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Sample records for responses including cardiomyocyte

  1. Delayed Cardiomyocyte Response to Total Body Particle Radiation Exposure - Identification of Regulatory Gene Network [proton

    National Aeronautics and Space Administration — We examined molecular responses using transcriptome profiling in isolated left ventricular murine cardiomyocytes to 90 cGy 1 GeV proton (1H) and 15 cGy 1 GeV/nucleon...

  2. Delayed Cardiomyocyte Response to Total Body Particle Radiation Exposure - Identification of Regulatory Gene Network [iron

    National Aeronautics and Space Administration — We examined molecular responses using transcriptome profiling in isolated left ventricular murine cardiomyocytes to 90 cGy 1 GeV proton (1H) and 15 cGy 1 GeV/nucleon...

  3. Regenerative responses after mild heart injuries for cardiomyocyte proliferation in zebrafish

    Itou, Junji; Akiyama, Ryutaro; Pehoski, Steve; Yu, Xiaodan; Kawakami, Hiroko; Kawakami, Yasuhiko

    2014-01-01

    Background The zebrafish heart regenerates after various severe injuries. Common processes of heart regeneration are cardiomyocyte proliferation, activation of epicardial tissue and neovascularization. In order to further characterize heart regeneration processes, we introduced milder injuries and compared responses to those induced by ventricular apex resection, a widely used injury method. We used scratching of the ventricular surface and puncturing of the ventricle with a fine tungsten needle as injury inducing techniques. Results Scratching the ventricular surface induced subtle cardiomyocyte proliferation and responses of the epicardium. Endothelial cell accumulation was limited to the surface of the heart. Ventricular puncture induced cardiomyocyte proliferation, endocardial and epicardial activation and neo-vascularization, similar to the resection method. However, the degree of the responses was milder, correlating with milder injury. Sham operation induced epicardial aldh1a2 expression but not tbx18 and WT1. Conclusions Puncturing the ventricle induces responses equivalent to resection at milder degrees in a shorter time frame and would be used as simple injury model. Scratching the ventricle did not induce heart regeneration and would be used for studying wound responses to epicardium. PMID:25074230

  4. KCNQ channels are involved in the regulatory volume decrease response in primary neonatal rat cardiomyocytes

    Calloe, Kirstine; Nielsen, Morten Schak; Grunnet, Morten

    2007-01-01

    of neonatal rat cardiomyocytes was studied in intact single cells attached to coverslips, i.e. with an intact cytoskeleton. The potential contribution of KCNQ (Kv7) channels to the RVD response and the possible involvement of the F-actin cytoskeleton were investigated. The rate of RVD was significantly...... changes the structure of the F-actin cytoskeleton, leading to a more rounded cell shape, less pronounced F-actin stress fibers and patches of actin. In the presence of cytochalasin D (1 microM), a potent inhibitor of actin polymerization, the RVD response was strongly reduced, confirming a possible role...... for an intact F-actin cytoskeleton in linking cell swelling to activation of ion transport in neonatal rat cardiomyocytes. Udgivelsesdato: 2007-Jun...

  5. Nanofiber-structured hydrogel yarns with pH-response capacity and cardiomyocyte-drivability for bio-microactuator application.

    Wu, Shaohua; Duan, Bin; Qin, Xiaohong; Butcher, Jonathan T

    2017-09-15

    Polymeric hydrogels have great potential in soft biological micro-actuator applications. However, inappropriate micro-architecture, non-anisotropy, weak biomechanics, and inferior response behaviors limit their development. In this study, we designed and manufactured novel polyacrylonitrile (PAN)-based hydrogel yarns composed with uniaxially aligned nanofibers. The nanofibrous hydrogel yarns possessed anisotropic architecture and robust mechanical properties with flexibility, and could be assembled into defined scaffold structures by subsequent processes. The as-prepared hydrogel yarns showed excellent pH response behaviors, with around 100% maximum length and 900% maximum diameter changes, and the pH response was completed within several seconds. Moreover, the hydrogel yarns displayed unique cell-responsive abilities to promote the cell adhesion, proliferation, and smooth muscle differentiation of human adipose derived mesenchymal stem cells (HADMSC). Chicken cardiomyocytes were further seeded onto our nanofibrous hydrogel yarns to engineer living cell-based microactuators. Our results demonstrated that the uniaxially aligned nanofibrous networks within the hydrogel yarns were the key characteristics leading to the anisotropic organization of cardiac cells, and improved sarcomere organization, mimicking the cardiomyocyte bundles in the native myocardium. The construct is capable of sustaining spontaneous cardiomyocyte pumping behaviors for 7days. Our PAN-based nanofibrous hydrogel yarns are attractive for creating linear microactuators with pH-response capacity and biological microactuators with cardiomyocyte-drivability. A mechanically robust polyacrylonitrile-based nanofibrous hydrogel yarn is fabricated by using a modified electrospinning setup in combination with chemical modification processes. The as-prepared hydrogel yarn possesses a uniaxially aligned nanofiber microarchitecture and supports a rapid, pH-dependent expansion/contraction response within a few

  6. Cardiomyocyte Regeneration

    Toshio Nakanishi

    2013-01-01

    Full Text Available The heart was initially believed to be a terminally differentiated organ; once the cardiomyocytes died, no recovery could be made to replace the dead cells. However, around a decade ago, the concept of cardiac stem cells (CSCs in adult hearts was proposed. CSCs differentiate into cardiomyocytes, keeping the heart functioning. Studies have proved the existence of stem cells in the heart. These somatic stem cells have been studied for use in cardiac regeneration. Moreover, recently, induced pluripotent stem cells (iPSCs were invented, and methodologies have now been developed to induce stable cardiomyocyte differentiation and purification of mature cardiomyocytes. A reprogramming method has also been applied to direct reprogramming using cardiac fibroblasts into cardiomyocytes. Here, we address cardiomyocyte differentiation of CSCs and iPSCs. Furthermore, we describe the potential of CSCs in regenerative biology and regenerative medicine.

  7. Curcumin and its demethoxy derivatives possess p300 HAT inhibitory activity and suppress hypertrophic responses in cardiomyocytes

    Yoichi Sunagawa

    2018-04-01

    Full Text Available The natural compound, curcumin (CUR, possesses several pharmacological properties, including p300-specific histone acetyltransferase (HAT inhibitory activity. In our previous study, we demonstrated that CUR could prevent the development of cardiac hypertrophy by inhibiting p300-HAT activity. Other major curcuminoids isolated from Curcuma longa including demethoxycurcumin (DMC and bisdemethoxycurcumin (BDMC are structural analogs of CUR. In present study, we first confirmed the effect of these three curcuminoid analogs on p300-HAT activity and cardiomyocyte hypertrophy.Our results showed that DMC and BDMC inhibited p300-HAT activity and cardiomyocyte hypertrophy to almost the same extent as CUR. As the three compounds have structural differences in methoxy groups at the 3-position of their phenol rings, our results suggest that these methoxy groups are not involved in the inhibitory effects on p300-HAT activity and cardiac hypertrophy. These findings provide useful insights into the structure–activity relationship and biological activity of curcuminoids for p300-HAT activity and cardiomyocyte hypertrophy. Keywords: Curcumin, Demethoxycurcumin, Bisdemethoxycurcumin, p300, Cardiomyocyte hypertrophy

  8. Role of S100A1 in hypoxia-induced inflammatory response in cardiomyocytes via TLR4/ROS/NF-κB pathway.

    Yu, Jiangkun; Lu, Yanyu; Li, Yapeng; Xiao, Lili; Xing, Yu; Li, Yanshen; Wu, Leiming

    2015-09-01

    S100A1 plays a crucial role in hypoxia-induced inflammatory response in cardiomyocytes. However, the role of S100A1 in hypoxia-induced inflammatory response in cardiomyocytes is still unknown. enzyme-linked immunosorbent assay (ELISA) was performed for the determination of inflammatory cytokines. Immunocytochemistry and immunofluorescence, Western blot analysis and Real-time polymerase chain reaction (RT-PCR) were conducted to assess protein or mRNA expressions. Fluorogenic probe dihydroethidium (DHE) was used to evaluate the generation of reactive oxygen species (ROS) while Hoechst 33342 staining for apoptosis. Small interfering RNA (siRNA) for S100A1 was used to evaluate the role of S100A1. The levels of ROS and inflammatory cytokine including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-8 in H9c2 cells were increased remarkably by hypoxia. However, IL-37 protein or mRNA levels were decreased significantly. Both Toll-like receptor 4 (TLR4) inhibitor Ethyl (6R)-6-[N-(2-Chloro-4fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) treatment or siRNA S100A1 downregulated TLR4 expression and inflammatory cytokine level and mRNA in H9c2 cells, as well as weakening ROS and phospho-p65 Nuclear factor (NF)-κB levels. Further, S100A1 treatment significantly reduced TNF-α protein or mRNA level whereas enhanced IL-37 protein or mRNA level, and could attenuate ROS and phospho-p65 NF-κB levels. Our results demonstrate that S100A1 can regulate the inflammatory response and oxidative stress in H9C2 cells via TLR4/ROS/NF-κB pathway. These findings provide an interesting strategy for protecting cardiomyocytes from hypoxia-induced inflammatory response. © 2015 Royal Pharmaceutical Society.

  9. Fractalkine depresses cardiomyocyte contractility.

    David Taube

    Full Text Available Our laboratory reported that male mice with cardiomyocyte-selective knockout of the prostaglandin E2 EP4 receptor sub-type (EP4 KO exhibit reduced cardiac function. Gene array on left ventricles (LV showed increased fractalkine, a chemokine implicated in heart failure. We therefore hypothesized that fractalkine is regulated by PGE2 and contributes to depressed contractility via alterations in intracellular calcium.Fractalkine was measured in LV of 28-32 week old male EP4 KO and wild type controls (WT by ELISA and the effect of PGE2 on fractalkine secretion was measured in cultured neonatal cardiomyocytes and fibroblasts. The effect of fractalkine on contractility and intracellular calcium was determined in Fura-2 AM-loaded, electrical field-paced cardiomyocytes. Cardiomyocytes (AVM from male C57Bl/6 mice were treated with fractalkine and responses measured under basal conditions and after isoproterenol (Iso stimulation.LV fractalkine was increased in EP4 KO mice but surprisingly, PGE2 regulated fractalkine secretion only in fibroblasts. Fractalkine treatment of AVM decreased both the speed of contraction and relaxation under basal conditions and after Iso stimulation. Despite reducing contractility after Iso stimulation, fractalkine increased the Ca(2+ transient amplitude but decreased phosphorylation of cardiac troponin I, suggesting direct effects on the contractile machinery.Fractalkine depresses myocyte contractility by mechanisms downstream of intracellular calcium.

