DTB or a metabolite. In this regard, two pathways for the metabolism of DTB were proposed based on the results of thin-layer chromatography of urine samples from rats treated with either 14 C- or 35 S-DTB. One pathway involved the reversible oxidation of DTB to the disulfide-containing compound thiuret, and the other involved the replacement of a sulfur atom with oxygen to form monothiobiuret. Thiuret, but not monothiobiuret, possessed comparable toxicity to STB. This further suggested that redox cycling between DTB and thiuret could be an important contributing factor to the toxicity of DTB

  7. Sexual dimorphism in hybrids rats.

    Science.gov (United States)

    Garcia-Falgueras, Alicia; Pinos, Helena; Fernández, Rosa; Collado, Paloma; Pasaro, Eduardo; Segovia, Santiago; Guillamon, Antonio

    2006-12-06

    Laboratory rat strains descend from Wistar rats as a consequence of artificial selection. Previously we reported that the medial posterior division of the bed nucleus of the stria terminalis (BSTMP) was sexually dimorphic in Wistar and Long-Evans strains while the medial anterior division (BSTMA) and the locus coeruleus (LC) only showed sex differences in the ancestor Wistar strain. The lateral posterior division (BSTLP) was isomorphic in both strains. The present work studies the number of neurons in the BSTMP, BSTMA, BSTLP and LC of male and female Wistar and Long-Evans rats (F(0)) and their hybrid F(1) and F(2) generations. The BSTMP is sexually dimorphic in the F(0), F(1) and F(2) generations while sex differences in the LC are only seen in F(0) Wistar rats but not in the F(0) Long-Evans or the F(1) and F(2) hybrid generations. Sex differences in the BSTMA are seen in F(0) Wistar but not in F(0) Long-Evans rats and completely disappear in the F(2) generations. The number of neurons in the LC of both males and females decreased in heterozygotic individuals (F(1)) but increased in homozygotic (F(2)). However, the number of neurons in the BSTMP changes significantly over the generations, although the ratio of neurons (female/male) is stable and unaffected in homo- or heterozygosis. Thus, the mechanism that regulates the neuronal female/male ratio would be different from the one that controls the number of neurons. The facts that sex differences in the BSTMP are not affected by homo- or heterozygosis and that they are seen in several mammalian orders suggest the existence of a "fixed" type of brain sex differences in the Mammalia Class.

  8. Effects of heroin on rat prosocial behavior.

    Science.gov (United States)

    Tomek, Seven E; Stegmann, Gabriela M; Olive, M Foster

    2018-05-04

    Opioid use disorders are characterized in part by impairments in social functioning. Previous research indicates that laboratory rats, which are frequently used as animal models of addiction-related behaviors, are capable of prosocial behavior. For example, under normal conditions, when a 'free' rat is placed in the vicinity of rat trapped in a plastic restrainer, the rat will release or 'rescue' the other rat from confinement. The present study was conducted to determine the effects of heroin on prosocial behavior in rats. For 2 weeks, rats were given the opportunity to rescue their cagemate from confinement, and the occurrence of and latency to free the confined rat was recorded. After baseline rescuing behavior was established, rats were randomly selected to self-administer heroin (0.06 mg/kg/infusion i.v.) or sucrose pellets (orally) for 14 days. Next, rats were retested for rescuing behavior once daily for 3 days, during which they were provided with a choice between freeing the trapped cagemate and continuing to self-administer their respective reinforcer. Our results indicate that rats self-administering sucrose continued to rescue their cagemate, whereas heroin rats chose to self-administer heroin and not rescue their cagemate. These findings suggest that rats with a history of heroin self-administration show deficits in prosocial behavior, consistent with specific diagnostic criteria for opioid use disorder. Behavioral paradigms providing a choice between engaging in prosocial behavior and continuing drug use may be useful in modeling and investigating the neural basis of social functioning deficits in opioid addiction. © 2018 Society for the Study of Addiction.

  9. Dynamics of the Skeletal Muscle Secretome during Myoblast Differentiation

    DEFF Research Database (Denmark)

    Henningsen, Jeanette; Rigbolt, Kristoffer T G; Blagoev, Blagoy

    2010-01-01

    During recent years, increased efforts have focused on elucidating the secretory function of skeletal muscle. Through secreted molecules, skeletal muscle affects local muscle biology in an auto/paracrine manner as well as having systemic effects on other tissues. Here we used a quantitative...... proteomics platform to investigate the factors secreted during the differentiation of murine C2C12 skeletal muscle cells. Using triple encoding stable isotope labeling by amino acids in cell culture, we compared the secretomes at three different time points of muscle differentiation and followed the dynamics...... of the skeletal muscle as a prominent secretory organ. In addition to previously reported molecules, we identified many secreted proteins that have not previously been shown to be released from skeletal muscle cells nor shown to be differentially released during the process of myogenesis. We found 188...

  10. Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts

    DEFF Research Database (Denmark)

    Le Bihan, Marie-Catherine; Barrio, Inigo; Mortensen, Tenna Pavia

    2015-01-01

    Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the ...

  11. Genome Editing in Rats Using TALE Nucleases.

    Science.gov (United States)

    Tesson, Laurent; Remy, Séverine; Ménoret, Séverine; Usal, Claire; Thinard, Reynald; Savignard, Chloé; De Cian, Anne; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio

    2016-01-01

    The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

  12. Cerveau isolé and pretrigeminal rats.

    Science.gov (United States)

    Zernicki, B; Gandolfo, G; Glin, L; Gottesmann, C

    1984-01-01

    Cortical and hippocampal EEG activity was analysed in 14 cerveau isole and 8 pretrigerninal rats. In the acute stage, waking EEG patterns were absent in the cerveau isole, whereas sleep EEG patterns were absent in the pretrigeminal preparations. However, already on the second day the EEG waking-sleep cycle recovered in the majority of rats. Paradoxically, stimuli directed to the caudal part of preparations evoked stronger cortical and hippocampal EEG arousal than olfactory and visual stimuli. The behavior of the caudal part was observed in 25 preparations. Although in abortive form, the rats did show some locomotor and grooming behavior, and could be fed orally. The peripheral events of paradoxical sleep appeared only on the fourth or fifth day of survival of the cerveau isole rats. It is concluded that the activity of the isolated cerebrum of the rat is similar to that of cat preparations, but that functions of the caudal neuraxis are superior in rats.

  13. Pubertal neurocranium growth in thymectomized rats.

    Science.gov (United States)

    Rino, W; Teixeira, D

    1979-01-01

    Differences in neurocranium growth at puberty were studied in rats of both sexes thymectomized and sham-thymectomized at 2, 4, 6, 8, 10, 12 and 14 days of age and in controls of matched age and sex; skull length, width and height, and skull base length and face length were measured. The neurocranium of the thymectomized rats was significantly smaller than that of the sham-thymectomized and control rats of both sexes and in all age-groups.

  14. Treatment of diabetic rats with encapsulated islets

    OpenAIRE

    Sweet, Ian R; Yanay, Ofer; Waldron, Lanaya; Gilbert, Merle; Fuller, Jessica M; Tupling, Terry; Lernmark, Ake; Osborne, William R A

    2008-01-01

    Immunoprotection of islets using bioisolator systems permits introduction of allogeneic cells to diabetic patients without the need for immunosuppression. Using TheraCyte? immunoisolation devices, we investigated two rat models of type 1 diabetes mellitus (T1DM), BB rats and rats made diabetic by streptozotocin (STZ) treatment. We chose to implant islets after the onset of diabetes to mimic the probable treatment of children with T1DM as they are usually diagnosed after disease onset. We enca...

  15. Rat bite fever in a pet lover.

    Science.gov (United States)

    Cunningham, B B; Paller, A S; Katz, B Z

    1998-02-01

    Rat-bite fever is an uncommon bacterial illness resulting from infection with Streptobacillus moniliformis that is often transmitted by the bite of a rat. The cutaneous findings in rat-bite fever are nonspecific but have been described as maculopapular or petechial. We describe a 9-year-old girl with acrally distributed hemorrhagic pustules, fever, and arthralgias. Diagnosis was delayed because of difficulty in identifying the pathologic organism. She was successfully treated with 10 days of ceftriaxone.

  16. Phosphodiesterases in the rat ovary

    DEFF Research Database (Denmark)

    Petersen, Tonny Studsgaard; Stahlhut, Martin; Andersen, Claus Yding

    2015-01-01

    that augmented cAMP levels stimulate primordial follicle growth. The present study examined the gene expression, enzyme activity and immunolocalization of the different cAMP hydrolysing PDEs families in the rat ovary. Further, the effect of PDE4 inhibition on primordial follicle activation in cultured neonatal......Phosphodiesterases (PDEs) are important regulators of the intracellular cAMP concentration, which is a central second messenger that affects a multitude of intracellular functions. In the ovaries, cAMP exerts diverse functions, including regulation of ovulation and it has been suggested...... rat ovaries was also evaluated. We found varied expression of all eight families in the ovary with Pde7b and Pde8a having the highest expression each accounting for more than 20% of the total PDE mRNA. PDE4 accounted for 15-26% of the total PDE activity. Immunoreactive PDE11A was found in the oocytes...

  17. Isolation of rat adrenocortical mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Solinas, Paola [Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Department of Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Fujioka, Hisashi [Electron Microscopy Facility, Department of Pharmacology, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Tandler, Bernard [Department of Biological Sciences, School of Dental Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Hoppel, Charles L., E-mail: charles.hoppel@case.edu [Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Department of Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer A method for isolation of adrenocortical mitochondria from the adrenal gland of rats is described. Black-Right-Pointing-Pointer The purified isolated mitochondria show excellent morphological integrity. Black-Right-Pointing-Pointer The properties of oxidative phosphorylation are excellent. Black-Right-Pointing-Pointer The method increases the opportunity of direct analysis of adrenal mitochondria from small animals. -- Abstract: This report describes a relatively simple and reliable method for isolating adrenocortical mitochondria from rats in good, reasonably pure yield. These organelles, which heretofore have been unobtainable in isolated form from small laboratory animals, are now readily accessible. A high degree of mitochondrial purity is shown by the electron micrographs, as well as the structural integrity of each mitochondrion. That these organelles have retained their functional integrity is shown by their high respiratory control ratios. In general, the biochemical performance of these adrenal cortical mitochondria closely mirrors that of typical hepatic or cardiac mitochondria.

  18. Renal function in streptozotocin-diabetic rats

    DEFF Research Database (Denmark)

    Jensen, P K; Christiansen, J S; Steven, K

    1981-01-01

    to the rise in kidney glomerular filtration rate (diabetic rats: 37.0 nl/min; control rats: 27.9 nl/min). Likewise renal plasma flow was significantly higher in the diabetic rats (4.1 ml/min) than in the control group (3.0 ml/min). Glomerular capillary pressure was identical in both groups (56.0 and 56.0 mm......-1mmHg-1). Kidney weight was significantly higher in the diabetic rats (1.15 g; control rats: 0.96 g) while body weight was similar in both groups (diabetic rats: 232 g; control rats: 238 g). Calculations indicate that the increases in transglomerular hydraulic pressure, renal plasma flow......Renal function was examined with micropuncture methods in the insulin-treated streptozotocin-diabetic rat. Kidney glomerular filtration rate was significantly higher in the diabetic rats (1.21 ml/min) than in the control group (0.84 ml/min) Nephron glomerular filtration rate increased in proportion...

  19. X-ray lethality in diabetic rats

    International Nuclear Information System (INIS)

    Cember, H.; Thorson, T.M. Jr.

    1978-01-01

    Rats were made diabetic with streptozotocin and were irradiated with X-rays at various exposure levels in order to determine the LD-50/30 day dose. Non-diabetic control rats were exposed in a similar manner. The LD-50 exposures for the diabetic rats and the control rats were 436 R, and 617 R respectively. In view of the high prevalence of diabetes among the adult population, this finding may have important implications for diabetic workers who may be exposed accidentally to high levels of ionizing radiation

  20. Automatic Training of Rat Cyborgs for Navigation.

    Science.gov (United States)

    Yu, Yipeng; Wu, Zhaohui; Xu, Kedi; Gong, Yongyue; Zheng, Nenggan; Zheng, Xiaoxiang; Pan, Gang

    2016-01-01

    A rat cyborg system refers to a biological rat implanted with microelectrodes in its brain, via which the outer electrical stimuli can be delivered into the brain in vivo to control its behaviors. Rat cyborgs have various applications in emergency, such as search and rescue in disasters. Prior to a rat cyborg becoming controllable, a lot of effort is required to train it to adapt to the electrical stimuli. In this paper, we build a vision-based automatic training system for rat cyborgs to replace the time-consuming manual training procedure. A hierarchical framework is proposed to facilitate the colearning between rats and machines. In the framework, the behavioral states of a rat cyborg are visually sensed by a camera, a parameterized state machine is employed to model the training action transitions triggered by rat's behavioral states, and an adaptive adjustment policy is developed to adaptively adjust the stimulation intensity. The experimental results of three rat cyborgs prove the effectiveness of our system. To the best of our knowledge, this study is the first to tackle automatic training of animal cyborgs.

  1. Toxicity and repellency to rats of actidione

    Science.gov (United States)

    Traub, R.; DeWitt, J.B.; Welch, J.F.; Newman, D.

    1950-01-01

    The antibiotic actidione was found to be highly repellent to laboratory rats and to significantly reduce gnawing attacks upon treated paperboards. Rats refused to accept food or water containing this material even under conditions of acute starvation and died of starvation and thirst,rather than accept water containing l.0 mg. of actidione per liter. The compound is highly toxic to .rats with the minimum .lethal dose by oral administration being approximately l.0 mg./Kg body weight. Paperboard treated with the compound resisted gnawing attacks by specially trained and motivated rats for periods of two hundred hours, although similar .untreated boards were pierced within thirty-to sixty minutes.

  2. [Pharmacokinetics of crocetin in rats].

    Science.gov (United States)

    Liu, Tong-zheng; Qian, Zhi-yu

    2002-05-01

    To develop an HPLC method for the determination of crocetin in rat plasma and study the pharmacokinetics in rats. Hypersil C18 column (5 microns, 4.6 mm x 200 mm) was used at column temperature 30 degrees C. The mobile phase consisted of methanol-water-acetic acid (75:24.5:0.5) at the flow rate of 1.0 mL.min-1. The UV detection wave length was 423 nm. The calibration curve was linear (gamma = 0.9996) in the range from 0.49 microgram.mL-1 to 7.87 micrograms.mL-1 for crocetin. The mean recovery was 105.2%. The lowest detectable concentration of crocetin was 0.14 microgram.mL-1 (S/N = 3). The RSDs of within-day and between-day were all less than 5%. The plasma crocetin was steady. The HPLC method of determination of crocetin in the plasma was established. After single dose of 50 mg.kg-1 ig in 10 rats, the main pharmacokinetic parameters were estimated as follows: T1/2 alpha (30 +/- 6) min, Tmax(65 +/- 16) min, Cmax(5.0 +/- 1.0) microgram.mL-1, AUC0-T(845 +/- 109) microgram.min.mL-1, Vd(5.0 +/- 0.8) L.kg-1. Crocetin was shown to be absorbed into the blood through the gastrointestinal tract. This method is quick, precise and reliable. Crocetin was shown to be quickly absorbed in rats.

  3. Stevia preferences in Wistar rats.

    Science.gov (United States)

    Núñez Martínez, Paula; Argüelles Luis, Juan; Perillán Méndez, Carmen

    2016-11-01

    The Stevia rebaudiana plant is likely to become a major source of high-potency sweetener for the growing natural-food market. S. rebaudiana is the source of a number of sweet diterpenoid glycosides, but the major sweet constituents are rebaudioside A and stevioside. These two constituents have similar pharmacokinetic and metabolic profiles in rats and humans, and thus, studies carried out with either steviol glycoside are relevant to both. Other studies illustrate the diversity of voluntary sweet intake in mammals. This study was done using a series of two-bottle tests that compared a wide range of sweetener concentrations versus saccharin concentrations and versus water. Wistar rats displayed preferences for stevia extract and pure rebaudioside A solutions over water at a range of concentrations (0.001% to 0.3%), and their intake peak occurred at 0.1% concentration. They also preferred solutions prepared with a commercial rebaudioside A plus erythritol mixture to water, and their peak was at 2% concentration. The present study provides new information about the responses of Wistar rats to stevia compounds and commercial stevia products such as Truvia. These results could help with the appropriate dosage selection for focused behavioral and physiological studies on stevia.

  4. Bifurcate effects of glucose on caspase-independent cell death during hypoxia

    International Nuclear Information System (INIS)

    Aki, Toshihiko; Nara, Akina; Funakoshi, Takeshi; Uemura, Koichi

    2010-01-01

    We investigated the effect of glucose on hypoxic death of rat cardiomyocyte-derived H9c2 cells and found that there is an optimal glucose concentration for protection against hypoxic cell death. Hypoxic cell death in the absence of glucose is accompanied by rapid ATP depletion, release of apoptosis-inducing factor from mitochondria, and nuclear chromatin condensation, all of which are inhibited by glucose in a dose-dependent manner. In contrast, excessive glucose also induces hypoxic cell death that is not accompanied by these events, suggesting a change in the mode of cell death between hypoxic cells with and without glucose supplementation.

  5. Determination of rat vertebral bone compressive fatigue properties in untreated intact rats and zoledronic-acid-treated, ovariectomized rats

    NARCIS (Netherlands)

    Brouwers, J.E.M.; Ruchselman, M.; Rietbergen, van B.; Bouxsein, M.L.

    2009-01-01

    Summary Compressive fatigue properties of whole vertebrae, which may be clinically relevant for osteoporotic vertebral fractures, were determined in untreated, intact rats and zoledronic-acid-treated, ovariectomized rats. Typical fatigue behavior was found and was similar to that seen in other

  6. Modulation of rat behaviour by using a rat-like robot

    International Nuclear Information System (INIS)

    Shi, Qing; Ishii, Hiroyuki; Kinoshita, Shinichi; Takanishi, Atsuo; Okabayashi, Satoshi; Iida, Naritoshi; Kimura, Hiroshi; Shibata, Shigenobu

    2013-01-01

    In this paper, we study the response of a rat to a rat-like robot capable of generating different types of behaviour (stressful, friendly, neutral). Experiments are conducted in an open-field where a rat-like robot called WR-4 is put together with live rats. The activity level of each rat subject is evaluated by scoring its locomotor activity and frequencies of performing rearing (rising up on its hind limbs) and body grooming (body cuddling and head curling) actions, whereas the degree of preference of that is indicated by the robot–rat distance and the frequency of contacting WR-4. The moving speed and behaviour of WR-4 are controlled in real-time based on the feedback from rat motion. The activity level and degree of preference of rats for each experimental condition are analysed and compared to understand the influence of robot behaviour. The results of this study show that the activity level and degree of preference of the rat decrease when exposed to a stressful robot, and increase when the robot exhibit friendly behaviour, suggesting that a rat-like robot can modulate rat behaviour in a controllable, predictable way. (paper)

  7. Grooming behavior of spontaneously hypertensive rats

    NARCIS (Netherlands)

    Buuse, M. van den; Jong, Wybren de

    1987-01-01

    In an open field spontaneously hypertensive rats (SHR) exhibited lower scores for grooming when compared to their normotensive controls, the Wistar Kyoto rats (WKY). After i.c.v. injection of 1 μg ACTH1–24 cumulative 50-min grooming scores were lower in SHR. Analysis of subscores indicated that the

  8. Morphological and neurohistological changes in adolescent rats ...

    African Journals Online (AJOL)

    Pregnancy was confirmed and the pregnant rats were divided into 3 groups based on the 3 trimesters (A, B, C), with each group having a control and a treated subgroup. The Control Groups (A1, B1, ... offspring of tobacco smokers. Keywords: Cortex, Histology, Prenatal nicotine, Adolescent rats, Neurological abnormalities ...

  9. Target-selected mutagenesis of the rat

    NARCIS (Netherlands)

    Smits, B.M.; Mudde, J.B.; Plasterk, R.; Cuppen, E.

    2004-01-01

    The rat is one of the most extensively studied model organisms, and with its genome being sequenced, tools to manipulate gene function in vivo have become increasingly important. We here report proof of principle for target-selected mutagenesis as a reverse genetic or knockout approach for the rat.

  10. Optical coherence tomography of the rat cochlea

    NARCIS (Netherlands)

    Wong, B. J. F.; de Boer, JF; Park, B.H.; Chen, ZP; Nelson, JS

    2000-01-01

    Optical coherence tomography (OCT) was used to image the internal structure of a rat cochlea (ex vivo). Immediately following sacrifice, the temporal bone of a Sprague-Dawley rat was harvested. Axial OCT cross sectional images lover regions of interest, 1x1 mm-2x8 mm) were obtained with a spatial

  11. Same-Different Categorization in Rats

    Science.gov (United States)

    Wasserman, Edward A.; Castro, Leyre; Freeman, John H.

    2012-01-01

    Same-different categorization is a fundamental feat of human cognition. Although birds and nonhuman primates readily learn same-different discriminations and successfully transfer them to novel stimuli, no such demonstration exists for rats. Using a spatial discrimination learning task, we show that rats can both learn to discriminate arrays of…

  12. in Alloxan-induced Diabetic Rats

    African Journals Online (AJOL)

    HP

    Group 4: Diabetic rats that were administered. 500 mg/kg body weight extracts. Group 5: Diabetic rats that were administered. 300 mg/kg body weight of metformin. The drug and extracts treatment was done for a period of 21 days using orogastric tube. Collection of blood samples. Following 21 days of extract administration, ...

  13. ischemic brain injury in neonatal rats

    African Journals Online (AJOL)

    Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, ... Methods: Forty-eight rats (P7-pups) were randomly assigned to one of four groups: ... Keywords: Hypoxic–ischemic brain injury, α-Lipoic acid, Cerebral infarct area, Edema, Antioxidants, .... Of the 48 rats initially used in the current study, 5.

  14. Bromsulphalein (BSP) clearance in ageing rats

    NARCIS (Netherlands)

    Hollander, C.F.; Leeuw-Israel, F.R. de; Arp-Neefjes, J.M.

    1968-01-01

    Liver function in ageing rats was studied, using the bromsulphalein (BSP) clearance test. The test was done on ultramicro scale. This made it possible to repeat the test several times in the same animal and to start a longitudinal study. In 3-month-old rats the BSP retentions, measured 15, 30 and 45

  15. Adrenergic blockade in diabetic and uninephrectomized rats

    DEFF Research Database (Denmark)

    Thulesen, J; Poulsen, Steen Seier; Jørgensen, P E

    1999-01-01

    The present study reports on the effects of adrenergic blocking agents on the renal growth and on the renal content and urinary excretion of epidermal growth factor (EGF) in streptozotocin-induced diabetic or uninephrectomized rats. Diabetic and uninephrectomized rats were allocated to groups...... treated with either saline or adrenergic antagonists and compared to controls and sham-operated controls, respectively. 24-hour urine samples were obtained on days 7, 14, and 21 and renal tissue samples on day 21. The 24-hour urinary excretion of EGF from controls and saline-treated diabetic rats...... was comparable. In adrenergic antagonist treated diabetic rats, it was reduced by at least 40% throughout the study period. Uninephrectomy caused a 50% reduction in the urinary excretion of EGF. This was not influenced by treatment with an adrenergic antagonist. After 3 weeks, saline-treated diabetic rats had...

  16. Transfer RNA methylases in rat placenta

    International Nuclear Information System (INIS)

    Jagtiani, S.K.; Narurkar, L.M.; Narurkar, M.V.

    1977-01-01

    Presence of tRNA methylases (5-adenosylmethionine : tRNA methyltransferases) was demonstrated at various stages of gestation in rat placenta, the enzyme being 50-100% higher than that of adult rat liver during early gestation. Placental tRNA methylases were shown to differ from those of liver in the extent of methylation. Glycine methyltransferase (S-adenosylmethionine : glycine methyltransferase), a regulatory enzyme in adult rat liver, was absent in placenta throughout gestation. The placental tRNA methylases could be inhibited in vitro by semipurified glycine methyltransferase from adult rat liver. The high placental tRNA methylase activity was comparable with the inhibitor-free enzyme activity of the adult rat liver. S-adenosyl-[Me- 14 C]-methionine was used in the investigation. (author)

  17. Development of ELISA kit for rat albumin

    International Nuclear Information System (INIS)

    Yuan Zhigang; Han Shiquan; Liu Yibing; Xu Wenge; Jia Juanjuan

    2009-01-01

    The Anti-rat albumin serum was prepared by immunized the sheep with rat albumin. A ELISA method was established for rat albumin. The measurement range of the assay was 1-50 mg/L, sensitivity of the assay was 0.42 mg/L, recovery rate was 85.0%-106.0%. Intra-and inter-assay variation coefficients were <8.9% and <12.8% respectively. The correlation coefficients between measured and expected values were 0.999 after serial dilution of the urine samples with high concentrations of rat albumin. A good correlation was observed between the ELISA and RIA methods, and the kit for rat albumin might provide a convenience in exploitation of renal drugs and experimental injury of the kidney. (authors)

  18. Microtubular stability affects pVHL-mediated regulation of HIF-1alpha via the p38/MAPK pathway in hypoxic cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Miao Teng

    Full Text Available BACKGROUND: Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL, as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. METHODOLOGY/PRINCIPAL FINDINGS: In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4 overexpression plasmids and microtubule-depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.

  19. Metabolism of methylphenidate in dog and rat

    International Nuclear Information System (INIS)

    Egger, H.; Bartlett, F.; Dreyfuss, R.; Karliner, J.

    1981-01-01

    The urinary metabolites of methylphenidate in the dog and rat were investigated. After oral administration of 14C-labeled methylphenidate, approximately 86% and 63% of the dose was recovered in the urine of the dog and rat, respectively. Less than 1% of the dose was excreted as unchanged drug. Metabolism involved oxidation, hydrolysis, and conjugation processes. The primary hydrolytic product was alpha-phenyl-2-piperidineacetic acid (24%, dog; 35-40%, rat). The primary metabolites of oxidation were methyl 6-oxo-alpha-phenyl-2-piperidineacetate (3%, dog; 1.5%, rat) and the glucuronide of alpha-(p-hydroxyphenyl)-2-piperidineacetic acid (10%, rat). The former also underwent extensive biotransformation, including: 1) hydrolysis to the lactam acid (27%, dog; 7-10%, rat) and subsequent carboxylic acid O-glucuronidation (15%, dog); or 2) hydroxylation at the 5-position (1%, dog; 2%, rat) and subsequent hydrolysis (4%, dog; 15-17%, rat); or 3) 5-O-glucuronidation (12%, dog). Additional minor metabolites from methyl-6-oxo-alpha-phenyl-2-piperidineacetate were the phenolic O-glucuronide of methyl alpha-(p-hydroxyphenyl)-6-oxo-2-piperidineacetate (1%, dog), and the 4-O-glucuronide of methyl 4-hydroxy-6-oxo-alpha-phenyl-2-piperidineacetate (1%, dog), and the taurine amide conjugate of alpha-(p-hydroxyphenyl)-6-oxo-2-piperidineacetic acid (1%, dog). Additional products from methylphenidate conjugation included methyl 1-carbamoyl-alpha-phenyl-2-piperidineacetate (1%, dog or rat) and its carboxylic acid hydrolysis product (1%, rat). The chirality of the major metabolites isolated from dog urine showed that metabolism was partially stereoselective in all investigated cases, except in the formation of alpha-phenyl-2-piperidineacetic acid

  20. Genetic susceptibility to mammary carcinogenesis in rats

    Energy Technology Data Exchange (ETDEWEB)

    Kamiya, Kenji; Nitta, Yumiko [Hiroshima Univ. (Japan). Research Inst. for Radiation Biology and Medicine

    1999-06-01

    The Copenhagen (COP) rat strain has previously been shown to be genetically resistant to chemical induction of breast cancer, while Wistar/Furth (WF) and Fischer 344 (F344) animals are relatively susceptible. We have compared the carcinogenic response of these three strains of rats to N-methyl-N-nitrosourea (MNU) with that to {sup 60}Co gamma rays. High incidences of mammary carcinomas were induced by MNU in the F344 and WF rats (100%), whereas the COP strain proved resistant (11.8%). In contrast, radiation-induced mammary carcinomas in COP rats developed in a similar incidence (37.0%) to those in the F344 (22.6%) and WF (26.9%) strains. The low incidence of papillary carcinomas in MNU-treated COP rats appeared to be directly related to the COP genetic resistance controlled by the Mcs genes. Ionizing radiation did, however, induce papillary carcinomas in all the three strains of rats. These carcinomas were more differentiated than MNU-induced cancers with regard to the two mammary differentiation markers, rat milk fat globule membrane (R-MFGM) and {alpha}-smooth muscle actin ({alpha}-SMA). Furthermore, ionizing radiation but not MNU induced mammary adenomas in all three strains, especially in COP rats. Such adenomas had differentiation marker profiles similar to these of carcinomas induced by {sup 60}Co gamma rays. When transplanted into syngenic hosts, growth of adenomas was 17 {beta}-estradiol (E{sub 2})-dependent and they progressed to carcinomas. Furthermore, one microcarcinoma was observed to develop from adenoma tissue in a radiation-exposed COP rat. The findings suggest that radiation and chemical carcinogens are likely to induce mammary cancers through different pathways or from different cell populations. The induction of relatively high incidences of mammary carcinomas and adenomas by radiation in COP rats may correlate with the genetically modulated and highly differentiated physiological status of their mammary glands. (author)

  1. Social exclusion intensifies anxiety-like behavior in adolescent rats.

    Science.gov (United States)

    Lee, Hyunchan; Noh, Jihyun

    2015-05-01

    Social connection reduces the physiological reactivity to stressors, while social exclusion causes emotional distress. Stressful experiences in rats result in the facilitation of aversive memory and induction of anxiety. To determine the effect of social interaction, such as social connection, social exclusion and equality or inequality, on emotional change in adolescent distressed rats, the emotional alteration induced by restraint stress in individual rats following exposure to various social interaction circumstances was examined. Rats were assigned to one of the following groups: all freely moving rats, all rats restrained, rats restrained in the presence of freely moving rats and freely moving rats with a restrained rat. No significant difference in fear-memory and sucrose consumption between all groups was found. Change in body weight significantly increased in freely moving rats with a restrained rat, suggesting that those rats seems to share the stressful experience of the restrained rat. Interestingly, examination of the anxiety-like behavior revealed only rats restrained in the presence of freely moving rats to have a significant increase, suggesting that emotional distress intensifies in positions of social exclusion. These results demonstrate that unequally excluded social interaction circumstances could cause the amplification of distressed status and anxiety-related emotional alteration. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Oxidative stress of crystalline lens in rat menopausal model

    OpenAIRE

    Acer, Semra; Pekel, Gökhan; Küçükatay, Vural; Karabulut, Aysun; Yağcı, Ramazan; Çetin, Ebru Nevin; Akyer, Şahika Pınar; Şahin, Barbaros

    2016-01-01

    ABSTRACT Purpose: To evaluate lenticular oxidative stress in rat menopausal models. Methods: Forty Wistar female albino rats were included in this study. A total of thirty rats underwent oophorectomy to generate a menopausal model. Ten rats that did not undergo oophorectomy formed the control group (Group 1). From the rats that underwent oophorectomy, 10 formed the menopause control group (Group 2), 10 were administered a daily injection of methylprednisolone until the end of the study (Gro...