  10. A novel miR-371a-5p-mediated pathway, leading to BAG3 upregulation in cardiomyocytes in response to epinephrine, is lost in Takotsubo cardiomyopathy.

    d'Avenia, M; Citro, R; De Marco, M; Veronese, A; Rosati, A; Visone, R; Leptidis, S; Philippen, L; Vitale, G; Cavallo, A; Silverio, A; Prota, C; Gravina, P; De Cola, A; Carletti, E; Coppola, G; Gallo, S; Provenza, G; Bossone, E; Piscione, F; Hahne, M; De Windt, L J; Turco, M C; De Laurenzi, V

    2015-10-29

    Molecular mechanisms protecting cardiomyocytes from stress-induced death, including tension stress, are essential for cardiac physiology and defects in these protective mechanisms can result in pathological alterations. Bcl2-associated athanogene 3 (BAG3) is expressed in cardiomyocytes and is a component of the chaperone-assisted autophagy pathway, essential for homeostasis of mechanically altered cells. BAG3 ablation in mice results in a lethal cardiomyopathy soon after birth and mutations of this gene have been associated with different cardiomyopathies including stress-induced Takotsubo cardiomyopathy (TTC). The pathogenic mechanism leading to TTC has not been defined, but it has been suggested that the heart can be damaged by excessive epinephrine (epi) spillover in the absence of a protective mechanism. The aim of this study was to provide more evidence for a role of BAG3 in the pathogenesis of TTC. Therefore, we sequenced BAG3 gene in 70 TTC patients and in 81 healthy donors with the absence of evaluable cardiovascular disease. Mutations and polymorphisms detected in the BAG3 gene included a frequent nucleotide change g2252c in the BAG3 3'-untranslated region (3'-UTR) of Takotsubo patients (PBAG3 upregulation on exposure to epi through an ERK-dependent upregulation of miR-371a-5p. In conclusion, the presence of a g2252c polymorphism in the BAG3 3'-UTR determines loss of miR-371a-5p binding and results in an altered response to epi, potentially representing a new molecular mechanism that contributes to TTC pathogenesis.

  11. Induction of a Radio-Adaptive Response by Low-dose Gamma Irradiation in Mouse Cardiomyocytes

    Westby, Christian M.; Seawright, John W.; Wu, Honglu

    2011-01-01

    One of the most significant occupational hazards to an astronaut is the frequent exposure to radiation. Commonly associated with increased risk for cancer related morbidity and mortality, radiation is also known to increase the risk for cardiovascular related disorders including: pericarditis, hypertension, and heart failure. It is believed that these radiation-induced disorders are a result of abnormal tissue remodeling. It is unknown whether radiation exposure promotes remodeling through fibrotic changes alone or in combination with programmed cell death. Furthermore, it is not known whether it is possible to mitigate the hazardous effects of radiation exposure. As such, we assessed the expression and mechanisms of radiation-induced tissue remodeling and potential radio-adaptive responses of p53-mediated apoptosis and fibrosis pathways along with markers for oxidative stress and inflammation in mice myocardium. 7 week old, male, C57Bl/6 mice were exposed to 6Gy (H) or 5cGy followed 24hr later with 6Gy (LH) 137Cs gamma radiation. Mice were sacrificed and their hearts extirpated 4, 24, or 72hr after final irradiation. Real Time - Polymerase Chain Reaction was used to evaluate target genes. Apoptotic genes Bad and Bax, pro-cell survival genes Bcl2 and Bcl2l2, fibrosis gene Vegfa, and oxidative stress genes Sod2 and GPx4 showed a reduced fold regulation change (Bad,-6.18; Bax,-6.94; Bcl2,-5.09; Bcl2l2,-4.03; Vegfa, -11.84; Sod2,-5.97; GPx4*,-28.72; * = Bonferroni adjusted p-value present compared to 24hr control. These data suggest a general reduction in genetic expression 4hrs after a high dose of gamma radiation. However, pre-exposure to 5cGy gamma radiation appears to facilitate a radio-adaptive response that mitigates the reduction in genetic expression associated with single high-dose gamma radiation exposure. This radio-adaptive response may serve as a potential countermeasure to radiation-induced myocardial remodeling and preserve the cardiovascular health of

  12. Genetic enrichment of cardiomyocytes derived from mouse ...

    Genetic enrichment of cardiomyocytes derived from mouse embryonic stem cells. WJ He, SC Li, LL Ye, H Liu, QW Wang, WD Han, XB Fu, ZL Chen. Abstract. Pluripotent embryonic stem cells (ESC) have the ability to differentiate into a variety of cell lineages in vitro, including cardiomyocytes. Successful applications of ...

  13. Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover.

    Richardson, Gavin D

    2016-05-23

    Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4',6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

  14. Effects of gamma-ray radiation on activity and apoptosis of rat cardiomyocytes in vitro

    Hu Shunying; Jiang Changsheng; Chen Guowei; Duan Haifeng; Wang Rongliang; Wu Bin; Guo Zikuan; Wang Lisheng

    2007-01-01

    Objective: It is reported that radiation-induced myocardial degeneration in the rat is preceded by changes in capillary structure and function. The aim of the present study is to investigate direct effect of gamma ray radiation on activity and apoptosis of cultured rat cardiomyocytes in vitro. Methods: The study was performed using primary cell cultures of cardiomyocytes isolated from hearts of now-born rats. After being cultured for 72h in vitro, cardiomyocytes were irradiated with single dose of 5 Gy, 10 Gy, 20 Gy of gamma ray respectively. At 48h post-irradiation, the concentration of LDH in the supernatant of cell culture was tested using methods introduced by International Federation of clinical chemistry (IFCC), and apoptosis was determined with flow cytometry. The viability of myocytes was determined with crystal violet test and MTT test at 48h and 120h post-irradiation respectively. Results: LDH concentration in the supernatant of cell culture of cardiomyocytes were increased significantly with the irradiation dose augment. Flow cytometry confirmed the induction of apoptosis in response to different gamma ray doses irradiation at 48h after irradiation. The viable cardiomyocytes irradiated by gamma ray were significantly declined at 120h after irradiation compared to un-irradiated cells, however there were no significant difference between two groups at 48h post-irradiation. Dose-effect relationship was demonstrated between cardiomyocyte apoptosis, viability and irradiation dose in the study. Conclusion: The study demonstrates gamma ray radiation can cause direct damage to cultured cardiomyocytes, including inhibiting activity and inducing apoptosis of cardiomyocytes in vitro, which shows dose effect relationship. The mechanism of gamma ray irradiation induced injury to cardiomyocytes should be investigated further. (authors)

  15. Regulation of cardiomyocyte autophagy by calcium.

    Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F G; Hill, Joseph A; Lavandero, Sergio

    2016-04-15

    Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy. Copyright © 2016 the American Physiological Society.

  16. Overexpression of KCNJ2 in induced pluripotent stem cell-derived cardiomyocytes for the assessment of QT-prolonging drugs

    Min Li

    2017-06-01

    Full Text Available Human induced pluripotent stem cell (hiPSC-derived cardiomyocytes hold great potentials to predict pro-arrhythmic risks in preclinical cardiac safety screening, although the hiPSC cardiomyocytes exhibit rather immature functional and structural characteristics, including spontaneous activity. Our physiological characterization and mathematical simulation showed that low expression of the inward-rectifier potassium (IK1 channel is a determinant of spontaneous activity. To understand impact of the low IK1 expression on the pharmacological properties, we tested if transduction of hiPSC-derived cardiomyocytes with KCNJ2, which encodes the IK1 channel, alters pharmacological response to cardiac repolarization processes. The transduction of KCNJ2 resulted in quiescent hiPSC-derived cardiomyocytes, which need pacing to elicit action potentials. Significant prolongation of paced action potential duration in KCNJ2-transduced hiPSC-derived cardiomyocytes was stably measured at 0.1 μM E-4031, although the same concentration of E-4031 ablated firing of non-treated hiPSC-derived cardiomyocytes. These results in single cells were confirmed by mathematical simulations. Using the hiPSC-derived cardiac sheets with KCNJ2-transduction, we also investigated effects of a range of drugs on field potential duration recorded at 1 Hz. The KCNJ2 overexpression in hiPSC-derived cardiomyocytes may contribute to evaluate a part of QT-prolonging drugs at toxicological concentrations with high accuracy.

  17. Dedifferentiation and proliferation of mammalian cardiomyocytes.

    Yiqiang Zhang

    2010-09-01

    Full Text Available It has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle.Here we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1 cardiomyocyte purification from rat hearts, and 2 genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs, while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit(+.Contradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness

  18. Doxorubicin Blocks Cardiomyocyte Autophagic Flux by Inhibiting Lysosome Acidification.

    Li, Dan L; Wang, Zhao V; Ding, Guanqiao; Tan, Wei; Luo, Xiang; Criollo, Alfredo; Xie, Min; Jiang, Nan; May, Herman; Kyrychenko, Viktoriia; Schneider, Jay W; Gillette, Thomas G; Hill, Joseph A

    2016-04-26

    The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1. Beclin 1(+/-) mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity. © 2016 American Heart Association, Inc.