  3. Dithiobiuret metabolism in the rat

    International Nuclear Information System (INIS)

    Williams, K.D.; Porter, W.R.; Peterson, R.E.

    1982-01-01

    Our main objective was to describe the metabolism of dithiobiuret (DTB) in the adult, male rat. Based on the thin-layer chromatographic analysis of urine from animals treated with [ 14 C] or [ 35 S] labeled DTB, two pathways for metabolism are proposed. One pathway is reversible and involves the oxidation of DTB to thiuret and the reduction of thiuret back to DTB. The other pathway consists of the desulfurization of DTB to monothiobiuret. The liver appears to desulfurate DTB because DTB-derived [35S] was eliminated from the liver more rapidly than [ 14 C]. The liver was the only tissue where the elimination kinetics of [ 35 S] and [ 14 C] DTB were different. DTB-derived radioactivity in urine that co-chromatographed with DTB, monothiobiuret, thiuret and sulfate was quantitated along with that of three uncharacterized metabolites. The presence of these unknown metabolites suggests that DTB metabolism is complex. The present study is the first description of the metabolic fate of DTB in the rat and serves as a starting point for determining whether DTB neurotoxicity is caused by the parent compound or a metabolite

  4. Lessons From Experiments in Rats

    Directory of Open Access Journals (Sweden)

    Albert Gramsbergen

    2001-01-01

    Full Text Available In this essay a few relevant aspects of the neural and behavioral development of the brain in the human and in the rat are reviewed and related to the consequences of lesions in the central and peripheral nervous system at early and later age. Movements initially are generated by local circuits in the spinal cord and without the involvement of descending projections. After birth, both in humans and in rats it seems that the devlopment of postural control is the limiting factor for several motor behaviors to mature. Strong indications exist that the cerebellum is significantly involved in this control. Lesions in the CNS at early stages interfere with fundamental processes of neural development, such as the establishment of fiber connections and cell death patterns. Consequently, the functional effects are strongly dependent on the stage of development. The young and undisturbed CNS, on the other hand, has a much greater capacity than the adult nervous system for compensating abnormal reinnervation in the peripheral nervous system. Animal experiments indicated that the cerebellar cortex might play an important part in this compensation. This possibility should be investigated further as it might offer important perspectives for treatment in the human.

  5. Autoprotection in acetaminophen intoxication in rats

    DEFF Research Database (Denmark)

    Dalhoff, K; Laursen, H; Bangert, K

    2001-01-01

    and liver tissue were collected before and 12, 24, 36, and 48 hr after the toxic dose and were analysed for hepatic glutathione and cysteine contents, hepatic glutathione-S-transferase and blood alanine aminotransferase activity, as well as acetaminophen concentration in plasma. Steady-state mRNA levels......Autoprotection by acetaminophen, i.e. increased resistance to toxic effects caused by pretreatment, is a well-known phenomenon. The purpose of the present work was to identify mechanisms for increased acetaminophen tolerance induced by pretreatment of rats. One group of female Wistar rats...... (pretreated rats) received acetaminophen orally in increasing doses (1 to 4.3 g/kg) twice a week for 3 weeks, one group (naïve rats) received the vehicle. At time zero pretreated rats received a toxic dose of 7.5 g/kg (100% lethal in naïve rats), and naïve rats received a toxic dose of 4.3 g/kg. Blood...

  6. Cerebral ammonia metabolism in hyperammonemic rats

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, A J; Mora, S N; Cruz, N F; Gelbard, A S

    1985-06-01

    The short-term metabolic fate of blood-borne (/sup 13/N)ammonia was determined in the brains of chronically (8- or 14-week portacaval-shunted rats) or acutely (urease-treated) hyperammonemic rats. Using a freeze-blowing technique it was shown that the overwhelming route for metabolism of blood-borne (/sup 13/N)ammonia in normal, chronically hyperammonemic and acutely hyperammonemic rat brain was incorporation into glutamine (amide). However, the rate of turnover of (/sup 13/N)ammonia to L-(amide-/sup 13/N)glutamine was slower in the hyperammonemic rat brain than in the normal rat brain. The activities of several enzymes involved in cerebral ammonia and glutamate metabolism were also measured in the brains of 14-week portacaval-shunted rats. The rat brain appears to have little capacity to adapt to chronic hyperammonemia because there were no differences in activity compared with those of weight-matched controls for the following brain enzymes involved in glutamate/ammonia metabolism: glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, glutamine transaminase, glutaminase, and glutamate decarboxylase. The present findings are discussed in the context of the known deleterious effects on the CNS of high ammonia levels in a variety of diseases.

  7. Abstract numerical discrimination learning in rats.

    Science.gov (United States)

    Taniuchi, Tohru; Sugihara, Junko; Wakashima, Mariko; Kamijo, Makiko

    2016-06-01

    In this study, we examined rats' discrimination learning of the numerical ordering positions of objects. In Experiments 1 and 2, five out of seven rats successfully learned to respond to the third of six identical objects in a row and showed reliable transfer of this discrimination to novel stimuli after being trained with three different training stimuli. In Experiment 3, the three rats from Experiment 2 continued to be trained to respond to the third object in an object array, which included an odd object that needed to be excluded when identifying the target third object. All three rats acquired this selective-counting task of specific stimuli, and two rats showed reliable transfer of this selective-counting performance to test sets of novel stimuli. In Experiment 4, the three rats from Experiment 3 quickly learned to respond to the third stimulus in object rows consisting of either six identical or six different objects. These results offer strong evidence for abstract numerical discrimination learning in rats.

  8. Total parenteral nutrition in diabetic rats

    International Nuclear Information System (INIS)

    Norcross, E.D.; Stein, T.P.

    1986-01-01

    Parenteral Nutrition with hypertonic glucose is frequently given to diabetic patients. Large amounts of insulin can be required. The purpose of this investigation was to develop a totally parenterally nourished diabetic rat model. 200 g Female Sprague Dawley rats were made diabetic by i.v. injection of streptozotocin (50 mg/kg). Rats were then allowed to recover for at least 1 week before undergoing surgical insertion of a central venous catheter for parenteral feeding. TPN was begun 3 days after surgery. Prior to this they were allowed unlimited access to food and water. Control (non-streptozotocin treated) rats were run at the same time. Protein turnover was investigated by using 15 N glycine. Preliminary results: diabetic rats given mostly fat as a calorie source survived well in the absence of exogenous insulin whereas those that were given glucose only as their non-protein calorie source showed poor survival even with exogenous insulin. N balance and protein turnover in the lipid treated diabetic rats were comparable to the non-diabetic control rats

  9. Embryogenesis-promoting factors in rat serum.

    Science.gov (United States)

    Katoh, M; Kimura, R; Shoji, R

    1998-06-15

    Regarding whole rat embryo cultures in vitro, rat serum as a culture medium is known to support the normal growth of rat embryos in the organogenesis phase. The purpose of the present study was to isolate the embryogenesis-promoting factors from rat serum as a first step in the development of a defined serum-free medium for a whole embryo culture system. Pooled rat serum after heat inactivation was fractionated into three major peaks (frA, containing a region of void volume, frB, and frC) by gel filtration. The 9.5-day rat embryos that were cultivated for 48 hr in essential salt medium containing frB (with a molecular size range of 100-500 kDa) revealed normal growth. Three proteins (27 kDa, 76 kDa, and 190 kDa) that had the embryogenesis-promoting effects were isolated from 3-hr delayed centrifuged rat serum by the ion exchange chromatography. The 76-kDa protein was found to be rat transferrin by immunoblotting. The 27-kDa protein was identified as apo-AI (the major apoprotein of high-density lipoprotein) by immunoblotting. High-density lipoprotein obtained from pooled rat serum by a NaBr density gradient ultracentrifugation was found to have a positive effect on embryogenesis. The 10-kDa protein was also identified as alpha 1-inhibitor 3 by immunoblotting. In addition, the embryogenesis-promoting effect of the fraction containing 27-kDa and 190-kDa proteins declined within a short period of storage at -20 degrees C. This decrease was countered by supplementing its fraction (D-2) with albumin isolated from rat serum. These results in the present study suggest that transferrin, high-density lipoprotein, and alpha 1-inhibitor 3 in rat serum may be embryogenesis-promoting factors, and that albumin appeared to play a role in the embryogenesis of rat embryos in whole embryo cultures.

  10. Establishment of a novel dwarf rat strain: cartilage calcification insufficient (CCI) rats.

    Science.gov (United States)

    Tanaka, Masami; Watanabe, Minoru; Yokomi, Izuru; Matsumoto, Naoki; Sudo, Katsuko; Satoh, Hitoshi; Igarashi, Tsuneo; Seki, Azusa; Amano, Hitoshi; Ohura, Kiyoshi; Ryu, Kakei; Shibata, Shunichi; Nagayama, Motohiko; Tanuma, Jun-ichi

    2015-01-01

    Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats.

  11. Establishment of a novel dwarf rat strain: cartilage calcification insufficient (CCI) rats

    Science.gov (United States)

    TANAKA, Masami; WATANABE, Minoru; YOKOMI, Izuru; MATSUMOTO, Naoki; SUDO, Katsuko; SATOH, Hitoshi; IGARASHI, Tsuneo; SEKI, Azusa; AMANO, Hitoshi; OHURA, Kiyoshi; RYU, Kakei; SHIBATA, Shunichi; NAGAYAMA, Motohiko; TANUMA, Jun-ichi

    2014-01-01

    Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats. PMID:25736479

  12. Ontogenic changes in selenite metabolism in rats

    International Nuclear Information System (INIS)

    Ostadalova, I.; Babicky, A.; Kopoldova, J.

    1982-01-01

    Radioselenium concentration and excretion was studied after administration of 75 Se-labelled selenite to male rats during ontogeny. The concentration of radioselenium in individual organs decreases with increasing age. The largest differences between young and adults were in the quantity and quality of excreted substances. During 2 h after the administration of 20 μmol selenite/kg young rats excreted 2.4% of the dose, essentially in the urine only, whilst adults excreted a total of 11%, distributed equally in breath and urine. The part excreted as methylated metabolites was 0.1% of the administered dose in young and 6.3% in adult rats. These results support the hypothesis that the differences in the sensitivity to the toxic action of selenite between young and adult rats can be due to ontogenic differences in selenium metabolism. (orig.)

  13. Tissue disposition of bifenthrin in the rat

    Data.gov (United States)

    U.S. Environmental Protection Agency — Tissue disposition of bifenthrin in the rat and oral and intravenous administration. This dataset is associated with the following publication: Hughes , M., D. Ross...

  14. Dietary GABA and food selection by rats.

    Science.gov (United States)

    Tews, J K; Repa, J J; Harper, A E

    1986-01-01

    To obtain further information pertaining to amino acid-induced alterations in feeding behavior, studies were performed to examine the food choices made by rats fed low protein diets made more or less aversive by the addition of various amino acids. When rats were allowed to choose between two diets, they preferred a low protein control, threonine-imbalanced or nonprotein diet to one containing 2.5% gamma-aminobutyric acid (GABA). Acceptance increased when GABA content was lowered to 1.5%; rats preferred this diet when the alternative diet was made sufficiently aversive. There were large individual differences among rats selecting from pairs of unacceptable diets. Avoidance of, or preference for, a given diet is clearly affected by the relative aversive qualities of the offered pair of diets.

  15. Experimental Salmonella typhimurium infections in rats. I

    DEFF Research Database (Denmark)

    Hougen, H P; Jensen, E T; Klausen, B

    1989-01-01

    The course of experimentally induced Salmonella typhimurium infection was studied in three groups of inbred LEW rats: homozygous +/+, athymic rnu/rnu and isogeneic thymus-grafted rnu/rnu rats. In the first experiment the animals were inoculated intraperitoneally with 10(8) bacteria and all animals...... became severely septicemic and died within a week of inoculation, irrespective of presence or absence of thymus. In the second experiment the animals were inoculated with 10(6) bacteria, and both euthymic and thymus-grafted animals responded with high titres of anti bacterial antibodies while these were...... very low in the athymic nude animals. Polyclonal antibody production was only observed in the euthymic animals and only regarding IgG. Athymic rats were not able to clear the infection, while the thymus-grafted animals reacted like euthymic rats: Very few animals housed the bacteria four weeks after...

  16. Black ginseng extract ameliorates hypercholesterolemia in rats

    Directory of Open Access Journals (Sweden)

    Evelyn Saba

    2016-04-01

    Conclusion: Administration of BG extracts to Sprague Dawley rats fed with high-cholesterol diet ameliorated hypercholesterolemia, which was mediated via modulation of cholesterol-metabolizing marker genes. This data throw a light on BG's cardioprotective effects.

  17. Reactive Protein Synthesis in Pregnant Rats

    African Journals Online (AJOL)

    olayemitoyin

    Department of Physiology, College of Medicine, University of Lagos, Nigeria. Summary: Genistein ... Oral exposure of pregnant rats to genistein precipitated hypothyroidism, altered some metabolic hormones with a ... consumption. Exposure to ...

  18. Tritium metabolism in rat tissues

    International Nuclear Information System (INIS)

    Takeda, H.

    1982-01-01

    As part of a series of studies designed to evaluate the relative radiotoxicity of various tritiated compounds, metabolism of tritium in rat tissues was studied after administration of tritiated water, leucine, thymidine, and glucose. The distribution and retention of tritium varied widely, depending on the chemical compound administered. Tritium introduced as tritiated water behaved essentially as body water and became uniformly distributed among the tissues. However, tritium administered as organic compounds resulted in relatively high incorporation into tissue constituents other than water, and its distribution differed among the various tissues. Moreover, the excretion rate of tritium from tissues was slower for tritiated organic compounds than for tritiated water. Administrationof tritiated organic compounds results in higher radiation doses to the tissues than does administration of tritiated water. Among the tritiated compounds examined, for equal radioactivity administered, leucine gave the highest radiation dose, followed in turn by thymidine, glucose, and water. (author)

  19. How rats combine temporal cues.

    Science.gov (United States)

    Guilhardi, Paulo; Keen, Richard; MacInnis, Mika L M; Church, Russell M

    2005-05-31

    The procedures for classical and operant conditioning, and for many timing procedures, involve the delivery of reinforcers that may be related to the time of previous reinforcers and responses, and to the time of onsets and terminations of stimuli. The behavior resulting from such procedures can be described as bouts of responding that occur in some pattern at some rate. A packet theory of timing and conditioning is described that accounts for such behavior under a wide range of procedures. Applications include the food searching by rats in Skinner boxes under conditions of fixed and random reinforcement, brief and sustained stimuli, and several response-food contingencies. The approach is used to describe how multiple cues from reinforcers and stimuli combine to determine the rate and pattern of response bouts.

  20. Epidermal growth factor in the rat prostate

    DEFF Research Database (Denmark)

    Tørring, Niels; Jørgensen, P E; Poulsen, Steen Seier

    1998-01-01

    Epidermal growth factor (EGF) induces proliferation in prostate epithelial and stromal cells in primary culture. This investigation was set up to characterize the time and spatial expression of EGF in the rat prostate.......Epidermal growth factor (EGF) induces proliferation in prostate epithelial and stromal cells in primary culture. This investigation was set up to characterize the time and spatial expression of EGF in the rat prostate....

  1. Epidermal growth factor reactivity in rat milk

    DEFF Research Database (Denmark)

    Raaberg, Lasse; Nexø, Ebba; Tollund, L

    1990-01-01

    whey elutes as a broad peak corresponding to a Stokes radius of 4.0 nm (an approximate molecular weight of 80 kDa). Almost no 6 kDa EGF is present. Judged by gel filtration of whey pre-incubated with 125I-EGF (6 kDa), no binding protein for EGF is present in rat whey. When rat milk is incubated...

  2. Genetically determined differences in the resistance to myocardial infarction in Wistar and August rats.

    Science.gov (United States)

    Belkina, L M; Saltykova, V A; Pshennikova, M G

    2001-06-01

    In intact August rats, the cardiac contractile function at rest was by 76% higher than in Wistar rats, while their hearts, both intact and after acute myocardial infarction, were more resistant to isometric load than the hearts of Wistar rats. Postinfarction mortality in August rats was 18% vs. 70% in Wistar rats. Adrenoreactivity of the myocardium in August rats was decreased compared to that in Wistar rats. These peculiarities can determine high resistance of August rats to myocardial infarction.

  3. Factors influencing zinc bioavailability in rats

    International Nuclear Information System (INIS)

    Mahalko, J.R.; Johnson, P.E.; Swan, P.B.

    1986-01-01

    The amount of Zn fed, its source, and the Zn status of experimental animals may affect Zn bioavailability. To test this, rats were fed doses of Zn from ZnCl 2 or from various foods labeled extrinsically. Three weeks before and after the test meal, rats were fed an AIN diet modified in Zn content. Absorption was calculated by monitoring whole body retention and extrapolating to zero time. In rats fed 12 ppm Zn and test doses of 6 to 275 μg, absorption decreased from 80 to 50%, and the amount absorbed increased quadratically (r 2 = 0.998), but turnover was unaffected. Rats fed 38 or 77 ppm Zn absorbed less of test doses of 290, 613, or 1700 μg Zn than did those fed 12 ppm, and their Zn turnover rate was higher. In two 2 x 7 factorial experiments, rats fed 12 or 38 ppm Zn were given 16 or 98 μg Zn from 7 Zn sources. Bioavailability from some foods was higher than from ZnCl 2 except in rats eating only 12 ppm Zn and receiving the small dose. There were greater differences in bioavailability among foods when tested at the higher Zn status or dose. This may explain inconsistencies seen in comparing Zn bioavailability by traditional growth assay with that seen in 65 Zn tracer studies. The authors conclude that Zn status of the experimental animal, as well as the amount of Zn and its source, will affect Zn bioavailability

  4. Modifying factors in rat mammary gland carcinogenesis

    International Nuclear Information System (INIS)

    Shellabarger, C.J.

    1975-01-01

    The spontaneous incidence of mammary adenocarcinomas and mammary fibroadenomas in rats was found to be related to the strain of rat studied. Strains of rats that are sensitive to chemical carcinogens in regard to induced mammary neoplasia tend to be the same strains of rats that are sensitive to radiation. Methylcholantrene (MCA) and x-rays appeared to act in an additive fashion on the induction of mammary adenocarcinomas when they were given together. Lactating and older rats lose responsiveness to chemical carcinogens but do not lose responsiveness to radiation. Radiation appears to act in a scopal fashion in the induction of mammary neoplasia. Mammary neoplasia induction was not changed when low LET radiation was split into 2 equal fractions and high LET radiation was more effective than low LET radiation in inducing mammary neoplasia. It is suggested that DMBA can act as an initiator for the induction of mammary adenocarcinomas, that phorbol can act as a promotor, and that viruses may induce mammary neoplasia. Diethylstilbestrol (DES) and radiation appeared to act synergistically in the induction of mammary adenocarcinomas in one strain of rat but not in another strain. (U.S.)

  5. Diuron-induced rat bladder epithelial cytotoxicity.

    Science.gov (United States)

    Da Rocha, Mitscheli S; Arnold, Lora L; Pennington, Karen L; Muirhead, David; Dodmane, Puttappa R; Anwar, Muhammad M; Battalora, Michael; De Camargo, João Lauro V; Cohen, Samuel M

    2012-12-01

    Diuron, a substituted urea herbicide, is carcinogenic to the rat urinary bladder at high dietary levels (2500 ppm). To further elucidate the mode of action, this study aimed to determine the time course and sequence of bladder cytotoxic and proliferative changes induced by diuron treatment of male Wistar rats. Rats were randomized into two groups (control and 2500 ppm diuron) and treated for 28 days. Ten rats from each group were terminated on each of study days 1, 3, 7, or 28. Scanning electron micro scopy (SEM) showed urothelial cell swelling beginning on day 1, and by day 28, showed extensive necrosis, exfoliation and piling up of cells suggestive of hyperplasia. No difference in the bromo deoxyuridine labeling index was detected. In a second experiment, rats were randomized into control and diuron-treated groups and treated for 7 days or 8 weeks. After 7 days, transmission electron microscopy showed cell degenerative changes and distention of the cytoplasm, organelles, and nuclei characteristic of cytolysis. This resulted in protrusion of the superficial cells into the lumen, corresponding to the cell swelling observed previously by SEM. After 8 weeks, bladders in the diuron-treated group showed an increased incidence of simple hyperplasia by light microscopy (6/10, p diuron exposure in rats.

  6. Radiation nephropathy in young and adult rats

    International Nuclear Information System (INIS)

    Jongejan, H.T.; van der Kogel, A.J.; Provoost, A.P.; Molenaar, J.C.

    1987-01-01

    The effects of bilateral kidney irradiation were compared in young and adult rats. During a 1 year period after a single dose of 0, 7.5, 10, 12.5, or 15 Gy on both kidneys, renal function (glomerular filtration rate and effective renal plasma flow), urine composition, and systolic blood pressure were measured periodically. The first changes after irradiation were observed in the glomerular filtration rate and urine osmolality. One month after 10, 12.5, and 15 Gy, glomerular filtration rate (GFR) and urine osmolality had declined below control values in the young rats. After this initial decline, renal function increased at control rate or even more during the third and fourth month after irradiation but decreased progressively thereafter. In the adult rats, GFR and urine osmolality started to decrease 3 months after 10, 12.5, and 15 Gy. A rise in systolic blood pressure and proteinuria started 2-3 months after 12.5 and 15 Gy in both age groups. Early changes in the glomerular filtration rate with a drop in urine osmolality in young rats, occurring during a period of rapid renal development indicated an irradiation-induced inhibition of glomerular and tubular development. Although renal function deteriorated at a later time in adult rats, dose-response relationships obtained in young and adult rats did not show significant differences

  7. Reproductive toxicity of Samanea tubulosa on rats

    Directory of Open Access Journals (Sweden)

    Maria Rafaella Luz de Araújo

    2015-12-01

    Full Text Available Samanea tubulosa is a plant used for medicinal and feeding purposes. However, ingestion of S. tubulosa pods has been associated with bovine abortion. Thus, the aim of this work was to investigate the effects of diet containing 5% of S. tubulosa pod meal on male and female Wistar rats. Diet was administered to male rats (n = 10 for 60 days before mating. Female rats (n = 10 received the treatment for 30 days, during cohabitation and from gestational day (GD 0 to GD20. Treated animals were mated with untreated rats. In male rats, plant consumption caused decreased food consumption and 20% fertility index reduction. Litters from treated males presented lower body weight and crown–rump length. Female rats treated with the plant increased water and food intake and body weight. Decreases in fertility, fecundity and gestation indices and increase of placenta weight and mean number of corpora lutea were found. Thus, owing to the possible general and reproductive toxic effects, long-term consumption of S. tubulosa is not recommended for phytotherapic or food purposes.

  8. Regulation of brain aromatase activity in rats

    International Nuclear Information System (INIS)

    Roselli, C.E.; Ellinwood, W.E.; Resko, J.A.

    1984-01-01

    The distribution and regulation of aromatase activity in the adult rat brain with a sensitive in vitro assay that measures the amount of 3 H 2 O formed during the conversion of [1 beta- 3 H]androstenedione to estrone. The rate of aromatase activity in the hypothalamus-preoptic area (HPOA) was linear with time up to 1 h, and with tissue concentrations up to 5 mgeq/200 microliters incubation mixture. The enzyme demonstrated a pH optimum of 7.4 and an apparent Michaelis-Menten constant (Km) of 0.04 microns. The greatest amount of aromatase activity was found in amygdala and HPOA from intact male rats. The hippocampus, midbrain tegmentum, cerebral cortex, cerebellum, and anterior pituitary all contained negligible enzymatic activity. Castration produced a significant decrease in aromatase activity in the HPOA, but not in the amygdala or cerebral cortex. The HPOAs of male rats contained significantly greater aromatase activity than the HPOAs of female rats. In females, this enzyme activity did not change during the estrous cycle or after ovariectomy. Administration of testosterone to gonadectomized male and female rats significantly enhanced HPOA aromatase activities to levels approximating those found in HPOA from intact males. Therefore, the results suggest that testosterone, or one of its metabolites, is a major steroidal regulator of HPOA aromatase activity in rats

  9. Radio protective effects of selenium on rats

    International Nuclear Information System (INIS)

    Bakir, A.; Alya, G

    2005-11-01

    Potential radio-protective effects of different selenium supplement concentrations of 4, 8, 15 and 30 ppm were evaluated in rats. Four groups of rats were administered different concentrations of selenium in drinking water for 30 days before irradiation starting from the ablactation which considered as day 0. The results showed that the sodium selenite of 4 ppm and 8 ppm enhance the 30-day survival of irradiated rats at 7 Gy ( sup 6 sup 0 Co source, whole body irradiation dose rate of 1 Gy x min sup - sup 1) compared to the control group. The mean cumulated probability of survival of rats was 69%+-6 (mean+-S.E.) and 77%+-6 in 4 and 8 ppm groups, respectively, versus 42%+-9 for control group (P<0.001). It was also indicated that sodium selenite with concentrations of 15 and 30 ppm had no significant reduction in mortality. The mean cumulated probability of survival of rats was 50%+-12 (P=0.39) and 49%+-14 (P=0.04), respectively. The toxic effects of selenium were observed at 15 ppm and 30 ppm, survivals after 30 days of selenium intake were 76% and 46%, respectively. It was concluded that 4 and 8 ppm sodium selenite have a radio-protective effect. 15 and 30 ppm sodium selenite had no radio-protective effects in rats, this may be due to a synergism of toxicity and radiation effects. (author)

  10. [Preventive effects of pueraria on presbycusis in rats].

    Science.gov (United States)

    Chen, Wangyan; Yao, Qi; Liu, Weihong; Zhang, Bibo; Wang, Ying; Liu, Bo

    2009-08-01

    To investigate the preventive effects of Pueraria on presbycusis in rats. Thirty-two 24-26 month old Wistar rats were randomly divided into four groups, and were treated with different dosages of Pueraria (1, 2, 4, 0 g x kg(-1) x d(-1)) separately for 4 weeks. Auditory brainstem response (ABR) was used to detect the change of hearing threshold of rats. Hemorheological items of rats were checked in each group. Compared with control group, the hearing threshold and hemorheological items of rats was significantly improved after treated with Pueraria (Ppresbycusis of rats.

  11. The endoplasmic reticulum stress-autophagy pathway is involved in apelin-13-induced cardiomyocyte hypertrophy in vitro

    Institute of Scientific and Technical Information of China (English)

    Feng XIE; Di WU; Shi-fang HUANG; Jian-gang CAO; He-ning LI; Lu HE; Mei-qing LIU; Lan-fang LI; Lin-xi CHEN

    2017-01-01

    Apelin is the endogenous ligand for the G protein-coupled receptor APJ,and plays important roles in the cardiovascular system.Our previous studies showed that apelin-13 promotes the hypertrophy of H9c2 rat cardiomyocytes through the PI3K-autophagy pathway.The aim of this study was to explore what roles ER stress and autophagy played in apelin-13-induced hypertrophy of cardiomyocytes in vitro.Treatment of H9c2 cells with apelin-13 (0.001-2 μJmol/L) dose-dependently increased the production of ROS and the expression levels of NADPH oxidase 4 (NOX4).Knockdown of Nox4 with siRNAs effectively prevented the reduction of GSH/GSSG ratio in apelin-13-treated cells.Furthermore,apelin-13 treatment dose-dependently increased the expression of Bip and CHOP,two ER stress markers,in the cells.Knockdown of APJ or Nox4 with the corresponding siRNAs,or application of NADPH inhibitor DPI blocked apelin-13-induced increases in Bip and CHOP expression.Moreover,apelin-13 treatment increased the formation of autophagosome and ER fragments and the LC3 puncta in the ER of the cells.Knockdown of APJ,Nox4,Bip or CHOP with the corresponding siRNAs,or application of DPI or salubrinal attenuated apelin-13-induced overexpression of LC3-Ⅱ/Ⅰ and beclin 1.Finally,knockdown of Nox4,Bip or CHOP with the corresponding siRNAs,or application of salubrinal significantly suppressed apelin-13-induced increases in the cell diameter,volume and protein contents.Our results demonstrate that ER stress-autophagy is involved in apelin-13-induced H9c2 cell hypertrophy.