  19. By Targeting Stat3 microRNA-17-5p Promotes Cardiomyocyte Apoptosis in Response to Ischemia Followed by Reperfusion

    Weijie Du

    2014-08-01

    Full Text Available Background: Several studies have confirmed the role of microRNAs in regulating ischemia/reperfusion-induced cardiac injury (I/R-I. MiR-17-5p has been regarded as an oncomiR in the development of cancer. However, its potential role in cardiomyocytes has not been exploited. The aim of this study is to investigate the role of miR-17-5p in I/R-I and the underlying mechanism through targeting Stat3, a key surviving factor in cardiomyocytes. Methods: MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide assay was used to detect the cell viability. ELISA and TUNEL were performed to measure apoptosis of neonatal rat ventricular cardiomyocytes (NRVCs. Infarct area was estimated by TTC (triphenyltetrazolium chloride and Evans blue staining. Western blot analysis was employed to detect the Stat3 and p-Stat3 levels and real-time RT-PCR was used to quantify miR-17-5p level. Results: The miR-17-5p level was significantly up-regulated in I/R-I mice and in NRVCs under oxidative stress. Overexpression of miR-17-5p aggravated cardiomyocyte injury with reduced cell viability and enhanced apoptotic cell death induced by H2O2, whereas inhibition of miR-17-5p by its antisense AMO-17-5p abrogated the deleterious changes. Moreover, the locked nucleic acid-modified antisense (LNA-anti-miR-17-5p markedly decreased the infarct area and apoptosis induced by I/R-I in mice. Furthermore, overexpression of miR-17-5p diminished the p-Stat3 level in response to H2O2. The results from Western blot analysis and luciferase reporter gene assay confirmed Stat3 as a target gene for miR-17-5p. Conclusion: Upregulation of miR-17-5p promotes apoptosis induced by oxidative stress via targeting Stat3, accounting partially for I/R-I.

  20. Phenotypic variability in LQT3 human induced pluripotent stem cell-derived cardiomyocytes and their response to antiarrhythmic pharmacologic therapy: An in silico approach.

    Paci, Michelangelo; Passini, Elisa; Severi, Stefano; Hyttinen, Jari; Rodriguez, Blanca

    2017-11-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are in vitro models with the clear advantages of their human origin and suitability for human disease investigations. However, limitations include their incomplete characterization and variability reported in different cell lines and laboratories. The purpose of this study was to investigate in silico ionic mechanisms potentially explaining the phenotypic variability of hiPSC-CMs in long QT syndrome type 3 (LQT3) and their response to antiarrhythmic drugs. Populations of in silico hiPSC-CM models were constructed and calibrated for control (n = 1,463 models) and LQT3 caused by I NaL channelopathy (n = 1,401 models), using experimental recordings for late sodium current (I NaL ) and action potentials (APs). Antiarrhythmic drug therapy was evaluated by simulating mexiletine and ranolazine multichannel effects. As in experiments, LQT3 hiPSC-CMs yield prolonged action potential duration at 90% repolarization (APD 90 ) (+34.3% than controls) and large electrophysiological variability. LQT3 hiPSC-CMs with symptomatic APs showed overexpression of I CaL , I K1 , and I NaL , underexpression of I Kr , and increased sensitivity to both drugs compared to asymptomatic LQT3 models. Simulations showed that both mexiletine and ranolazine corrected APD prolongation in the LQT3 population but also highlighted differences in drug response. Mexiletine stops spontaneous APs in more LQT3 hiPSC-CMs models than ranolazine (784/1,401 vs 53/1,401) due to its stronger action on I Na . In silico simulations demonstrate our ability to recapitulate variability in LQT3 and control hiPSC-CM phenotypes, and the ability of mexiletine and ranolazine to reduce APD prolongation, in agreement with experiments. The in silico models also identify potential ionic mechanisms of phenotypic variability in LQT3 hiPSC-CMs, explaining APD prolongation in symptomatic vs asymptomatic LQT3 hiPSC-CMs. Copyright © 2017 The Authors. Published by

  1. Tumor Necrosis Factor-α Produced in Cardiomyocytes Mediates a Predominant Myocardial Inflammatory Response to Stretch in Early Volume Overload

    Chen, Yuanwen; Pat, Betty; Zheng, Junying; Cain, Laura; Powell, Pamela; Shi, Ke; Sabri, Abdelkarim; Husain, Ahsan; Dell’Italia, Louis J

    2010-01-01

    Acute stretch caused by volume overload (VO) of aorto-caval fistula (ACF) induces a variety of myocardial responses including mast cell accumulation, matrix metalloproteinase (MMP) activation and collagen degradation, all of which are critical in dictating long term left ventricle (LV) outcome to VO. Meanwhile, these responses can be part of myocardial inflammation dictated by tumor necrosis factor-α (TNF-α) which is elevated after acute ACF. However, it is unknown whether TNF-α mediates a ma...

  2. Smart Farming: Including Rights Holders for Responsible Agricultural Innovation

    Kelly Bronson

    2018-01-01

    This article draws on the literature of responsible innovation to suggest concrete processes for including rights holders in the “smart” agricultural revolution. It first draws upon historical agricultural research in Canada to highlight how productivist values drove seed innovations with particular consequences for the distribution of power in the food system. Next, the article uses document analysis to suggest that a similar value framework is motivating public investment in smart farming i...

  3. Aggregated Demand Modelling Including Distributed Generation, Storage and Demand Response

    Marzooghi, Hesamoddin; Hill, David J.; Verbic, Gregor

    2014-01-01

    It is anticipated that penetration of renewable energy sources (RESs) in power systems will increase further in the next decades mainly due to environmental issues. In the long term of several decades, which we refer to in terms of the future grid (FG), balancing between supply and demand will become dependent on demand actions including demand response (DR) and energy storage. So far, FG feasibility studies have not considered these new demand-side developments for modelling future demand. I...

  4. A consistent response spectrum analysis including the resonance range

    Schmitz, D.; Simmchen, A.

    1983-01-01

    The report provides a complete consistent Response Spectrum Analysis for any component. The effect of supports with different excitation is taken into consideration, at is the description of the resonance ranges. It includes information explaining how the contributions of the eigenforms with higher eigenfrequencies are to be considered. Stocking of floor response spectra is also possible using the method described here. However, modified floor response spectra must now be calculated for each building mode. Once these have been prepared, the calculation of the dynamic component values is practically no more complicated than with the conventional, non-consistent methods. The consistent Response Spectrum Analysis can supply smaller and larger values than the conventional theory, a fact which can be demonstrated using simple examples. The report contains a consistent Response Spectrum Analysis (RSA), which, as far as we know, has been formulated in this way for the first time. A consistent RSA is so important because today this method is preferentially applied as an important tool for the earthquake proof of components in nuclear power plants. (orig./HP)

  5. Smart Farming: Including Rights Holders for Responsible Agricultural Innovation

    Kelly Bronson

    2018-02-01

    Full Text Available This article draws on the literature of responsible innovation to suggest concrete processes for including rights holders in the “smart” agricultural revolution. It first draws upon historical agricultural research in Canada to highlight how productivist values drove seed innovations with particular consequences for the distribution of power in the food system. Next, the article uses document analysis to suggest that a similar value framework is motivating public investment in smart farming innovations. The article is of interest to smart farming’s decision makers (from farmers to governance actors and a broader audience – anyone interested in engendering equity through innovation-led societal transitions.

  6. Pivotal role of cardiomyocyte TGF-β signaling in the murine pathological response to sustained pressure overload

    Koitabashi, Norimichi; Danner, Thomas; Zaiman, Ari L.; Pinto, Yigal M.; Rowell, Janelle; Mankowski, Joseph; Zhang, Dou; Nakamura, Taishi; Takimoto, Eiki; Kass, David A.

    2011-01-01

    The cardiac pathological response to sustained pressure overload involves myocyte hypertrophy and dysfunction along with interstitial changes such as fibrosis and reduced capillary density. These changes are orchestrated by mechanical forces and factors secreted between cells. One such secreted

  7. Hierarchical Energy Management of Microgrids including Storage and Demand Response

    Songli Fan

    2018-05-01

    Full Text Available Battery energy storage (BES and demand response (DR are considered to be promising technologies to cope with the uncertainty of renewable energy sources (RES and the load in the microgrid (MG. Considering the distinct prediction accuracies of the RES and load at different timescales, it is essential to incorporate the multi-timescale characteristics of BES and DR in MG energy management. Under this background, a hierarchical energy management framework is put forward for an MG including multi-timescale BES and DR to optimize operation with the uncertainty of RES as well as load. This framework comprises three stages of scheduling: day-ahead scheduling (DAS, hour-ahead scheduling (HAS, and real-time scheduling (RTS. In DAS, a scenario-based stochastic optimization model is established to minimize the expected operating cost of MG, while ensuring its safe operation. The HAS is utilized to bridge DAS and RTS. In RTS, a control strategy is proposed to eliminate the imbalanced power owing to the fluctuations of RES and load. Then, a decomposition-based algorithm is adopted to settle the models in DAS and HAS. Simulation results on a seven-bus MG validate the effectiveness of the proposed methodology.

  8. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

    Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

    2013-08-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Naturally Engineered Maturation of Cardiomyocytes

    Gaetano J. Scuderi

    2017-05-01

    Full Text Available Ischemic heart disease remains one of the most prominent causes of mortalities worldwide with heart transplantation being the gold-standard treatment option. However, due to the major limitations associated with heart transplants, such as an inadequate supply and heart rejection, there remains a significant clinical need for a viable cardiac regenerative therapy to restore native myocardial function. Over the course of the previous several decades, researchers have made prominent advances in the field of cardiac regeneration with the creation of in vitro human pluripotent stem cell-derived cardiomyocyte tissue engineered constructs. However, these engineered constructs exhibit a functionally immature, disorganized, fetal-like phenotype that is not equivalent physiologically to native adult cardiac tissue. Due to this major limitation, many recent studies have investigated approaches to improve pluripotent stem cell-derived cardiomyocyte maturation to close this large functionality gap between engineered and native cardiac tissue. This review integrates the natural developmental mechanisms of cardiomyocyte structural and functional maturation. The variety of ways researchers have attempted to improve cardiomyocyte maturation in vitro by mimicking natural development, known as natural engineering, is readily discussed. The main focus of this review involves the synergistic role of electrical and mechanical stimulation, extracellular matrix interactions, and non-cardiomyocyte interactions in facilitating cardiomyocyte maturation. Overall, even with these current natural engineering approaches, pluripotent stem cell-derived cardiomyocytes within three-dimensional engineered heart tissue still remain mostly within the early to late fetal stages of cardiomyocyte maturity. Therefore, although the end goal is to achieve adult phenotypic maturity, more emphasis must be placed on elucidating how the in vivo fetal microenvironment drives cardiomyocyte

  10. Including dynamic CO2 intensity with demand response

    Stoll, Pia; Brandt, Nils; Nordström, Lars

    2014-01-01

    Hourly demand response tariffs with the intention of reducing or shifting loads during peak demand hours are being intensively discussed among policy-makers, researchers and executives of future electricity systems. Demand response rates have still low customer acceptance, apparently because the consumption habits requires stronger incentive to change than any proposed financial incentive. An hourly CO 2 intensity signal could give customers an extra environmental motivation to shift or reduce loads during peak hours, as it would enable co-optimisation of electricity consumption costs and carbon emissions reductions. In this study, we calculated the hourly dynamic CO 2 signal and applied the calculation to hourly electricity market data in Great Britain, Ontario and Sweden. This provided a novel understanding of the relationships between hourly electricity generation mix composition, electricity price and electricity mix CO 2 intensity. Load shifts from high-price hours resulted in carbon emission reductions for electricity generation mixes where price and CO 2 intensity were positively correlated. The reduction can be further improved if the shift is optimised using both price and CO 2 intensity. The analysis also indicated that an hourly CO 2 intensity signal can help avoid carbon emissions increases for mixes with a negative correlation between electricity price and CO 2 intensity. - Highlights: • We present a formula for calculating hybrid dynamic CO 2 intensity of electricity generation mixes. • We apply the dynamic CO 2 Intensity on hourly electricity market prices and generation units for Great Britain, Ontario and Sweden. • We calculate the spearman correlation between hourly electricity market price and dynamic CO 2 intensity for Great Britain, Ontario and Sweden. • We calculate carbon footprint of shifting 1 kWh load daily from on-peak hours to off-peak hours using the dynamic CO 2 intensity. • We conclude that using dynamic CO 2 intensity for

  11. Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

    Fuegemann, Christopher J; Samraj, Ajoy K; Walsh, Stuart; Fleischmann, Bernd K; Jovinge, Stefan; Breitbach, Martin

    2010-12-01

    Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. © 2010 by John Wiley & Sons, Inc.

  12. (Re-)programming of subtype specific cardiomyocytes.

    Hausburg, Frauke; Jung, Julia Jeannine; Hoch, Matti; Wolfien, Markus; Yavari, Arash; Rimmbach, Christian; David, Robert

    2017-10-01

    Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. AKIP1 expression modulates mitochondrial function in rat neonatal cardiomyocytes

    Yu, Hongjuan; Tigchelaar, Wardit; Koonen, Debby P. Y.; Patel, Hemal H.; de Boer, Rudolf A.; van Gilst, Wiek H.; Westenbrink, B. Daan; Sillje, Herman H. W.

    2013-01-01

    A kinase interacting protein 1 (AKIP1) is a molecular regulator of protein kinase A and nuclear factor kappa B signalling. Recent evidence suggests AKIP1 is increased in response to cardiac stress, modulates acute ischemic stress response, and is localized to mitochondria in cardiomyocytes. The

  14. Hypoxia decreases creatine uptake in cardiomyocytes, while creatine supplementation enhances HIF activation.

    Santacruz, Lucia; Arciniegas, Antonio Jose Luis; Darrabie, Marcus; Mantilla, Jose G; Baron, Rebecca M; Bowles, Dawn E; Mishra, Rajashree; Jacobs, Danny O

    2017-08-01

    Creatine (Cr), phosphocreatine (PCr), and creatine kinases (CK) comprise an energy shuttle linking ATP production in mitochondria with cellular consumption sites. Myocytes cannot synthesize Cr: these cells depend on uptake across the cell membrane by a specialized creatine transporter (CrT) to maintain intracellular Cr levels. Hypoxia interferes with energy metabolism, including the activity of the creatine energy shuttle, and therefore affects intracellular ATP and PCr levels. Here, we report that exposing cultured cardiomyocytes to low oxygen levels rapidly diminishes Cr transport by decreasing V max and K m Pharmacological activation of AMP-activated kinase (AMPK) abrogated the reduction in Cr transport caused by hypoxia. Cr supplementation increases ATP and PCr content in cardiomyocytes subjected to hypoxia, while also significantly augmenting the cellular adaptive response to hypoxia mediated by HIF-1 activation. Our results indicate that: (1) hypoxia reduces Cr transport in cardiomyocytes in culture, (2) the cytoprotective effects of Cr supplementation are related to enhanced adaptive physiological responses to hypoxia mediated by HIF-1, and (3) Cr supplementation increases the cellular ATP and PCr content in RNCMs exposed to hypoxia. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  15. Importance of Thickness in Human Cardiomyocyte Network for Effective Electrophysiological Stimulation Using On-Chip Extracellular Microelectrodes

    Hamada, Tomoyo; Nomura, Fumimasa; Kaneko, Tomoyuki; Yasuda, Kenji

    2012-06-01

    We have developed a three-dimensionally controlled in vitro human cardiomyocyte network assay for the measurements of drug-induced conductivity changes and the appearance of fatal arrhythmia such as ventricular tachycardia/fibrillation for more precise in vitro predictive cardiotoxicity. To construct an artificial conductance propagation model of a human cardiomyocyte network, first, we examined the cell concentration dependence of the cell network heights and found the existence of a height limit of cell networks, which was double-layer height, whereas the cardiomyocytes were effectively and homogeneously cultivated within the microchamber maintaining their spatial distribution constant and their electrophysiological conductance and propagation were successfully recorded using a microelectrode array set on the bottom of the microchamber. The pacing ability of a cardiomyocyte's electrophysiological response has been evaluated using microelectrode extracellular stimulation, and the stimulation for pacing also successfully regulated the beating frequencies of two-layered cardiomyocyte networks, whereas monolayered cardiomyocyte networks were hardly stimulated by the external electrodes using the two-layered cardiomyocyte stimulation condition. The stability of the lined-up shape of human cardiomyocytes within the rectangularly arranged agarose microchambers was limited for a two-layered cardiomyocyte network because their stronger force generation shrunk those cells after peeling off the substrate. The results indicate the importance of fabrication technology of thickness control of cellular networks for effective extracellular stimulation and the potential concerning thick cardiomyocyte networks for long-term cultivation.

  16. Generation of Cardiomyocytes from Pluripotent Stem Cells.

    Nakahama, Hiroko; Di Pasquale, Elisa

    2016-01-01

    The advent of pluripotent stem cells (PSCs) enabled a multitude of studies for modeling the development of diseases and testing pharmaceutical therapeutic potential in vitro. These PSCs have been differentiated to multiple cell types to demonstrate its pluripotent potential, including cardiomyocytes (CMs). However, the efficiency and efficacy of differentiation vary greatly between different cell lines and methods. Here, we describe two different methods for acquiring CMs from human pluripotent lines. One method involves the generation of embryoid bodies, which emulates the natural developmental process, while the other method chemically activates the canonical Wnt signaling pathway to induce a monolayer of cardiac differentiation.

  17. MicroRNA-145 suppresses ROS-induced Ca2+ overload of cardiomyocytes by targeting CaMKIIδ

    Cha, Min-Ji; Jang, Jin-Kyung; Ham, Onju; Song, Byeong-Wook; Lee, Se-Yeon; Lee, Chang Yeon; Park, Jun-Hee; Lee, Jiyun; Seo, Hyang-Hee; Choi, Eunhyun; Jeon, Woo-min; Hwang, Hye Jin; Shin, Hyun-Taek

    2013-01-01

    Highlights: •CaMKIIδ mediates H 2 O 2 -induced Ca 2+ overload in cardiomyocytes. •miR-145 can inhibit Ca 2+ overload. •A luciferase assay confirms that miR-145 functions as a CaMKIIδ-targeting miRNA. •Overexpression of miR-145 regulates CaMKIIδ-related genes and ameliorates apoptosis. -- Abstract: A change in intracellular free calcium (Ca 2+ ) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca 2+ signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca 2+ -mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H 2 O 2 -mediated Ca 2+ overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca 2+ overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca 2+ -related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca 2+ overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses

  18. MicroRNA-145 suppresses ROS-induced Ca{sup 2+} overload of cardiomyocytes by targeting CaMKIIδ

    Cha, Min-Ji [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Jang, Jin-Kyung [College of Pharmacy, Sookmyung Women’s University, 52 HyoChangWon-Gil, Yongsan-ku, Seoul 140-742 (Korea, Republic of); Ham, Onju; Song, Byeong-Wook; Lee, Se-Yeon [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Lee, Chang Yeon; Park, Jun-Hee [Department of Integrated Omics for Biomedical Sciences, Graduate School, Yonsei University, 50 Yonsei-ro, Seodamun-gu, Seoul 120-759 (Korea, Republic of); Lee, Jiyun; Seo, Hyang-Hee [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Choi, Eunhyun [Severance Integrative Research Institute for Cerebral and Cardiovascular Disease, Yonsei University Health System, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Jeon, Woo-min [Department of Animal Resource, Sahmyook University, Seoul 139-742 (Korea, Republic of); Hwang, Hye Jin [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Shin, Hyun-Taek [College of Pharmacy, Sookmyung Women’s University, 52 HyoChangWon-Gil, Yongsan-ku, Seoul 140-742 (Korea, Republic of); and others

    2013-06-14

    Highlights: •CaMKIIδ mediates H{sub 2}O{sub 2}-induced Ca{sup 2+} overload in cardiomyocytes. •miR-145 can inhibit Ca{sup 2+} overload. •A luciferase assay confirms that miR-145 functions as a CaMKIIδ-targeting miRNA. •Overexpression of miR-145 regulates CaMKIIδ-related genes and ameliorates apoptosis. -- Abstract: A change in intracellular free calcium (Ca{sup 2+}) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca{sup 2+} signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca{sup 2+}-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H{sub 2}O{sub 2}-mediated Ca{sup 2+} overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca{sup 2+} overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca{sup 2+}-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca{sup 2+} overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

  19. Measuring Fast Calcium Fluxes in Cardiomyocytes

    Golebiewska, Urszula; Scarlata, Suzanne

    2011-01-01

    Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase Cβ- Gαq pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated Gαq7. This entrapment has the effect of stabilizing the activated state of Gαq and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad

  20. Innervating sympathetic neurons regulate heart size and the timing of cardiomyocyte cell cycle withdrawal.

    Kreipke, R E; Birren, S J

    2015-12-01

    Sympathetic drive to the heart is a key modulator of cardiac function and interactions between heart tissue and innervating sympathetic fibres are established early in development. Significant innervation takes place during postnatal heart development, a period when cardiomyocytes undergo a rapid transition from proliferative to hypertrophic growth. The question of whether these innervating sympathetic fibres play a role in regulating the modes of cardiomyocyte growth was investigated using 6-hydroxydopamine (6-OHDA) to abolish early sympathetic innervation of the heart. Postnatal chemical sympathectomy resulted in rats with smaller hearts, indicating that heart growth is regulated by innervating sympathetic fibres during the postnatal period. In vitro experiments showed that sympathetic interactions resulted in delays in markers of cardiomyocyte maturation, suggesting that changes in the timing of the transition from hyperplastic to hypertrophic growth of cardiomyocytes could underlie changes in heart size in the sympathectomized animals. There was also an increase in the expression of Meis1, which has been linked to cardiomyocyte cell cycle withdrawal, suggesting that sympathetic signalling suppresses cell cycle withdrawal. This signalling involves β-adrenergic activation, which was necessary for sympathetic regulation of cardiomyocyte proliferation and hypertrophy. The effect of β-adrenergic signalling on cardiomyocyte hypertrophy underwent a developmental transition. While young postnatal cardiomyocytes responded to isoproterenol (isoprenaline) with a decrease in cell size, mature cardiomyocytes showed an increase in cell size in response to the drug. Together, these results suggest that early sympathetic effects on proliferation modulate a key transition between proliferative and hypertrophic growth of the heart and contribute to the sympathetic regulation of adult heart size. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  1. Cardiomyocyte architectural plasticity in fetal, neonatal, and adult pig hearts delineated with diffusion tensor MRI.