  12. Diclofenac induces proteasome and mitochondrial dysfunction in murine cardiomyocytes and hearts.

    Science.gov (United States)

    Ghosh, Rajeshwary; Goswami, Sumanta K; Feitoza, Luis Felipe B B; Hammock, Bruce; Gomes, Aldrin V

    2016-11-15

    One of the most common nonsteroidal anti-inflammatory drugs (NSAIDs) used worldwide, diclofenac (DIC), has been linked to increased risk of cardiovascular disease (CVD). The molecular mechanism(s) by which DIC causes CVD is unknown. Proteasome activities were studied in hearts, livers, and kidneys from male Swiss Webster mice treated with either 100mg/kg DIC for 18h (acute treatment) or 10mg/kg DIC for 28days (chronic treatment). Cultured H9c2 cells and neonatal cardiomyocytes were also treated with different concentrations of DIC and proteasome function, cell death and ROS generation studied. Isolated mouse heart mitochondria were utilized to determine the effect of DIC on various electron transport chain complex activities. DIC significantly inhibited the chymotrypsin-like proteasome activity in rat cardiac H9c2 cells, murine neonatal cardiomyocytes, and mouse hearts, but did not affect proteasome subunit expression levels. Proteasome activity was also affected in liver and kidney tissues from DIC treated animals. The levels of polyubiquitinated proteins increased in hearts from DIC treated mice. Importantly, the levels of oxidized proteins increased while the β5i immunoproteasome activity decreased in hearts from DIC treated mice. DIC increased ROS production and cell death in H9c2 cells and neonatal cardiomyocytes while the cardioprotective NSAID, aspirin, had no effect on ROS levels or cell viability. DIC inhibited mitochondrial Complex III, a major source of ROS, and impaired mitochondrial membrane potential suggesting that mitochondria are the major sites of ROS generation. These results suggest that DIC induces cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. High Uric Acid Induces Insulin Resistance in Cardiomyocytes In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Li Zhi

    Full Text Available Clinical studies have shown hyperuricemia strongly associated with insulin resistance as well as cardiovascular disease. Direct evidence of how high uric acid (HUA affects insulin resistance in cardiomyocytes, but the pathological mechanism of HUA associated with cardiovascular disease remains to be clarified. We aimed to examine the effect of HUA on insulin sensitivity in cardiomyocytes and on insulin resistance in hyperuricemic mouse model. We exposed primary cardiomyocytes and a rat cardiomyocyte cell line, H9c2 cardiomyocytes, to HUA, then quantified glucose uptake with a fluorescent glucose analog, 2-NBDG, after insulin challenge and detected reactive oxygen species (ROS production. Western blot analysis was used to examine the levels of insulin receptor (IR, phosphorylated insulin receptor substrate 1 (IRS1, Ser307 and phospho-Akt (Ser473. We monitored the impact of HUA on insulin resistance, insulin signaling and IR, phospho-IRS1 (Ser307 and phospho-Akt levels in myocardial tissue of an acute hyperuricemia mouse model established by potassium oxonate treatment. HUA inhibited insulin-induced glucose uptake in H9c2 and primary cardiomyocytes. It increased ROS production; pretreatment with N-acetyl-L-cysteine (NAC, a ROS scavenger, reversed HUA-inhibited glucose uptake induced by insulin. HUA exposure directly increased the phospho-IRS1 (Ser307 response to insulin and inhibited that of phospho-Akt in H9C2 cardiomyocytes, which was blocked by NAC. Furthermore, the acute hyperuricemic mice model showed impaired glucose tolerance and insulin tolerance accompanied by increased phospho-IRS1 (Ser307 and inhibited phospho-Akt response to insulin in myocardial tissues. HUA inhibited insulin signaling and induced insulin resistance in cardiomyocytes in vitro and in vivo, which is a novel potential mechanism of hyperuricemic-related cardiovascular disease.

  14. Fish oil versus arachis oil food supplementation in relation to pregnancy duration in rats

    DEFF Research Database (Denmark)

    Olsen, S.F.; Hansen, Harald S.; Jensen, B.

    1990-01-01

    Throughout pregnancy, Lewis rats were fed standard rat chow supplemented with 15% (w/w) of either MaxEPA fish oil (FO) or arachis oil (AO); a third group was fed standard rat chow only (St) (n = 15, 15, and 16 rats, respectively). Compared to AO-rats, FO-rats had substantially higher levels of n-3...

  15. Increased radiosensitivity of cerebral capillaries in neonatal Gunn rats as compared to Sprague-Dawley rats

    International Nuclear Information System (INIS)

    Landolt, R.; Arn, D.

    1979-01-01

    The extent of petechial haemorrhages of the cerebral cortex examined between 14 hours and 4 days after X-irradiation to the head was compared in Sprague-Dawley and homozygous Gunn rats with congenital hyperbilirubinaemia. Animals 1 to 2 days old received single doses of either 250, 500 or 750 rad. By means of a special scoring scale the degree of the damage to the micro vasculature was semi-quantitatively estimated. In both strains a significant difference in effect was obtained between 250 and 500 rad, but not between 500 and 750 rad. The shape of the dose-effect curve in Gunn rats was similar to that of Sprague-Dawley rats, but displaced upwards. In Gunn rats the effect of 250 rad was greater that that of 750 rad in Sprague-Dawley rats. Possible radiosensitizing mechanisms are discussed with reference to the literature and these results. (author)

  16. Social structure predicts genital morphology in African mole-rats.

    Directory of Open Access Journals (Sweden)

    Marianne L Seney

    2009-10-01

    Full Text Available African mole-rats (Bathyergidae, Rodentia exhibit a wide range of social structures, from solitary to eusocial. We previously found a lack of sex differences in the external genitalia and morphology of the perineal muscles associated with the phallus in the eusocial naked mole-rat. This was quite surprising, as the external genitalia and perineal muscles are sexually dimorphic in all other mammals examined. We hypothesized that the lack of sex differences in naked mole-rats might be related to their unusual social structure.We compared the genitalia and perineal muscles in three African mole-rat species: the naked mole-rat, the solitary silvery mole-rat, and the Damaraland mole-rat, a species considered to be eusocial, but with less reproductive skew than naked mole-rats. Our findings support a relationship between social structure, mating system, and sexual differentiation. Naked mole-rats lack sex differences in genitalia and perineal morphology, silvery mole-rats exhibit sex differences, and Damaraland mole-rats are intermediate.The lack of sex differences in naked mole-rats is not an attribute of all African mole-rats, but appears to have evolved in relation to their unusual social structure and reproductive biology.

  17. Social structure predicts genital morphology in African mole-rats.

    Science.gov (United States)

    Seney, Marianne L; Kelly, Diane A; Goldman, Bruce D; Sumbera, Radim; Forger, Nancy G

    2009-10-15

    African mole-rats (Bathyergidae, Rodentia) exhibit a wide range of social structures, from solitary to eusocial. We previously found a lack of sex differences in the external genitalia and morphology of the perineal muscles associated with the phallus in the eusocial naked mole-rat. This was quite surprising, as the external genitalia and perineal muscles are sexually dimorphic in all other mammals examined. We hypothesized that the lack of sex differences in naked mole-rats might be related to their unusual social structure. We compared the genitalia and perineal muscles in three African mole-rat species: the naked mole-rat, the solitary silvery mole-rat, and the Damaraland mole-rat, a species considered to be eusocial, but with less reproductive skew than naked mole-rats. Our findings support a relationship between social structure, mating system, and sexual differentiation. Naked mole-rats lack sex differences in genitalia and perineal morphology, silvery mole-rats exhibit sex differences, and Damaraland mole-rats are intermediate. The lack of sex differences in naked mole-rats is not an attribute of all African mole-rats, but appears to have evolved in relation to their unusual social structure and reproductive biology.

  18. Cardioprotective Effects of 20(S-Ginsenoside Rh2 against Doxorubicin-Induced Cardiotoxicity In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Hongbo Wang

    2012-01-01

    Full Text Available Doxorubicin (DOX is considered as one of the best antineoplastic agents. However, its clinical use is restricted by its associated cardiotoxicity, which is mediated by the production of reactive oxygen species. In this study, 20(S-ginsenoside Rh2 (Rh2 was explored whether it had protective effects against DOX-induced cardiotoxicity. In vitro study on H9C2 cell line, as well as in vivo investigation in one mouse and one rat model of DOX-induced cardiomyopathy, was carried out. The results showed that pretreatment with Rh2 significantly increased the viability of DOX-injured H9C2 cells. In the mouse model, Rh2 could suppress the DOX-induced release of the cardiac enzymes into serum and improved the occurred pathological changes through ameliorating the decreased antioxidant biomolecules and the cumulated lipid peroxidation malondialdehyde in heart tissues. In the rat model, Rh2 could attenuate the change of ECG resulting from DOX administration. Furthermore, Rh2 enhanced the antitumor activity of DOX in A549 cells. Our findings thus demonstrated that Rh2 pretreatment could effectively alleviate heart injury induced by DOX, and Rh2 might act as a novel protective agent in the clinical usefulness of DOX.

  19. Ginkgolide B Exerts Cardioprotective Properties against Doxorubicin-Induced Cardiotoxicity by Regulating Reactive Oxygen Species, Akt and Calcium Signaling Pathways In Vitro and In Vivo.

    Science.gov (United States)

    Gao, Junqing; Chen, Tao; Zhao, Deqiang; Zheng, Jianpu; Liu, Zongjun

    2016-01-01

    The aim of this study was to evaluate the effect of Ginkgolide B (GB) on doxorubicin (DOX) induced cardiotoxicity in vitro and in vivo. Rat cardiomyocyte cell line H9c2 was pretreated with GB and subsequently subjected to doxorubicin treatment. Cell viability and cell apoptosis were assessed by MTT assay and Hoechst staining, respectively. Reactive oxygen species (ROS), Akt phosphorylation and intracellular calcium were equally determined in order to explore the underlying molecular mechanism. To verify the in vivo therapeutic effect of GB, we established a mouse model of cardiotoxicity and determined left ventricle ejection fraction (LVEF) and left ventricular mass (LVM). The in vitro experimental results indicated that pretreatment with GB significantly decreases the viability and apoptosis of H9c2 cells by decreasing ROS and intracellular calcium levels and activating Akt phosphorylation. In the in vivo study, we recorded an improved LVEF and a decreased LVM in the group of cardiotoxic rats treated with GB. Altogether, our findings anticipate that GB exerts a cardioprotective effect through possible regulation of the ROS, Akt and calcium pathways. The findings suggest that combination of GB with DOX in chemotherapy could help avoid the cardiotoxic side effects of GB.

  20. Automated registration of tail bleeding in rats.

    Science.gov (United States)

    Johansen, Peter B; Henriksen, Lars; Andresen, Per R; Lauritzen, Brian; Jensen, Kåre L; Juhl, Trine N; Tranholm, Mikael

    2008-05-01

    An automated system for registration of tail bleeding in rats using a camera and a user-designed PC-based software program has been developed. The live and processed images are displayed on the screen and are exported together with a text file for later statistical processing of the data allowing calculation of e.g. number of bleeding episodes, bleeding times and bleeding areas. Proof-of-principle was achieved when the camera captured the blood stream after infusion of rat whole blood into saline. Suitability was assessed by recording of bleeding profiles in heparin-treated rats, demonstrating that the system was able to capture on/off bleedings and that the data transfer and analysis were conducted successfully. Then, bleeding profiles were visually recorded by two independent observers simultaneously with the automated recordings after tail transection in untreated rats. Linear relationships were found in the number of bleedings, demonstrating, however, a statistically significant difference in the recording of bleeding episodes between observers. Also, the bleeding time was longer for visual compared to automated recording. No correlation was found between blood loss and bleeding time in untreated rats, but in heparinized rats a correlation was suggested. Finally, the blood loss correlated with the automated recording of bleeding area. In conclusion, the automated system has proven suitable for replacing visual recordings of tail bleedings in rats. Inter-observer differences can be eliminated, monotonous repetitive work avoided, and a higher through-put of animals in less time achieved. The automated system will lead to an increased understanding of the nature of bleeding following tail transection in different rodent models.

  1. Effect of radiation on rat skin collagen

    International Nuclear Information System (INIS)

    Nogami, Akira

    1980-01-01

    I. Albino male rats were exposed for 16 weeks to ultraviolet light (UVL) which has principle emission at 305 nm. There were no significant changes between control and UVL-exposed skins in the total hydroxyproline content. However, a little increase of citrate-soluble collagen, a little decrease of insoluble collagen and a decrease of aldehyde content in soluble collagen were observed with UVL exposure. Total acid glycosaminoglycan in skin increased 30% or more from control. These results show that the effect of UVL on rat skin in vivo was merely inflammation phenomenon and that the 'aging' process of skin was not caused in our experimental conditions. II. The effects of radiation on the solubility of rat skin collagen were examined under various conditions. 1) When intact rats were exposed to a single dose of radiation from 43 kVp X-ray source, the solubility in skin collagen did not change at 4,000 R dosage, while in irradiation of 40,000 R a decreased solubility in collagen was observed. When rats were given 400 R a week for 12 weeks, there was no changes in the solubility of collagen during experimental period. 2) In vitro exposure to skins, an irradiation of 40,000 R from 43 kVp X-ray source caused a decrease in the solubility of collagen. While an irradiation of 40,000 R of dosage from 200 kVp X-ray source resulted in the increase in soluble collagen and the decrease in insoluble collagen. 3) When intact rats were given a single dose of 40,000 R from 60 Co- gamma -ray, insoluble collagen decreased in both young and adult rats. Similar changes in collagen solubility were observed in vitro gamma -irradiation. (author)

  2. [Pinealectomy and early castration in the female Wistar rat].

    Science.gov (United States)

    Slama-Scemama, A

    1976-05-17

    Pinealectomy does not significantly modify the level of pituitary and plasma gonadotropins in intact and in castrated female Rats from brith to 75 days of age. Only the weight of the thyroid gland is higher in pinealectomized rats.

  3. Patterns of blood pressure variability in normotensive and hypertensive rats

    DEFF Research Database (Denmark)

    Holstein-Rathlou, N H; He, J; Wagner, A J

    1995-01-01

    We sought patterns in mean arterial pressure of normotensive rats and alterations in chronic hypertension. Pressure was recorded for 4-6 days by telemetry from conscious, unrestrained rats and sampled digitally at 3 Hz, using normotensive Sprague-Dawley rats, spontaneously hypertensive rats (SHR)...... the day; less pronounced in 2K,1C; and not detectable in SHR. There are regular patterns of blood pressure fluctuations and specific modifications to the patterns by different forms of hypertension.......We sought patterns in mean arterial pressure of normotensive rats and alterations in chronic hypertension. Pressure was recorded for 4-6 days by telemetry from conscious, unrestrained rats and sampled digitally at 3 Hz, using normotensive Sprague-Dawley rats, spontaneously hypertensive rats (SHR...