    Zhang, Lei; Allen, John; Hu, Lingzhi; Caruthers, Shelton D; Wickline, Samuel A; Chen, Junjie

    2013-01-15

    Cardiomyocyte organization is a critical determinant of coordinated cardiac contractile function. Because of the acute opening of the pulmonary circulation, the relative workload of the left ventricle (LV) and right ventricle (RV) changes substantially immediately after birth. We hypothesized that three-dimensional cardiomyocyte architecture might be required to adapt rapidly to accommodate programmed perinatal changes of cardiac function. Isolated fixed hearts from pig fetuses or pigs at midgestation, preborn, postnatal day 1 (P1), postnatal day 5, postnatal day 14 (P14), and adulthood (n = 5 for each group) were acquired for diffusion-weighted magnetic resonance imaging. Cardiomyocyte architecture was visualized by three-dimensional fiber tracking and was quantitatively evaluated by the measured helix angle (α(h)). Upon the completion of MRI, hearts were sectioned and stained with hematoxylin/eosin (H&E) to evaluate cardiomyocyte alignment, with picrosirius red to evaluate collagen content, and with anti-Ki67 to evaluate postnatal cell proliferation. The helical architecture of cardiomyocyte was observed as early as the midgestational period. Postnatal changes of cardiomyocyte architecture were observed from P1 to P14, which primary occurred in the septum and RV free wall (RVFW). In the septum, the volume ratio of LV- vs. RV-associated cardiomyocytes rapidly changed from RV-LV balanced pattern at birth to LV dominant pattern by P14. In the RVFW, subendocardial α(h) decreased by ~30° from P1 to P14. These findings indicate that the helical architecture of cardiomyocyte is developed as early as the midgestation period. Substantial and rapid adaptive changes in cardiac microarchitecture suggested considerable developmental plasticity of cardiomyocyte form and function in the postnatal period in response to altered cardiac mechanical function.

  2. Excitation model of pacemaker cardiomyocytes of cardiac conduction system

    Grigoriev, M.; Babich, L.

    2015-11-01

    Myocardium includes typical and atypical cardiomyocytes - pacemakers, which form the cardiac conduction system. Excitation from the atrioventricular node in normal conditions is possible only in one direction. Retrograde direction of pulses is impossible. The most important prerequisite for the work of cardiomyocytes is the anatomical integrity of the conduction system. Changes in contractile force of the cardiomyocytes, which appear periodically, are due to two mechanisms of self-regulation - heterometric and homeometric. Graphic course of the excitation pulse propagation along the heart muscle more accurately reveals the understanding of the arrhythmia mechanism. These models have the ability to visualize the essence of excitation dynamics. However, they do not have the proper forecasting function for result estimation. Integrative mathematical model enables further investigation of general laws of the myocardium active behavior, allows for determination of the violation mechanism of electrical and contractile function of cardiomyocytes. Currently, there is no full understanding of the topography of pacemakers and ionic mechanisms. There is a need for the development of direction of mathematical modeling and comparative studies of the electrophysiological arrangement of cells of atrioventricular connection and ventricular conduction system.

  3. Atrial natriuretic peptide regulates Ca channel in early developmental cardiomyocytes.

    Lin Miao

    Full Text Available BACKGROUND: Cardiomyocytes derived from murine embryonic stem (ES cells possess various membrane currents and signaling cascades link to that of embryonic hearts. The role of atrial natriuretic peptide (ANP in regulation of membrane potentials and Ca(2+ currents has not been investigated in developmental cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of ANP in regulating L-type Ca(2+ channel current (I(CaL in different developmental stages of cardiomyocytes derived from ES cells. ANP decreased the frequency of action potentials (APs in early developmental stage (EDS cardiomyocytes, embryonic bodies (EB as well as whole embryo hearts. ANP exerted an inhibitory effect on basal I(CaL in about 70% EDS cardiomyocytes tested but only in about 30% late developmental stage (LDS cells. However, after stimulation of I(CaL by isoproterenol (ISO in LDS cells, ANP inhibited the response in about 70% cells. The depression of I(CaL induced by ANP was not affected by either Nomega, Nitro-L-Arginine methyl ester (L-NAME, a nitric oxide synthetase (NOS inhibitor, or KT5823, a cGMP-dependent protein kinase (PKG selective inhibitor, in either EDS and LDS cells; whereas depression of I(CaL by ANP was entirely abolished by erythro-9-(2-Hydroxy-3-nonyl adenine (EHNA, a selective inhibitor of type 2 phosphodiesterase(PDE2 in most cells tested. CONCLUSION/SIGNIFICANCES: Taken together, these results indicate that ANP induced depression of action potentials and I(CaL is due to activation of particulate guanylyl cyclase (GC, cGMP production and cGMP-activation of PDE2 mediated depression of adenosine 3', 5'-cyclic monophophate (cAMP-cAMP-dependent protein kinase (PKA in early cardiomyogenesis.

  4. Nerves Regulate Cardiomyocyte Proliferation and Heart Regeneration.

    Mahmoud, Ahmed I; O'Meara, Caitlin C; Gemberling, Matthew; Zhao, Long; Bryant, Donald M; Zheng, Ruimao; Gannon, Joseph B; Cai, Lei; Choi, Wen-Yee; Egnaczyk, Gregory F; Burns, Caroline E; Burns, C Geoffrey; MacRae, Calum A; Poss, Kenneth D; Lee, Richard T

    2015-08-24

    Some organisms, such as adult zebrafish and newborn mice, have the capacity to regenerate heart tissue following injury. Unraveling the mechanisms of heart regeneration is fundamental to understanding why regeneration fails in adult humans. Numerous studies have revealed that nerves are crucial for organ regeneration, thus we aimed to determine whether nerves guide heart regeneration. Here, we show using transgenic zebrafish that inhibition of cardiac innervation leads to reduction of myocyte proliferation following injury. Specifically, pharmacological inhibition of cholinergic nerve function reduces cardiomyocyte proliferation in the injured hearts of both zebrafish and neonatal mice. Direct mechanical denervation impairs heart regeneration in neonatal mice, which was rescued by the administration of neuregulin 1 (NRG1) and nerve growth factor (NGF) recombinant proteins. Transcriptional analysis of mechanically denervated hearts revealed a blunted inflammatory and immune response following injury. These findings demonstrate that nerve function is required for both zebrafish and mouse heart regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Oxidative stress and cardiomyocyte necrosis with elevated serum troponins: pathophysiologic mechanisms.

    Robinson, Antwon D; Ramanathan, Kodangudi B; McGee, Jesse E; Newman, Kevin P; Weber, Karl T

    2011-08-01

    The progressive nature of heart failure is linked to multiple factors, including an ongoing loss of cardiomyocytes and necrosis. Necrotic cardiomyocytes leave behind several footprints: the spillage of their contents leading to elevations in serum troponins; and morphologic evidence of tissue repair with scarring. The pathophysiologic origins of cardiomyocyte necrosis relates to neurohormonal activation, including the adrenergic nervous system. Catecholamine-initiated excessive intracellular Ca accumulation and mitochondria Ca overloading in particular initiate a mitochondriocentric signal-transducer-effector pathway to necrosis and which includes the induction of oxidative stress and opening of their inner membrane permeability transition pore. Hypokalemia, ionized hypocalcemia and hypomagnesemia, where consequent elevations in parathyroid hormone further account for excessive intracellular Ca accumulation, hypozincemia and hyposelenemia each compromise metalloenzyme-based antioxidant defenses. The necrotic loss of cardiomyocytes and adverse structural remodeling of myocardium is related to the central role played by a mitochondriocentric pathway initiated by neurohormonal activation.

  6. Evidence for Cardiomyocyte Renewal in Humans

    Bergmann, O; Bhardwaj, R D; Bernard, S; Zdunek, S; Barnabe-Heider, F; Walsh, S; Zupicich, J; Alkass, K; Buchholz, B A; Druid, H; Jovinge, S; Frisen, J

    2008-10-14

    It has been difficult to establish whether we are limited to the heart muscle cells we are born with or if cardiomyocytes are generated also later in life. We have taken advantage of the integration of {sup 14}C, generated by nuclear bomb tests during the Cold War, into DNA to establish the age of cardiomyocytes in humans. We report that cardiomyocytes renew, with a gradual decrease from 1% turning over annually at the age of 20 to 0.3% at the age of 75. Less than 50% of cardiomyocytes are exchanged during a normal lifespan. The capacity to generate cardiomyocytes in the adult human heart suggests that it may be rational to work towards the development of therapeutic strategies aiming to stimulate this process in cardiac pathologies.

  7. High Uric Acid Induces Insulin Resistance in Cardiomyocytes In Vitro and In Vivo.

    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.