  4. Classical clinical signs in rats experimemtally infected with Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Nwoha Rosemary Ijeoma Ogechi

    2015-02-01

    Full Text Available Objective: To investigate clinical signs in Trypanosoma brucei infection in albino rats. Methods: Fourteen rats grouped into 2 with 7 rats in each group were used to determine classical clinical manifestation of Trypanosoma brucei infection in rats. Group A rats were uninfected control and Group B rats were infected with Trypanosoma brucei. Results: Parasitaemia was recorded in Group B by (3.86±0.34 d and the peak of parasitaemia was observed at Day 5 post infection. Classical signs observed included squint eyes, raised whiskers, lethargy, no weight loss, pyrexia, isolation from the other rats, and starry hair coat. Conclusions: These signs could be diagnostic or aid in diagnosis of Trypanosoma brucei infection in rats.

  5. [Pituitary function of dysgenesic femal rats. Studies with grafting method].

    Science.gov (United States)

    Vanhems, E; Busquet, J

    1975-01-01

    Misulban administered to pregnant rats on the 15th day of gestation provoked gonadal dysgenesia in the offspring. Study of the pituitary function of dysgenesic female rats, realized by grafting method, showed gonadotrophic hypersecretion.

  6. Water metabolism and modification of tritium excretion in the rat

    International Nuclear Information System (INIS)

    Ichimasa, Y.; Akita, Y.

    1982-01-01

    1. The intake and excretion of tritium were studied in rats exposed to tritiated water vapor. The metabolism of tritium was also investigated in rats given single administrations of tritiated water and in rats given daily administrations (per os or i.p.). The results were essentially in accord with those reported previously. 2. Amounts of drinking water consumed and urine excreted by rats drinking water with 0.15% saccharin were 1.5 to 2 times higher than in rats drinking tap water. The tritium activity in various tissues of rats drinking water with 0.15% saccharin decreased to about half of that of rats drinking tap water. A similar tendency was observed also in rats drinking beer. The diuretic agent sodium acetazolamide also enhanced the urinary excretion of tritium. (author)

  7. Uptake of trace elements in adult and suckling rat lenses

    International Nuclear Information System (INIS)

    Nabekura, Tomohiro; Ito, Yoshimasa; Minami, Takeshi; Hirunuma, Rieko; Enomoto, Shuichi

    2001-01-01

    The uptake of trace elements in the lens was compared in adult and suckling rat lenses. Multitracers, including 15 trace elements, As, Be, Co, Fe, Mn, Rb, Rh, Ru, Sc, Se, Sr, Y, V, Zn, and Zr, were incubated with the lenses for 4 hr and their concentrations in the lens were measured. A high uptake rate of Zn was observed in the lenses of both adult and suckling rats in comparison with those of the other elements, and the Zn concentration in the lens of suckling rats was higher than that of adult rats. The uptake rate of Sr was higher in adult rats than in suckling rats. On the other contrary, Rb and Se concentrations in the lens were higher in suckling rats than in adult rats. The present study suggests that the different mechanisms depending on development serve to transport trace elements into the lens. (author)

  8. Absorption of plutonium in the iron-deficient rat

    International Nuclear Information System (INIS)

    Ragan, H.A.

    1977-01-01

    Iron deficiency did not enhance absorption of plutonium following intragastric gavage of rats. Absorption of plutonium citrate in both control and iron-deficient rats was about 0.03% of the administered dose

  9. (Urginea Altissima), Against the Field Rat, Arvicanthis Abyssinicus

    African Journals Online (AJOL)

    Bernt Lindtjorn

    the field rat, Arvicanthis abyssincus with the aim of developing locally based ... inhabited by humans and is commonly found in open ... rat, A. abyssinicus in a choice and non-choice tests. ..... sowing control of house mice (Mus domesticus):.

  10. Biochemical and Haematological Indices of Weanly Albino Rats Fed ...

    African Journals Online (AJOL)

    acer

    ABSTRACT: Malnutrition is a public health problem in Nigeria accounting for more than 50% of ... weanly albino rats using nutritional, biochemical ... groundnut (16%), soy beans (16%), crayfish ... consumption was observed in rats on PC and.

  11. Ulinastatin Reduces T Cell Apoptosis in Rats with Severe Acute ...

    African Journals Online (AJOL)

    in rats with severe acute pancreatitis (SAP) and to elucidate its underlying molecular mechanism. Methods: Thirty .... on T lymphocytes apoptosis in SAP rat model and elucidated ..... oxygen radicals, the exhaustion of adenine nucleotide and ...

  12. Healing effect of Shaoshang Yuhe yihao on burns in rats

    African Journals Online (AJOL)

    tissue expressions of transforming growth factor-beta (TGF-β) were determined along with skin histopathology. ... Polygonum cuspidatum Sieb. et Zucc., Aloe vera var. chinensis ... anesthetizing the rats after the rat hair was shaved. The top of ...

  13. Biological effects of 137Cs, incorporated into organism of rats

    International Nuclear Information System (INIS)

    Monakhov, A.S.; Strekalov, S.A.; Sokolov, A.V.; Aver'yanova, T.K.

    1987-01-01

    Results of investigating mutagenous and hemotoxic effects of 137 Cs on blood lymphocytes of rats are presented. 137 Cs was orally administrated into organism of rats as 270 kBq/g chloride solution. 137 Cs mutagenous effect was studied on metaphase plates of rat blood lymphocytes in course of rats lifetime experiment. It is stated that 137 Cs inducing severe disturbances of genetic material in a great quantity of blood lymphocytes, causes their total killing

  14. Tumorigenicity of sodium ascorbate in male rats.

    Science.gov (United States)

    Cohen, S M; Anderson, T A; de Oliveira, L M; Arnold, L L

    1998-06-15

    Sodium ascorbate, like other sodium salts such as saccharin, glutamate, and bicarbonate, produces urinary alterations when fed at high doses to rats, which results in mild superficial urothelial cytotoxicity and regeneration but not tumors in a standard 2-year bioassay. Sodium saccharin was shown to produce a low incidence of bladder tumors in rats if administered in a two-generation bioassay. In the present study, we evaluated sodium ascorbate in a two-generation bioassay that involved feeding to the male and female parental F344 rats for 4 weeks before mating, feeding the dams during gestation and lactation, and then feeding the weaned (at 28 days of age) male F1 generation rats for the remainder of their lifetime (up to 128 weeks of the experiment). Dietary levels of 1.0, 5.0, and 7.0% sodium ascorbate were tested. At 5.0 and 7.0% sodium ascorbate, there was an increase in urinary bladder urothelial papillary and nodular hyperplasia and the induction of a few papillomas and carcinomas. There was a dose-responsive increase in renal pelvic calcification and hyperplasia and inhibition of the aging nephropathy of rats even at the level of 1% sodium ascorbate. Because the short-term urothelial effects of sodium ascorbate in rats are inhibited by treatments producing urinary acidification to pH sodium ascorbate to evaluate the long-term effects. The combination of 7.0% sodium ascorbate plus 2.78% NH4Cl in the diet was toxic, and the group was terminated early during the course of the experiment. The group fed 5.0% sodium ascorbate plus 2.04% NH4Cl showed complete inhibition of the urothelial effects of sodium ascorbate and significant inhibition of its renal effects. We also demonstrated the presence of a calcium phosphate-containing urinary precipitate in rats fed sodium ascorbate at all doses, in a dose-responsive manner. The formation of the precipitate was inhibited by coadministration with NH4Cl. The proliferative effects of sodium ascorbate on the male rat

  15. From engineering to editing the rat genome.

    Science.gov (United States)

    Meek, Stephen; Mashimo, Tomoji; Burdon, Tom

    2017-08-01

    Since its domestication over 100 years ago, the laboratory rat has been the preferred experimental animal in many areas of biomedical research (Lindsey and Baker The laboratory rat. Academic, New York, pp 1-52, 2006). Its physiology, size, genetics, reproductive cycle, cognitive and behavioural characteristics have made it a particularly useful animal model for studying many human disorders and diseases. Indeed, through selective breeding programmes numerous strains have been derived that are now the mainstay of research on hypertension, obesity and neurobiology (Okamoto and Aoki Jpn Circ J 27:282-293, 1963; Zucker and Zucker J Hered 52(6):275-278, 1961). Despite this wealth of genetic and phenotypic diversity, the ability to manipulate and interrogate the genetic basis of existing phenotypes in rat strains and the methodology to generate new rat models has lagged significantly behind the advances made with its close cousin, the laboratory mouse. However, recent technical developments in stem cell biology and genetic engineering have again brought the rat to the forefront of biomedical studies and enabled researchers to exploit the increasingly accessible wealth of genome sequence information. In this review, we will describe how a breakthrough in understanding the molecular basis of self-renewal of the pluripotent founder cells of the mammalian embryo, embryonic stem (ES) cells, enabled the derivation of rat ES cells and their application in transgenesis. We will also describe the remarkable progress that has been made in the development of gene editing enzymes that enable the generation of transgenic rats directly through targeted genetic modifications in the genomes of zygotes. The simplicity, efficiency and cost-effectiveness of the CRISPR/Cas gene editing system, in particular, mean that the ability to engineer the rat genome is no longer a limiting factor. The selection of suitable targets and gene modifications will now become a priority: a challenge where

  16. Opiates and cerebral functional activity in rats

    International Nuclear Information System (INIS)

    Trusk, T.C.

    1986-01-01

    Cerebral activity was measured using the free-fatty acid [1- 14 C] octanoate as a fast functional tracer in conscious, unrestrained rats 5 minutes after intravenous injection of heroin, cocaine or saline vehicle. Regional changes of octanoate labeling density in the autoradiograms relative to saline-injected animals were used to determine the functional activity effects of each drug. Heroin and cocaine each produced a distinctive pattern of activity increases and suppression throughout the rat brain. Similar regional changes induced by both drugs were found in limbic brain regions implicated in drug reinforcement. Labeled octanoate autoradiography was used to measure the cerebral functional response to a tone that had previously been paired to heroin injections. Rats were trained in groups of three consisting of one heroin self-administration animal, and two animals receiving yoked infusion of heroin or saline. A tone was paired with each infusion during training. Behavioral experiments in similarly trained rats demonstrated that these training conditions impart secondary reinforcing properties to the tone in animals previously self-administering heroin, while the tone remains behaviorally neutral in yoked-infusion rats. Cerebral functional activity was measured during presentation of the tone without drug infusion. Octanoate labeling density changed in fifteen brain areas in response to the tone previously paired to heroin without response contingency. Labeling density was significantly modified in sixteen regions as a result of previously pairing the tone to response-contingent heroin infusions

  17. Working Memory Systems in the Rat.

    Science.gov (United States)

    Bratch, Alexander; Kann, Spencer; Cain, Joshua A; Wu, Jie-En; Rivera-Reyes, Nilda; Dalecki, Stefan; Arman, Diana; Dunn, Austin; Cooper, Shiloh; Corbin, Hannah E; Doyle, Amanda R; Pizzo, Matthew J; Smith, Alexandra E; Crystal, Jonathon D

    2016-02-08

    A fundamental feature of memory in humans is the ability to simultaneously work with multiple types of information using independent memory systems. Working memory is conceptualized as two independent memory systems under executive control [1, 2]. Although there is a long history of using the term "working memory" to describe short-term memory in animals, it is not known whether multiple, independent memory systems exist in nonhumans. Here, we used two established short-term memory approaches to test the hypothesis that spatial and olfactory memory operate as independent working memory resources in the rat. In the olfactory memory task, rats chose a novel odor from a gradually incrementing set of old odors [3]. In the spatial memory task, rats searched for a depleting food source at multiple locations [4]. We presented rats with information to hold in memory in one domain (e.g., olfactory) while adding a memory load in the other domain (e.g., spatial). Control conditions equated the retention interval delay without adding a second memory load. In a further experiment, we used proactive interference [5-7] in the spatial domain to compromise spatial memory and evaluated the impact of adding an olfactory memory load. Olfactory and spatial memory are resistant to interference from the addition of a memory load in the other domain. Our data suggest that olfactory and spatial memory draw on independent working memory systems in the rat. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Gastric secretion elicited by conditioning in rats.

    Science.gov (United States)

    Caboclo, José Liberato Ferreira; Cury, Francico de Assis; Borin, Aldenis Albanese; Caboclo, Luís Otávio Sales Ferreira; Ribeiro, Maria Fernanda Sales Caboclo; de Freitas, Pedro José; Andersson, Sven

    2009-01-01

    To investigate whether interdigestive gastric acid secretion can be controlled by a possible memory-related cortical mechanism. To evaluate gastric secretion in rats, we used a methodology that allows gastric juice collection in rats in their habitual conditions (without any restraining) by pairing sound as the conditioning stimulus (CS) and food as the unconditioning stimulus (US). The levels of gastric acid secretion under basal conditions and under sound stimulation were recorded and the circulating gastrin levels determined. When the gastric juice was collected in the course of the conditioning procedure, the results showed that under noise stimulation a significant increase in gastric acid secretion occurred after 10 days of conditioning (p<0.01). The significance was definitively demonstrated after 13 days of conditioning (p<0.001). Basal secretions of the conditioned rats reached a significant level after 16 days of conditioning. The levels of noise-stimulated gastric acid secretion were the highest so far described in physiological experiments carried out in rats and there were no significant increases in the circulating gastrin levels. The results point to the important role played by cortical structures in the control of interdigestive gastric acid secretion in rats. If this mechanism is also present in humans, it may be involved in diseases caused by inappropriate gastric acid secretion during the interprandial periods.

  19. Opiates and cerebral functional activity in rats

    Energy Technology Data Exchange (ETDEWEB)

    Trusk, T.C.

    1986-01-01

    Cerebral activity was measured using the free-fatty acid (1-/sup 14/C) octanoate as a fast functional tracer in conscious, unrestrained rats 5 minutes after intravenous injection of heroin, cocaine or saline vehicle. Regional changes of octanoate labeling density in the autoradiograms relative to saline-injected animals were used to determine the functional activity effects of each drug. Heroin and cocaine each produced a distinctive pattern of activity increases and suppression throughout the rat brain. Similar regional changes induced by both drugs were found in limbic brain regions implicated in drug reinforcement. Labeled octanoate autoradiography was used to measure the cerebral functional response to a tone that had previously been paired to heroin injections. Rats were trained in groups of three consisting of one heroin self-administration animal, and two animals receiving yoked infusion of heroin or saline. A tone was paired with each infusion during training. Behavioral experiments in similarly trained rats demonstrated that these training conditions impart secondary reinforcing properties to the tone in animals previously self-administering heroin, while the tone remains behaviorally neutral in yoked-infusion rats. Cerebral functional activity was measured during presentation of the tone without drug infusion. Octanoate labeling density changed in fifteen brain areas in response to the tone previously paired to heroin without response contingency. Labeling density was significantly modified in sixteen regions as a result of previously pairing the tone to response-contingent heroin infusions.

  20. Hematological changes in opium addicted diabetic rats.

    Science.gov (United States)

    Asadikaram, Gholamreza; Sirati-Sabet, Majid; Asiabanha, Majid; Shahrokhi, Nader; Jafarzadeh, Abdollah; Khaksari, Mohammad

    2013-01-01

    Chronic opioid treatment in animal models has shown to alter hematological parameters. The aim of this study was to evaluate the biological effects of opium on the number of peripheral blood cells and red blood cells (RBCs) indices in diabetic rats. Peripheral blood samples were collected from diabetic, opium-addicted, diabetic opium-addicted and normal male and female rats and hematological parameters were measured. The mean number of white blood cells (WBCs) was significantly higher in diabetic opium-addict females compared to diabetic non-addict female group. In both male and female, the mean number of neutrophils was significantly higher and the mean number of lymphocytes was lower in diabetic opium-addicted rats than those observed in diabetic non-addicted group. In diabetic opium-addicted male group the mean counts of RBC significantly increased as compared with diabetic male group. However, in diabetic addicted female, the mean number of RBCs was significantly lower than diabetic non-addicted female group. In both males and females, the mean number of platelets was significantly lower in diabetic addict rats compared to diabetic non-addict group. Generally, the results indicated that opium addiction has different effects on male and female rats according to the number of WBC, RBC and RBC indices. It could also be concluded that in the opium-addicts the risk of infection is enhanced due to the weakness of immune system as a result of the imbalance effect of opium on the immune cells.

  1. Cardiopulmonary Changes with Moderate Decompression in Rats

    Science.gov (United States)

    Robinson, R.; Little, T.; Doursout, M.-F.; Butler, B. D.; Chelly, J. E.

    1996-01-01

    Sprague-Dawley rats were compressed to 616 kPa for 120 min then decompressed at 38 kPa/min to assess the cardiovascular and pulmonary responses to moderate decompression stress. In one series of experiments the rats were chronically instrumented with Doppler ultrasonic probes for simultaneous measurement of blood pressure, cardiac output, heart rate, left and right ventricular wall thickening fraction, and venous bubble detection. Data were collected at base-line, throughout the compression/decompression protocol, and for 120 min post decompression. In a second series of experiments the pulmonary responses to the decompression protocol were evaluated in non-instrumented rats. Analyses included blood gases, pleural and bronchoalveolar lavage (BAL) protein and hemoglobin concentration, pulmonary edema, BAL and lung tissue phospholipids, lung compliance, and cell counts. Venous bubbles were directly observed in 90% of the rats where immediate post-decompression autopsy was performed and in 37% using implanted Doppler monitors. Cardiac output, stroke volume, and right ventricular wall thickening fractions were significantly decreased post decompression, whereas systemic vascular resistance was increased suggesting a decrease in venous return. BAL Hb and total protein levels were increased 0 and 60 min post decompression, pleural and plasma levels were unchanged. BAL white blood cells and neutrophil percentages were increased 0 and 60 min post decompression and pulmonary edema was detected. Venous bubbles produced with moderate decompression profiles give detectable cardiovascular and pulmonary responses in the rat.

  2. Somatostatin ontogenesis in the gastrointestinal and pancreatic tract: study in normal rats and during a induced diabetes in neonates rats

    International Nuclear Information System (INIS)

    Cunha, M.C.

    1980-01-01

    The ontogenic studies of somatostatin of pancreas, ileum and duodenum of Wistar rats and the rats with induced diabetes were done. The radioimmunologic method to dose the somatostatin was used. (L.M.J.)

  3. Phenotypic Characterization of LEA Rat: A New Rat Model of Nonobese Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Tadashi Okamura

    2013-01-01

    Full Text Available Animal models have provided important information for the genetics and pathophysiology of diabetes. Here we have established a novel, nonobese rat strain with spontaneous diabetes, Long-Evans Agouti (LEA rat derived from Long-Evans (LE strain. The incidence of diabetes in the males was 10% at 6 months of age and 86% at 14 months, while none of the females developed diabetes. The blood glucose level in LEA male rats was between 200 and 300 mg/dl at 120 min according to OGTT. The glucose intolerance in correspondence with the impairment of insulin secretion was observed in male rats, which was the main cause of diabetes in LEA rats. Histological examination revealed that the reduction of β-cell mass was caused by progressive fibrosis in pancreatic islets in age-dependent manner. The intracytoplasmic hyaline droplet accumulation and the disappearance of tubular epithelial cell layer associated with thickening of basement membrane were evident in renal proximal tubules. The body mass index and glycaemic response to exogenous insulin were comparable to those of control rats. The unique characteristics of LEA rat are a great advantage not only to analyze the progression of diabetes, but also to disclose the genes involved in type 2 diabetes mellitus.

  4. Effect of Yushen zhuyun decoction on rats with diminished ovarian ...

    African Journals Online (AJOL)

    Purpose: To investigate the effect of Yushen zhuyun decoction (YSZYF) on rats with diminished ovarian reserve (DOR). Methods: High-performance liquid chromatography (HPLC) was used to determine the major phytochemical constituents of YSZYF. Rats with DOR (DOR rats) were prepared by administration of ...

  5. Haematological and Serum Biochemical Variables in rats Treated ...

    African Journals Online (AJOL)

    The haematology and serum biochemical effects of oral administration of the ethanolic extract of the root of Moringa oleifera at 50, 100 and 150 mg/kg were investigated in 30 mated female Wistar rats. The rats were assigned into five groups of six rats each. Group A was given 50mg/kg of the extract; group B, 100mg/kg; ...

  6. Activation of peripheral leukocytes in rat pregnancy and experimental preeclampsia

    NARCIS (Netherlands)

    Faas, MM; Schuiling, GA; Linton, EA; Sargent, IL; Redman, CWG

    OBJECTIVE: The aim of this study was to search for activation markers of peripheral leukocytes in experimental preeclampsia in the rat. STUDY DESIGN: Experimental preeclampsia was induced in 14-day-pregnant rats by infusion of endotoxin (1.0 mu g/kg body weight). For comparison, rats with normal

  7. Effects of Culling on Leptospira interrogans Carriage by Rats

    Science.gov (United States)

    Byers, Kaylee A.; Donovan, Christina M.; Bidulka, Julie J.; Stephen, Craig; Patrick, David M.; Himsworth, Chelsea G.

    2018-01-01

    We found that lethal, urban rat control is associated with a significant increase in the odds that surviving rats carry Leptospira interrogans. Our results suggest that human interventions have the potential to affect and even increase the prevalence of zoonotic pathogens within rat populations. PMID:29350160

  8. ( Cola Nitida Rubra ) on Reproductive Hormones in Rats

    African Journals Online (AJOL)

    Our previous study suggests that aqueous extract of kola nut had effect on reproductive hormones in male rats. This study evaluates the effects of kola nut extract on plasma level of testosterone and luteinizing hormones in male rats. 30 adult male rats were used. These were divided into three groups: group A served as ...

  9. Effect of thiabendazole on some rat hepatic xenobiotic metabolising enzymes

    NARCIS (Netherlands)

    Price, R.J.; Scott, M.P.; Walters, D.G.; Stierum, R.H.; Groten, J.P.; Meredith, C.; Lake, B.G.

    2004-01-01

    The effect of thiabendazole (TB) on some rat hepatic xenobiotic metabolising enzymes has been investigated. Male Sprague-Dawley rats were fed control diet or diets containing 102-5188 ppm TB for 28 days. As a positive control for induction of hepatic xenobiotic metabolism, rats were also fed diets

  10. An essential role of Nrf2 in American ginseng-mediated anti-oxidative actions in cardiomyocytes.

    Science.gov (United States)

    Li, Jinqing; Ichikawa, Tomonaga; Jin, Yu; Hofseth, Lorne J; Nagarkatti, Prakash; Nagarkatti, Mitzi; Windust, Anthony; Cui, Taixing

    2010-07-20

    Ginseng has been used as a folk medicine for thousands of years in Asia, and has become a popular herbal medicine world-wide. Recent studies have revealed that ginseng, including American ginseng, exerts antioxidant effects in the cardiovascular system; however, the underlying mechanisms are not fully understood. Thus, we investigated role of Nrf2, a master transcription factor of endogenous anti-oxidative defense systems, in the regulation of American ginseng-mediated anti-oxidative actions in cardiomyocytes. A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. H9C2 cells, a rat cardiomyocyte cell line, were exposed to angiotensin II (Ang II) or tumor necrosis factor alpha (TNFalpha) to induce oxidative stress that was examined by measuring formation of reactive oxygen and nitrogen species. Oxidative stress-induced cell death was induced by exogenous addition of hydrogen peroxide (H(2)O(2)). Proteins were measured by Western blot and mRNA expression was determined by quantitative real time PCR. Nrf2-driven transcriptional activity was assessed by antioxidant response element (ARE)-luciferase reporter assay. Direct Nrf2 binding to its target gene promoters was determined by chromatin immunoprecipitation assay. Adenoviral over-expression of Nrf2 shRNA was utilized to knock down Nrf2 in H9C2 cells. Immunochemical staining was applied for Nrf2 expression in the heart. American ginseng induced dramatic increases in Nrf2 protein expression, Nrf2 nuclear translocation, Nrf2 transcriptional activity, direct Nrf2 binding to its target gene promoters, and expression of a group of anti-oxidative genes driven by Nrf2 in H9C2 cells. In addition, American ginseng inhibited Ang II- or TNFalpha-induced free radical formation and H(2)O(2)-induced cell death in H9C2 cells over-expressed with control shRNA but not in the cells over-expressed with Nrf2 shRNA. Finally, oral

  11. Immature rats show ovulatory defects similar to those in adult rats lacking prostaglandin and progesterone actions

    Directory of Open Access Journals (Sweden)

    Sanchez-Criado Jose E

    2004-09-01

    Full Text Available Abstract Gonadotropin-primed immature rats (GPIR constitute a widely used model for the study of ovulation. Although the equivalence between the ovulatory process in immature and adult rats is generally assumed, the morphological and functional characteristics of ovulation in immature rats have been scarcely considered. We describe herein the morphological aspects of the ovulatory process in GPIR and their response to classical ovulation inhibitors, such as the inhibitor of prostaglandin (PG synthesis indomethacin (INDO and a progesterone (P receptor (PR antagonist (RU486. Immature Wistar rats were primed with equine chorionic gonadotropin (eCG at 21, 23 or 25 days of age, injected with human chorionic gonadotropin (hCG 48 h later, and sacrificed 16 h after hCG treatment, to assess follicle rupture and ovulation. Surprisingly, GPIR showed age-related ovulatory defects close similar to those in adult rats lacking P and PG actions. Rats primed with eCG at 21 or 23 days of age showed abnormally ruptured corpora lutea in which the cumulus-oocyte complex (COC was trapped or had been released to the ovarian interstitum, invading the ovarian stroma and blood and lymphatic vessels. Supplementation of immature rats with exogenous P and/or PG of the E series did not significantly inhibit abnormal follicle rupture. Otherwise, ovulatory defects were practically absent in rats primed with eCG at 25 days of age. GPIR treated with INDO showed the same ovulatory alterations than vehicle-treated ones, although affecting to a higher proportion of follicles. Blocking P actions with RU486 increased the number of COC trapped inside corpora lutea and decreased ovulation. The presence of ovulatory defects in GPIR, suggests that the capacity of the immature ovary to undergo the coordinate changes leading to effective ovulation is not fully established in Wistar rats primed with eCG before 25 days of age.

  12. Regulation of Taurine transporter activity in cultured rat retinal ganglion cells and rat retinal Muller Cells

    International Nuclear Information System (INIS)

    Eissa, Laila A.; Smith, Sylvia B.; El-sherbeny, Amira A.

    2006-01-01

    Diabetic retinopathy is one of the most common complications of diabetes. The amino acid taurine is believed to play an antioxidant protective role in diabetic retinopathy through the scavenging of the reactive species. It is not well established whether taurine uptake is altered in retina cells during diabetic conditions. Thus, the present study was designed to investigate the changes in taurine transport in cultures of rat retinal Muller cells and rat retinal ganglion cells under conditions associated with diabetes. Taurine was abundantly taken up by retinal Muller cells and rat retinal ganglion cells under normal glycemic condition. Taurine was actively transported to rat Muller cells and rat retinal ganglion cells in a Na and Cl dependant manner. Taurine uptake further significantly elevated in both type of cells after the incubation with high glucose concentration. This effect could be attributed to the increase in osmolarity. Because Nitric Oxide (NO) is a molecule implicated in the pathogenesis of diabetes, we also determined the activity of taurine transporter in cultured rat retinal Muller cells and rat retinal ganglion cells in the presence of the NO donors, SIN-1 and SNAP. Taurine uptake was elevated above control value after 24-h incubation with low concentration of NO donors. We finally investigated the ability of neurotoxic glutamate to change taurine transporter activity in both types of cells. Uptake of taurine was significantly increased in rat retinal ganglion cells when only incubated with high concentration of glutamate. Our data provide evidence that taurine transporter is present in cultured rat retinal ganglion and Muller cells and is regulated by hyperosmolarity. The data are relevant to disease such as diabetes and neuronal degeneration where retinal cell volume may dramatically change. (author)

  13. Threonine phosphorylation of rat liver glycogen synthase

    International Nuclear Information System (INIS)

    Arino, J.; Arro, M.; Guinovart, J.J.

    1985-01-01

    32 P-labeled glycogen synthase specifically immunoprecipitated from 32 P-phosphate incubated rat hepatocytes contains, in addition to [ 32 P] phosphoserine, significant levels of [ 32 P] phosphothreonine. When the 32 P-immunoprecipitate was cleaved with CNBr, the [ 32 P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 in vitro. After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32 P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the ''in vivo'' phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase

  14. Iron appetite and latent learning in rats.

    Science.gov (United States)

    Woods, S C; Vasselli, J R; Milam, K M

    1977-11-01

    Two experiments are reported which show that rats are capable of forming an association between the presence of iron in a solution when it is not specifically needed and a subsequent state of iron deficiency. Specifically, rats were trained to lever press for water while thirsty. One group received ferrous ions in addition to the water. When these rats were subsequently rendered iron deficient, they lever pressed more under extinction conditions as a graded function of lower hemoglobin levels. Controls that either did not receive ferrous ions during training or received solutions other than ferrous solutions during training did not respond this way under extinction conditions. This is therefore a type of latent learning previously demonstrated only for sodium appetite.

  15. Metabolic neural mapping in neonatal rats

    International Nuclear Information System (INIS)

    DiRocco, R.J.; Hall, W.G.

    1981-01-01

    Functional neural mapping by 14 C-deoxyglucose autoradiography in adult rats has shown that increases in neural metabolic rate that are coupled to increased neurophysiological activity are more evident in axon terminals and dendrites than neuron cell bodies. Regions containing architectonically well-defined concentrations of terminals and dendrites (neuropil) have high metabolic rates when the neuropil is physiologically active. In neonatal rats, however, we find that regions containing well-defined groupings of neuron cell bodies have high metabolic rates in 14 C-deoxyglucose autoradiograms. The striking difference between the morphological appearance of 14 C-deoxyglucose autoradiograms obtained from neonatal and adult rats is probably related to developmental changes in morphometric features of differentiating neurons, as well as associated changes in type and locus of neural work performed

  16. Helminth parasites in black rats (Rattus rattus) and brown rats (Rattus norvegicus) from different environments in the Netherlands

    NARCIS (Netherlands)

    Franssen, Frits; Swart, Arno; van Knapen, Frans|info:eu-repo/dai/nl/070114749; van der Giessen, Joke

    2016-01-01

    BACKGROUND: Rattus norvegicus (brown rat) and Rattus rattus (black rat) are known carriers of bacteria, viruses, and parasites of zoonotic and veterinary importance. Moreover, rats may play a role in the transmission of muscle larvae of the zoonotic nematode Trichinella spiralis to farm animals. We

  17. Neutron irradiation of rat embryos in utero

    International Nuclear Information System (INIS)

    Vogel, H.H. Jr.