  8. Electrophysiological properties and calcium handling of embryonic stem cell-derived cardiomyocytes

    Jae Boum Youm

    2016-03-01

    Full Text Available Embryonic stem cell-derived cardiomyocytes (ESC-CMs hold great interest in many fields of research including clinical applications such as stem cell and gene therapy for cardiac repair or regeneration. ESC-CMs are also used as a platform tool for pharmacological tests or for investigations of cardiac remodeling. ESC-CMs have many different aspects of morphology, electrophysiology, calcium handling, and bioenergetics compared with adult cardiomyocytes. They are immature in morphology, similar to sinus nodal-like in the electrophysiology, higher contribution of trans-sarcolemmal Ca2+ influx to Ca2+ handling, and higher dependence on anaerobic glycolysis. Here, I review a detailed electrophysiology and Ca2+ handling features of ESC-CMs during differentiation into adult cardiomyocytes to gain insights into how all the developmental changes are related to each other to display cardinal features of developing cardiomyocytes.

  9. In vitro model to study the effects of matrix stiffening on Ca2+ handling and myofilament function in isolated adult rat cardiomyocytes.

    van Deel, Elza D; Najafi, Aref; Fontoura, Dulce; Valent, Erik; Goebel, Max; Kardux, Kim; Falcão-Pires, Inês; van der Velden, Jolanda

    2017-07-15

    This paper describes a novel model that allows exploration of matrix-induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function. Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca 2+ handling and myofilament function. Cell shortening and Ca 2+ handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix. Matrix stiffness-impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness. Matrix stiffness-induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. Extracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte-matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix-induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca 2+ handling but does not alter myofilament-generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness-induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness-induced changes in cardiomyocyte

  10. Apoptosis of rats’ cardiomyocytes after chronic energy drinks consumption

    Slawinski Miroslaw Aleksander

    2018-03-01

    Full Text Available Energy drinks (ED are beverages containing caffeine, taurine, vitamins, herbal extracts, and sugar or sweeteners. They are marketed as capable of improving stamina, athletic performance and concentration, moreover, as serving as a source of energy. Still, there are very few papers describing the impact of ED on cell biology – including cell apoptosis within tissues. Therefore, in our study, we assessed the symptoms of rat cardiomyocytes apoptosis after 8 weeks consumption of ED.

  11. Phosphorylation of protein kinase C sites Ser42/44 decreases Ca2+-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes

    Wijnker, P.J.M.; Sequeira Oliveira, V.; Witjas-Paalberends, E.R.; Foster, D.B.; dos Remedios, C.G.; Murphy, A.M.; Stienen, G.J.M.; van der Velden, J.

    2014-01-01

    Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal

  12. Adrenaline and reactive oxygen species elicit proteome and energetic metabolism modifications in freshly isolated rat cardiomyocytes

    Costa, Vera Marisa; Silva, Renata; Tavares, Ludgero Canario; Vitorino, Rui; Amado, Francisco; Carvalho, Felix; Bastos, Maria de Lourdes; Carvalho, Marcia; Carvalho, Rui Albuquerque; Remiao, Fernando

    2009-01-01

    The sustained elevation of plasma and interstitial catecholamine levels, namely adrenaline (ADR), and the generation of reactive oxygen species (ROS) are well recognized hallmarks of several cardiopathologic conditions, like cardiac ischemia/reperfusion (I/R) and heart failure (HF). The present work aimed to investigate the proteomics and energetic metabolism of cardiomyocytes incubated with ADR and/or ROS. To mimic pathologic conditions, freshly isolated calcium-tolerant cardiomyocytes from adult rat were incubated with ADR alone or in the presence of a system capable of generating ROS [(xanthine with xanthine oxidase) (XXO)]. Two-dimensional electrophoresis with matrix-assisted laser desorption/ionization and time-of-flight mass spectrometer analysis were used to define protein spot alterations in the cardiomyocytes incubated with ADR and/or ROS. Moreover, the energetic metabolism and the activity of mitochondrial complexes were evaluated by nuclear magnetic resonance and spectrophotometric determinations, respectively. The protein extract was mainly constituted by cardiac mitochondrial proteins and the alterations found were included in five functional classes: (i) structural proteins, notably myosin light chain-2; (ii) redox regulation proteins, in particular superoxide dismutase (SOD); (iii) energetic metabolism proteins, encompassing ATP synthase alpha chain and dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex; (iv) stress response proteins, like the heat shock proteins; and (v) regulatory proteins, like cytochrome c and voltage-dependent anion channel 1. The XXO system elicited alterations in cardiac contractile proteins, as they showed high levels of cleavage, and also altered energetic metabolism, through increased lactate and alanine levels. The cardiomyocytes incubation with ADR resulted in an accentuated increase in mitochondrial complexes activity and the decrease in alanine/lactate ratio, thus reflecting a high

  13. Defects of mtDNA Replication Impaired Mitochondrial Biogenesis During Trypanosoma cruzi Infection in Human Cardiomyocytes and Chagasic Patients: The Role of Nrf1/2 and Antioxidant Response

    Wan, Xianxiu; Gupta, Shivali; Zago, Maria P.; Davidson, Mercy M.; Dousset, Pierre; Amoroso, Alejandro; Garg, Nisha Jain

    2012-01-01

    Background Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)–regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. Methods and Results We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi–infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)–regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. Conclusions The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi–infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for

  14. Genetic enrichment of cardiomyocytes derived from mouse ...

    Jane

    2011-06-22

    Jun 22, 2011 ... Pluripotent embryonic stem cells (ESC) have the ability to differentiate into a ... We describe a simple method to generate relatively pure cardiomyocytes from mouse ... In this study, we described the generation of transgenic.

  15. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B.; Rivkees, Scott A.

    2014-01-01

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20–60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3–65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. PMID:25354728

  16. Cation dyshomeostasis and cardiomyocyte necrosis: the Fleckenstein hypothesis revisited

    Borkowski, Brian J.; Cheema, Yaser; Shahbaz, Atta U.; Bhattacharya, Syamal K.; Weber, Karl T.

    2011-01-01

    An ongoing loss of cardiomyocytes to apoptotic and necrotic cell death pathways contributes to the progressive nature of heart failure. The pathophysiological origins of necrotic cell loss relate to the neurohormonal activation that accompanies acute and chronic stressor states and which includes effector hormones of the adrenergic nervous system. Fifty years ago, Albrecht Fleckenstein and coworkers hypothesized the hyperadrenergic state, which accompanies such stressors, causes cardiomyocyte necrosis based on catecholamine-initiated excessive intracellular Ca2+ accumulation (EICA), and mitochondrial Ca2+ overloading in particular, in which the ensuing dysfunction and structural degeneration of these organelles leads to necrosis. In recent years, two downstream factors have been identified which, together with EICA, constitute a signal–transducer–effector pathway: (i) mitochondria-based induction of oxidative stress, in which the rate of reactive oxygen metabolite generation exceeds their rate of detoxification by endogenous antioxidant defences; and (ii) the opening of the mitochondrial inner membrane permeability transition pore (mPTP) followed by organellar swelling and degeneration. The pathogenesis of stress-related cardiomyopathy syndromes is likely related to this pathway. Other factors which can account for cytotoxicity in stressor states include: hypokalaemia; ionized hypocalcaemia and hypomagnesaemia with resultant elevations in parathyroid hormone serving as a potent mediator of EICA; and hypozincaemia with hyposelenaemia, which compromise antioxidant defences. Herein, we revisit the Fleckenstein hypothesis of EICA in leading to cardiomyocyte necrosis and the central role played by mitochondria. PMID:21398641

  17. Disturbance of cardiac gene expression and cardiomyocyte structure predisposes Mecp2-null mice to arrhythmias

    Hara, Munetsugu; Takahashi, Tomoyuki; Mitsumasu, Chiaki; Igata, Sachiyo; Takano, Makoto; Minami, Tomoko; Yasukawa, Hideo; Okayama, Satoko; Nakamura, Keiichiro; Okabe, Yasunori; Tanaka, Eiichiro; Takemura, Genzou; Kosai, Ken-ichiro; Yamashita, Yushiro; Matsuishi, Toyojiro

    2015-01-01

    Methyl-CpG-binding protein 2 (MeCP2) is an epigenetic regulator of gene expression that is essential for normal brain development. Mutations in MeCP2 lead to disrupted neuronal function and can cause Rett syndrome (RTT), a neurodevelopmental disorder. Previous studies reported cardiac dysfunction, including arrhythmias in both RTT patients and animal models of RTT. In addition, recent studies indicate that MeCP2 may be involved in cardiac development and dysfunction, but its role in the developing and adult heart remains unknown. In this study, we found that Mecp2-null ESCs could differentiate into cardiomyocytes, but the development and further differentiation of cardiovascular progenitors were significantly affected in MeCP2 deficiency. In addition, we revealed that loss of MeCP2 led to dysregulation of endogenous cardiac genes and myocardial structural alterations, although Mecp2-null mice did not exhibit obvious cardiac functional abnormalities. Furthermore, we detected methylation of the CpG islands in the Tbx5 locus, and showed that MeCP2 could target these sequences. Taken together, these results suggest that MeCP2 is an important regulator of the gene-expression program responsible for maintaining normal cardiac development and cardiomyocyte structure. PMID:26073556

  18. Impact of calcium-sensitive dyes on the beating properties and pharmacological responses of human iPS-derived cardiomyocytes using the calcium transient assay.