    1978-01-01

    In the rat radiation is most effective in producing congenital anomalies during the organ-forming period (days 9 to 13), which is approximately equivalent to the 14th to 50th days of human pregnancy. We have exposed female Sprague--Dawley rats on the 18th day of pregnancy to single whole-body doses of fission neutrons (20 to 150 rads). After 20 rads there was a small decrease in body weight which lasted from birth to weaning. During this period 9% of the irradiated rats died compared with 4% of the controls. After 50 rads, 65/275 (23.6%) of the rats died between birth and weaning, and the body-weight loss of the survivors was increased. After 100 rads, 62/133 (47%) died at birth or day 1 and 103/133 (77.4%) died before weaning. A large and significant decrease in body weight persisted in the survivors. After 150 rads of fission neutrons, all 95 rats died within 48 hr of birth. From cross-fostering experiments, we believe this is a direct effect of radiation on the embryos and not an indirect action through the mother or her milk. The LD 50 for the period from birth to weaning is approximately 75 rads of fission neutrons. Studies of organ weight were conducted daily for the first week after birth in an attempt to find the cause of radiation mortality. Body weight of the irradiated animals averaged only about one-half that of the controls. The liver, kidney, brain, and testes of the neutron-irradiated rats weighed significantly less than those of the controls. The weights of the spleen, lungs, duodenum, and stomach were decreased but not significantly. The bone marrow appeared depleted in the irradiated long bones, but the spleen maintained active hematopoiesis 1 to 2 months after neutron exposure

  18. Oxidative stress in normal and diabetic rats.

    Science.gov (United States)

    Torres, M D; Canal, J R; Pérez, C

    1999-01-01

    Parameters related to oxidative stress were studied in a group of 10 Wistar diabetic rats and 10 control rats. The levels of total erythrocyte catalase activity in the diabetic animals were significantly (pC18:2) ratios. Greater vitaminE/triglyceride (TG) ratio, however, appeared in the control group. The corresponding vitamin A ratios (vitaminA/TG, vitaminA/PUFA, vitaminA/C 18:2) were higher in the control group. Our work corroborates the findings that fatty acid metabolism presents alterations in the diabetes syndrome and that the antioxidant status is affected.

  19. Renal alterations in prediabetic rats with periodontitis

    DEFF Research Database (Denmark)

    Andersen, Carla Cruvinel Pontes; Holmstrup, Palle; Buschard, Karsten

    2008-01-01

    BACKGROUND: Periodontitis was shown to have an impact on glucose levels in prediabetic and diabetic rats. The Zucker fatty rat (ZFR) is a well-characterized model of prediabetes presenting with impaired glucose tolerance, hyperinsulinemia, dyslipidemia, and moderate hypertension. The aim...... IValpha1, fibronectin, and nephrin. Urinary albumin excretion and creatinine clearance were also evaluated. RESULTS: In prediabetic ZFRs, periodontitis was associated with kidney hypertrophy (P = 0.03) and a tendency for increased glomerular volume (P = 0.06). In lean littermates, elevated fibronectin m...

  20. Immunity to Fasciola hepatica in the rat

    International Nuclear Information System (INIS)

    Armour, J.; Dargie, J.D.

    1974-01-01

    Experiments were carried out which demonstrated an acquired immunity to Fasciola hapatica in the rat. It was shown that this immunity could be transferred to recipients using either lymphoid cells or serum from infected donor rats. The extent of the protection obtained by cells appeared to be related to the quantity and persistence of the antigenic stimulus in the donor. Likewise, the degree of immunity conferred by immune serum was dependent upon the volume transferred. The significance of these results in relation to the mechanism of immunity to fascioliasis is discussed

  1. Characterization of rat lung ICAM-1

    DEFF Research Database (Denmark)

    Beck-Schimmer, B; Schimmer, R C; Schmal, H

    1998-01-01

    studies, rat pulmonary artery endothelial cells (RPAEC), rat alveolar macrophages and aortic rings were stimulated (as described below) and evaluated for ICAM-1 expression. TREATMENT: RPAEC and macrophages were stimulated with lipopolysaccharide (LPS) and recombinant murine tumour necrosis factor alpha...... peaked at 4 h, while lung ICAM- I protein peaked at 6 h. CONCLUSIONS: Quantitation of ICAM-1 expression in vitro and in vivo suggests that ICAM-1 plays a central role in two lung inflammatory models. Furthermore, lung ICAM-1 upregulation involves at least two cell types: vascular endothelial cells...

  2. Absorption of pentacaine from ulcerous rat stomach

    International Nuclear Information System (INIS)

    Tomcikova, O.; Babulova, A.; Durisova, M.; Trnovec, T.; Benes, L.

    1985-01-01

    Pentacaine is a local anaesthetic which exhibited positive effects on healing of model ulcers in the rat stomach. The in situ disappearance of pentacaine from the ulcerous and intact rat stomach was studied. Gastric ulcers were produced by oral administration of phenylbutazone (200 mg/kg) 3.5 h before absorption experiment. Pentacaine exhibited a biexponential decrease from the lumen of the stomach, the rate of which was essentially the same in both groups. The total amount of pentacaine absorbed was small because of extremly low absorption rate. (author)

  3. Fenbendazole treatment and litter size in rats.

    Science.gov (United States)

    Johnston, Nancy A; Bieszczak, Jeremiah R; Verhulst, Steven; Disney, Kimberly E; Montgomery, Kyle E; Toth, Linda A

    2006-11-01

    Fenbendazole is commonly used in laboratory animal medicine as an anthelmintic for elimination of pinworms. It is generally regarded as a safe drug with minimal side effects. In our facility, 2 breeding colonies of rats were treated with fenbendazole to eliminate pinworms. Analysis of the breeding records revealed that feeding Sprague-Dawley rats a diet containing fenbendazole on a continuous basis for 7 consecutive weeks was associated with a significant reduction in litter size. Although the mechanism underlying this effect is unknown, the finding prompts caution when using fenbendazole to treat valuable breeding colonies or strains that are poor breeders.

  4. The oncogenic action of ionizing radiation on rat skin

    International Nuclear Information System (INIS)

    Burns, F.J.

    1991-01-01

    Progress has occurred in several areas corresponding to the specific aims of the proposal: (1) Progression and multiple events in radiation carcinogenesis of rat skin as a function of LET; (2) cell cycle kinetics of irradiated rat epidermis as determined by double labeling and double emulsion autoradiography; (3) oncogene activation detected by in situ hybridization in radiation-induced rat skin tumors; (4) amplification of the c-myc oncogene in radiation-induced rat skin tumors as a function of LET; and (5) transformation of rat skin keratinocytes by ionizing radiation in combination with c-Ki-ras and c-myc oncogenes. 111 refs., 13 figs., 12 tabs

  5. Rats demonstrate helping behavior toward a soaked conspecific.

    Science.gov (United States)

    Sato, Nobuya; Tan, Ling; Tate, Kazushi; Okada, Maya

    2015-09-01

    Helping behavior is a prosocial behavior whereby an individual helps another irrespective of disadvantages to him or herself. In the present study, we examined whether rats would help distressed, conspecific rats that had been soaked with water. In Experiment 1, rats quickly learned to liberate a soaked cagemate from the water area by opening the door to allow the trapped rat into a safe area. Additional tests showed that the presentation of a distressed cagemate was necessary to induce rapid door-opening behavior. In addition, it was shown that rats dislike soaking and that rats that had previously experienced a soaking were quicker to learn how to help a cagemate than those that had never been soaked. In Experiment 2, the results indicated that rats did not open the door to a cagemate that was not distressed. In Experiment 3, we tested behavior when rats were forced to choose between opening the door to help a distressed cagemate and opening a different door to obtain a food reward. Irrespective of how they learned to open the door, in most test trials, rats chose to help the cagemate before obtaining a food reward, suggesting that the relative value of helping others is greater than the value of a food reward. These results suggest that rats can behave prosocially and that helper rats may be motivated by empathy-like feelings toward their distressed cagemate.

  6. [Subcutaneous transplants of juvenile rat testicular tissues continue to develop and secret androgen in adult rats].

    Science.gov (United States)

    Yu, Zhou; Wang, Tong; Cui, Jiangbo; Song, Yajuan; Ma, Xianjie; Su, Yingjun; Peng, Pai

    2017-12-01

    Objective To explore the effects of subcutaneous microenvironment of adult rats on survival, development and androgen secretion of Leydig cells of transplanted juvenile rat testis. Methods Healthy adult SD rats were randomly divided into control group, sham group, castrated group and non-castrated group. Rats in the control group were kept intact, no testis was transplanted subcutaneously after adult recipients were castrated in the sham group; 5-7-day juvenile rat testes were transplanted subcutaneously in the castrated group, with one testis per side; Testes resected from juvenile rats were directly transplanted subcutaneously on both sides of the recipients in the non-castrated group. The grafts were obtained and weighed 4 weeks later. Then the histological features of the grafts were examined by HE staining; the expression and distribution of hydroxysteroid 17-beta dehydrogenase 1 (HSD-17β1) were investigated by immunohistochemistry; and the serum androgen level was determined by ELISA. Results The average mass of grafts obtained from the castrated group was significantly higher than that of the non-castrated group. Immunohistochemistry indicated that Leydig cells were visible in the tissues from both the castrated and non-castrated groups, but the number of HSD-17β1-posotive cells in the castrated group was larger than that in the non-castrated group. ELISA results showed that the serum androgen level was higher in the control group and non-castrated group than in the sham group and castrated group, and compared with the sham group, the serum androgen level in the castrated group was significantly higher. Conclusion The juvenile rat testis subcutaneously transplanted could further develop under the adult recipient rat skin, and the Leydig cells of grafts harbored the ability to produce and secret androgen.

  7. Effect of x-irradiation in rats bearing walker-256-carcinosarcoma and normal rats

    International Nuclear Information System (INIS)

    Ehara, Kazuhiko

    1978-01-01

    Serum protein fractions and total proteins were studied with bloods obtained from the rats exposed each to the partial-, whole-bodies and the transplanted tumors (Walker-256-carcinosarcoma transplanted in the right hind leg). The electrophoretic variation induced in the sera of tumor-bearing rats (Group II), and the content of total proteins decreased. Early irradiation to the tumor part of rats less induced the variations of the electrophoretic pattern and the decrease of the amount of serum total proteins. When the distant metastasis appeared during irradiation treatment, the electrophoretic patterns and content of total proteins changed proportionally to the variation in sera of Group II. On the other hand, the γ-globulin (G) fraction increased in the long-term survival rat. The separation of the rat serum β-G into two peaks of β 1 - and β 2 -G was shown only in Group IV (late irradiation to the right hind leg). This finding supposed that some factors involve in the sera of rats with transplanted primary tumor grown up to a fixed size and guessed the appearance of the distant metastasis during x-irradiation. The percentages of the albumin and γ-G decreased slightly and those of the α 1 -, α 2 - and β-G increased slightly in the rats with 300 rad partial-body (the right hind leg) x-irradiation daily for 20 days. The remarkable decrease of the albumin and γ-G, the increase of the α 1 - and β-G, the marked increase of the α 2 -G and the decrease of serum total proteins were demonstrated for the sera of rats with 1,000 rad whole-body x-irradiation at a time. These phenomena seem to be related to the destructive and reticuloendothelial injury by the exposure. (auth.)

  8. Palmitoylated PrRP analog decreases body weight in DIO rats but not in ZDF rats

    Czech Academy of Sciences Publication Activity Database

    Holubová, Martina; Zemenová, Jana; Mikulášková, Barbora; Panajotová, V.; Stöhr, J.; Haluzík, M.; Kuneš, Jaroslav; Železná, Blanka; Maletínská, Lenka

    2016-01-01

    Roč. 229, č. 2 (2016), s. 85-96 ISSN 0022-0795 R&D Projects: GA ČR(CZ) GA15-08679S; GA TA ČR(CZ) TE01020028 Institutional support: RVO:61388963 Keywords : prolactin-releasing peptide * lipidization * diet-induced obesity * ZDF rats * food intake * rat Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.706, year: 2016

  9. Palmitoylated PrRP analog decreases body weight in DIO rats but not in ZDF rats

    Czech Academy of Sciences Publication Activity Database

    Holubová, M.; Zemenová, J.; Mikulášková, Barbora; Panajotová, V.; Stöhr, J.; Haluzík, M.; Kuneš, Jaroslav; Železná, B.; Maletínská, L.

    2016-01-01

    Roč. 229, č. 2 (2016), s. 85-96 ISSN 0022-0795 R&D Projects: GA TA ČR(CZ) TE01020028; GA ČR(CZ) GA15-08679S Institutional support: RVO:67985823 Keywords : prolactin-releasing peptide * lipidization * diet-induced obesity * ZDF rats * food intake rat Subject RIV: ED - Physiology Impact factor: 4.706, year: 2016

  10. Efficacy and Safety of Gwakhyangjeonggi-San Retention Enema in Normal Rats and Spontaneously Hypertensive Rats

    Directory of Open Access Journals (Sweden)

    Eunyoung Song

    2013-01-01

    Full Text Available The purpose of this study is to establish a protocol of retention-enema experiments and evaluate the antihypertensive effect and the safety of Gwakhyangjeonggi-san retention enema. Normal and spontaneously hypertensive rats (SHRs were divided into treatment and control groups, respectively. We applied the Gwakhyangjeonggi-san extract by decoction and 0.9% NaCl in each group, estimated the blood pressure and body weight, and performed HPLC analysis. ALT, AST, BUN, and creatinine were examined. The systolic blood pressure within each group in normal rats differed significantly in time effect, and so did the diastolic blood pressure in the treatment group of normal rats. The systolic, diastolic, and mean blood pressure showed significant differences in group effect in the treatment group of the SHRs. The time effect of the body weight in both groups of normal rats differed significantly, so did group × time and time effects in both groups of SHRs. AST, ALT, BUN, and creatinine showed no significant difference between groups. We concluded that the Gwakhyangjeonggi-san retention enema has a hypotensive effect in normal rats within the regular range of blood pressure, but an antihypertensive effect in SHRs. Also, the intervention is safe and does not affect the liver and kidney functions in normal rats.

  11. Rats do not eat alone in public: Food-deprived rats socialize rather than competing for baits.

    Directory of Open Access Journals (Sweden)

    Omri Weiss

    Full Text Available Limited resources result in competition among social animals. Nevertheless, social animals also have innate preferences for cooperative behavior. In the present study, 12 dyads of food-deprived rats were tested in four successive trials, and then re-