    Kopljar, Ivan; Hermans, An N; Teisman, Ard; Gallacher, David J; Lu, Hua Rong

    Calcium-based screening of hiPS-CMs is a useful preclinical safety evaluation platform with the ability to generate robust signals that facilitates high-throughput screening and data analysis. However, due to the potential inherent toxicities, it is important to understand potential effects of different calcium-sensitive dyes on the hiPS-CMs model. We compared three calcium-sensitive fluorescence dyes (Cal520, ACTOne and Calcium 5) for their impact on the variability, the beating properties and the pharmacological responses of hiPS-CMs using the Hamamatsu FDSS/μCell imaging platform. Direct effects of three dyes on the electrophysiological properties of hiPS-CMs were evaluated with the multi-electrode array (MEA) Axion Maestro platform. We propose a specific experimental protocol for each dye which gives the most optimal assay conditions to minimize variability and possible adverse effects. We showed that Cal520 had the smallest effect on hiPS-CMs together with the longest-lasting stable amplitude signal (up to 4 h). Although all dyes had a (minor) acute effect on hiPS-CMs, in the form of reduced beat rate and prolonged field potential duration, the selection of the dye did not influence the pharmacological response of four cardioactive drugs (dofetilide, moxifloxacin, nimodipine and isoprenaline). In conclusion, we have documented that different calcium sensitive dyes have only minor direct (acute) effects on hiPS-CMs with Cal520 showing the least effects and the longest lasting signal amplitude. Importantly, drug-induced pharmacological responses in hiPS-CMs were comparable between the three dyes. These findings should help further improve the robustness of the hiPS-CMs-based calcium transient assay as a predictive, preclinical cardiac safety evaluation tool. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Identification, Selection, and Enrichment of Cardiomyocyte Precursors

    Bianca Ferrarini Zanetti

    2013-01-01

    Full Text Available The large-scale production of cardiomyocytes is a key step in the development of cell therapy and tissue engineering to treat cardiovascular diseases, particularly those caused by ischemia. The main objective of this study was to establish a procedure for the efficient production of cardiomyocytes by reprogramming mesenchymal stem cells from adipose tissue. First, lentiviral vectors expressing neoR and GFP under the control of promoters expressed specifically during cardiomyogenesis were constructed to monitor cell reprogramming into precardiomyocytes and to select cells for amplification and characterization. Cellular reprogramming was performed using 5′-azacytidine followed by electroporation with plasmid pOKS2a, which expressed Oct4, Sox2, and Klf4. Under these conditions, GFP expression began only after transfection with pOKS2a, and less than 0.015% of cells were GFP+. These GFP+ cells were selected for G418 resistance to find molecular markers of cardiomyocytes by RT-PCR and immunocytochemistry. Both genetic and protein markers of cardiomyocytes were present in the selected cells, with some variations among them. Cell doubling time did not change after selection. Together, these results indicate that enrichment with vectors expressing GFP and neoR under cardiomyocyte-specific promoters can produce large numbers of cardiomyocyte precursors (CMPs, which can then be differentiated terminally for cell therapy and tissue engineering.

  20. MicroRNA-1 overexpression blunts cardiomyocyte hypertrophy elicited by thyroid hormone.

    Diniz, Gabriela Placoná; Lino, Caroline Antunes; Moreno, Camila Rodrigues; Senger, Nathalia; Barreto-Chaves, Maria Luiza Morais

    2017-12-01

    It is well-known that increased thyroid hormone (TH) levels induce cardiomyocyte growth. MicroRNAs (miRNAs) have been identified as key players in cardiomyocyte hypertrophy, which is associated with increased risk of heart failure. In this study, we evaluated the miR-1 expression in TH-induced cardiac hypertrophy, as well as the potential involvement of miR-1 in cardiomyocyte hypertrophy elicited by TH in vitro. The possible role of type 1 angiotensin II receptor (AT1R) in the effect promoted by TH in miR-1 expression was also evaluated. Neonatal rat cardiac myocytes (NRCMs) were treated with T 3 for 24 hr and Wistar rats were subjected to hyperthyroidism for 14 days combined or not with AT1R blocker. Real Time RT-PCR analysis indicated that miR-1 expression was decreased in cardiac hypertrophy in response to TH in vitro and in vivo, and this effect was unchanged by AT1R blocker. In addition, HDAC4, which is target of miR-1, was increased in NRCMs after T 3 treatment. A gain-of-function study revealed that overexpression of miR-1 prevented T 3 -induced cardiomyocyte hypertrophy and reduced HADC4 mRNA levels in NRCMs. In vivo experiments confirmed the downregulation of miR-1 in cardiac tissue from hyperthyroid animals, which was accompanied by increased HDAC4 mRNA levels. In addition, HDAC inhibitor prevented T 3 -induced cardiomyocyte hypertrophy. Our data reveal a new mechanistic insight into cardiomyocyte growth in response to TH, suggesting that miR-1 plays a role in cardiomyocyte hypertrophy induced by TH potentially via targeting HADC4. © 2017 Wiley Periodicals, Inc.

  1. Sphingosine-1-phosphate promotes the differentiation of human umbilical cord mesenchymal stem cells into cardiomyocytes under the designated culturing conditions

    Zhang Henggui

    2011-06-01

    Full Text Available Abstract Background It is of growing interest to develop novel approaches to initiate differentiation of mesenchymal stem cells (MSCs into cardiomyocytes. The purpose of this investigation was to determine if Sphingosine-1-phosphate (S1P, a native circulating bioactive lipid metabolite, plays a role in differentiation of human umbilical cord mesenchymal stem cells (HUMSCs into cardiomyocytes. We also developed an engineered cell sheet from these HUMSCs derived cardiomyocytes by using a temperature-responsive polymer, poly(N-isopropylacrylamide (PIPAAm cell sheet technology. Methods Cardiomyogenic differentiation of HUMSCs was performed by culturing these cells with either designated cardiomyocytes conditioned medium (CMCM alone, or with 1 μM S1P; or DMEM with 10% FBS + 1 μM S1P. Cardiomyogenic differentiation was determined by immunocytochemical analysis of expression of cardiomyocyte markers and patch clamping recording of the action potential. Results A cardiomyocyte-like morphology and the expression of α-actinin and myosin heavy chain (MHC proteins can be observed in both CMCM culturing or CMCM+S1P culturing groups after 5 days' culturing, however, only the cells in CMCM+S1P culture condition present cardiomyocyte-like action potential and voltage gated currents. A new approach was used to form PIPAAm based temperature-responsive culture surfaces and this successfully produced cell sheets from HUMSCs derived cardiomyocytes. Conclusions This study for the first time demonstrates that S1P potentiates differentiation of HUMSCs towards functional cardiomyocytes under the designated culture conditions. Our engineered cell sheets may provide a potential for clinically applicable myocardial tissues should promote cardiac tissue engineering research.

  2. The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold and heat

    Kazuo eNakashima

    2014-05-01

    Full Text Available Drought negatively impacts plant growth and the productivity of crops around the world. Understanding the molecular mechanisms in the drought response is important for improvement of drought tolerance using molecular techniques. In plants, abscisic acid (ABA is accumulated under osmotic stress conditions caused by drought, and has a key role in stress responses and tolerance. Comprehensive molecular analyses have shown that ABA regulates the expression of many genes under osmotic stress conditions, and the ABA-responsive element (ABRE is the major cis-element for ABA-responsive gene expression. Transcription factors (TFs are master regulators of gene expression. ABRE-binding protein (AREB and ABRE-binding factor (ABF TFs control gene expression in an ABA-dependent manner. SNF1-related protein kinases 2, group A 2C-type protein phosphatases, and ABA receptors were shown to control the ABA signaling pathway. ABA-independent signaling pathways such as dehydration-responsive element-binding protein (DREB TFs and NAC TFs are also involved in stress responses including drought, heat and cold. Recent studies have suggested that there are interactions between the major ABA signaling pathway and other signaling factors in stress responses. The important roles of these transcription factors in crosstalk among abiotic stress responses will be discussed. Control of ABA or stress signaling factor expression can improve tolerance to environmental stresses. Recent studies using crops have shown that stress-specific overexpression of TFs improves drought tolerance and grain yield compared with controls in the field.

  3. The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold, and heat.

    Nakashima, Kazuo; Yamaguchi-Shinozaki, Kazuko; Shinozaki, Kazuo

    2014-01-01

    Drought negatively impacts plant growth and the productivity of crops around the world. Understanding the molecular mechanisms in the drought response is important for improvement of drought tolerance using molecular techniques. In plants, abscisic acid (ABA) is accumulated under osmotic stress conditions caused by drought, and has a key role in stress responses and tolerance. Comprehensive molecular analyses have shown that ABA regulates the expression of many genes under osmotic stress conditions, and the ABA-responsive element (ABRE) is the major cis-element for ABA-responsive gene expression. Transcription factors (TFs) are master regulators of gene expression. ABRE-binding protein and ABRE-binding factor TFs control gene expression in an ABA-dependent manner. SNF1-related protein kinases 2, group A 2C-type protein phosphatases, and ABA receptors were shown to control the ABA signaling pathway. ABA-independent signaling pathways such as dehydration-responsive element-binding protein TFs and NAC TFs are also involved in stress responses including drought, heat, and cold. Recent studies have suggested that there are interactions between the major ABA signaling pathway and other signaling factors in stress responses. The important roles of these TFs in crosstalk among abiotic stress responses will be discussed. Control of ABA or stress signaling factor expression can improve tolerance to environmental stresses. Recent studies using crops have shown that stress-specific overexpression of TFs improves drought tolerance and grain yield compared with controls in the field.

  4. Transient Response Dynamic Module Modifications to Include Static and Kinetic Friction Effects

    Misel, J. E.; Nenno, S. B.; Takahashi, D.

    1984-01-01

    A methodology that supports forced transient response dynamic solutions when both static and kinetic friction effects are included in a structural system model is described. Modifications that support this type of nonlinear transient response solution are summarized for the transient response dynamics (TRD) NASTRAN module. An overview of specific modifications for the NASTRAN processing subroutines, INITL, TRD1C, and TRD1D, are described with further details regarding inspection of nonlinear input definitions to define the type of nonlinear solution required, along with additional initialization requirements and specific calculation subroutines to successfully solve the transient response problem. The extension of the basic NASTRAN nonlinear methodology is presented through several stages of development to the point where constraint equations and residual flexibility effects are introduced into the finite difference Newmark-Beta recurrsion formulas. Particular emphasis is placed on cost effective solutions for large finite element models such as the Space Shuttle with friction degrees of freedom between the orbiter and payloads mounted in the cargo bay. An alteration to the dynamic finite difference equations of motion is discussed, which allows one to include friction effects at reasonable cost for large structural systems such as the Space Shuttle. Data are presented to indicate the possible impact of transient friction loads to the payload designer for the Space Shuttle. Transient response solution data are also included, which compare solutions without friction forces and those with friction forces for payloads mounted in the Space Shuttle cargo bay. These data indicate that payload components can be sensitive to friction induced loads.

  5. Up-regulation of miR-26a promotes apoptosis of hypoxic rat neonatal cardiomyocytes by repressing GSK-3β protein expression.

    Suh, Jong Hui; Choi, Eunmi; Cha, Min-Ji; Song, Byeong-Wook; Ham, Onju; Lee, Se-Yeon; Yoon, Cheesoon; Lee, Chang-Yeon; Park, Jun-Hee; Lee, Sun Hee; Hwang, Ki-Chul

    2012-06-29

    Myocardial ischemia is the major cause of morbidity and mortality due to cardiovascular diseases. This disease is a severe stress condition that causes extensive biochemical changes which trigger cardiac cell death. Stress conditions such as deprivation of glucose and oxygen activate the endoplasmic reticulum in the cytoplasm of cells, including cardiomyocytes, to generate and propagate apoptotic signals in response to these conditions. microRNAs (miRNAs) are a class of small non-coding RNAs that mediate posttranscriptional gene silencing. The miRNAs play important roles in regulating cardiac physiological and pathological events such as hypertrophy, apoptosis, and heart failure. However, the roles of miRNAs in reactive oxygen species (ROS)-mediated injury on cardiomyocytes are uncertain. In this study, we identified at the apoptotic concentration of H(2)O(2), miR-26a expression was increased. To determine the potential roles of miR-26a in H(2)O(2)-mediated cardiac apoptosis, miR-26a expression was regulated by a miR-26a or an anti-miR-26a. Overexpression of miR-26a increased apoptosis as determined by upregulation of Annexin V/PI positive cell population, caspase-3 activity and expression of pro-apoptotic signal molecules, whereas inhibition of miR-26a reduced apoptosis. We identified GSK3B as a direct downstream target of miR-26a. Furthermore, miR-26a attenuated viability and increased caspase-3 activity in normal cardiomyocytes. This study demonstrates that miR-26a promotes ROS-induced apoptosis in cardiomyocytes. Thus, miR-26a affects ROS-mediated gene regulation and cellular injury response. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. The Adipokine Chemerin Induces Apoptosis in Cardiomyocytes

    Diego Rodríguez-Penas

    2015-08-01

    Full Text Available Background: The adipokine chemerin has been associated with cardiovascular disease. We investigated the effects of chemerin on viability and intracellular signalling in murine cardiomyocytes, and the effects of insulin and TNF-α on cardiomyocyte chemerin production. Methods: Hoechst dye vital staining and cell cycle analysis were used to analyse the viability of murine cardiac cells in culture. Western blot was used to explore the phosphorylation of AKT and caspase-9 activity in neonatal rat cardiomyocytes and HL-1 cells. Finally, RT-qPCR, ELISA and western blot were performed to examine chemerin and CMKLR1 expression after insulin and TNF-α treatment in cardiac cells. Results: Chemerin treatment increased apoptosis, reduced phosphorylation of AKT at Thr308 and increased caspase-9 activity in murine cardiomyocytes. Insulin treatment lowered chemerin and CMKLR1 mRNA and protein levels, and the amount of chemerin in the cell media, while TNF-α treatment increased chemerin mRNA and protein levels but decreased expression of the CMKLR1 gene. Conclusion: Chemerin induces apoptosis, reduces AKT phosphorylation and increases the cleavage of caspase-9 in murine cardiomyocytes. The expression of chemerin is regulated by important metabolic (insulin and inflammatory (TNF-α mediators at cardiac level. Our results suggest that chemerin could play a role in the physiopathology of cardiac diseases.

  7. Advanced neuroblastoma: improved response rate using a multiagent regimen (OPEC) including sequential cisplatin and VM-26.

    Shafford, E A; Rogers, D W; Pritchard, J

    1984-07-01

    Forty-two children, all over one year of age, were given vincristine, cyclophosphamide, and sequentially timed cisplatin and VM-26 (OPEC) or OPEC and doxorubicin (OPEC-D) as initial treatment for newly diagnosed stage III or IV neuroblastoma. Good partial response was achieved in 31 patients (74%) overall and in 28 (78%) of 36 patients whose treatment adhered to the chemotherapy protocol, compared with a 65% response rate achieved in a previous series of children treated with pulsed cyclophosphamide and vincristine with or without doxorubicin. Only six patients, including two of the six children whose treatment did not adhere to protocol, failed to respond, but there were five early deaths from treatment-related complications. Tumor response to OPEC, which was the less toxic of the two regimens, was at least as good as tumor response to OPEC-D. Cisplatin-induced morbidity was clinically significant in only one patient and was avoided in others by careful monitoring of glomerular filtration rate and hearing. Other centers should test the efficacy of OPEC or equivalent regimens in the treatment of advanced neuroblastoma.

  8. High-throughput cardiac safety evaluation and multi-parameter arrhythmia profiling of cardiomyocytes using microelectrode arrays

    Gilchrist, Kristin H., E-mail: kgilchrist@rti.org; Lewis, Gregory F.; Gay, Elaine A.; Sellgren, Katelyn L.; Grego, Sonia

    2015-10-15

    Microelectrode arrays (MEAs) recording extracellular field potentials of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) provide a rich data set for functional assessment of drug response. The aim of this work is the development of a method for a systematic analysis of arrhythmia using MEAs, with emphasis on the development of six parameters accounting for different types of cardiomyocyte signal irregularities. We describe a software approach to carry out such analysis automatically including generation of a heat map that enables quick visualization of arrhythmic liability of compounds. We also implemented signal processing techniques for reliable extraction of the repolarization peak for field potential duration (FPD) measurement even from recordings with low signal to noise ratios. We measured hiPS-CM's on a 48 well MEA system with 5 minute recordings at multiple time points (0.5, 1, 2 and 4 h) after drug exposure. We evaluated concentration responses for seven compounds with a combination of hERG, QT and clinical proarrhythmia properties: Verapamil, Ranolazine, Flecainide, Amiodarone, Ouabain, Cisapride, and Terfenadine. The predictive utility of MEA parameters as surrogates of these clinical effects were examined. The beat rate and FPD results exhibited good correlations with previous MEA studies in stem cell derived cardiomyocytes and clinical data. The six-parameter arrhythmia assessment exhibited excellent predictive agreement with the known arrhythmogenic potential of the tested compounds, and holds promise as a new method to predict arrhythmic liability. - Highlights: • Six parameters describing arrhythmia were defined and measured for known compounds. • Software for efficient parameter extraction from large MEA data sets was developed. • The proposed cellular parameter set is predictive of clinical drug proarrhythmia.

  9. Simulated selection responses for breeding programs including resistance and resilience to parasites in Creole goats.

    Gunia, M; Phocas, F; Gourdine, J-L; Bijma, P; Mandonnet, N

    2013-02-01

    The Creole goat is a local breed used for meat production in Guadeloupe (French West Indies). As in other tropical countries, improvement of parasite resistance is needed. In this study, we compared predicted selection responses for alternative breeding programs with or without parasite resistance and resilience traits. The overall breeding goal included traits for production, reproduction, and parasite resilience and resistance to ensure a balanced selection outcome. The production traits were BW and dressing percentage (DP). The reproduction trait was fertility (FER), which was the number of doe kiddings per mating. The resistance trait was worm fecal egg count (FEC), which is a measurement of the number of gastro-intestinal parasite eggs found in the feces. The resilience trait was the packed cell volume (PCV), which is a measurement of the volume of red blood cells in the blood. Dressing percentage, BW, and FEC were measured at 11 mo of age, which is the mating or selling age. Fertility and PCV were measured on females at each kidding period. The breeding program accounting for the overall breeding goal and a selection index including all traits gave annual selection responses of 800 g for BW, 3.75% for FER, 0.08% for DP, -0.005 ln(eggs/g) for FEC, and 0.28% for PCV. The expected selection responses for BW and DP in this breeding program were reduced by 2% and 6%, respectively, compared with a breeding program not accounting for FEC and PCV. The overall breeding program, proposed for the Creole breed, offers the best breeding strategy in terms of expected selection responses, making it possible to improve all traits together. It offers a good balance between production and adaptation traits and may present some interest for the selection of other goat breeds in the tropics.

  10. Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes.

    Yang, Xiulan; Pabon, Lil; Murry, Charles E

    2014-01-31

    The discovery of human pluripotent stem cells (hPSCs), including both human embryonic stem cells and human-induced pluripotent stem cells, has opened up novel paths for a wide range of scientific studies. The capability to direct the differentiation of hPSCs into functional cardiomyocytes has provided a platform for regenerative medicine, development, tissue engineering, disease modeling, and drug toxicity testing. Despite exciting progress, achieving the optimal benefits has been hampered by the immature nature of these cardiomyocytes. Cardiac maturation has long been studied in vivo using animal models; however, finding ways to mature hPSC cardiomyocytes is only in its initial stages. In this review, we discuss progress in promoting the maturation of the hPSC cardiomyocytes, in the context of our current knowledge of developmental cardiac maturation and in relation to in vitro model systems such as rodent ventricular myocytes. Promising approaches that have begun to be examined in hPSC cardiomyocytes include long-term culturing, 3-dimensional tissue engineering, mechanical loading, electric stimulation, modulation of substrate stiffness, and treatment with neurohormonal factors. Future studies will benefit from the combinatorial use of different approaches that more closely mimic nature's diverse cues, which may result in broader changes in structure, function, and therapeutic applicability.

  11. AKIP1 expression modulates mitochondrial function in rat neonatal cardiomyocytes.

    Hongjuan Yu

    Full Text Available A kinase interacting protein 1 (AKIP1 is a molecular regulator of protein kinase A and nuclear factor kappa B signalling. Recent evidence suggests AKIP1 is increased in response to cardiac stress, modulates acute ischemic stress response, and is localized to mitochondria in cardiomyocytes. The mitochondrial function of AKIP1 is, however, still elusive. Here, we investigated the mitochondrial function of AKIP1 in a neonatal cardiomyocyte model of phenylephrine (PE-induced hypertrophy. Using a seahorse flux analyzer we show that PE stimulated the mitochondrial oxygen consumption rate (OCR in cardiomyocytes. This was partially dependent on PE mediated AKIP1 induction, since silencing of AKIP1 attenuated the increase in OCR. Interestingly, AKIP1 overexpression alone was sufficient to stimulate mitochondrial OCR and in particular ATP-linked OCR. This was also true when pyruvate was used as a substrate, indicating that it was independent of glycolytic flux. The increase in OCR was independent of mitochondrial biogenesis, changes in ETC density or altered mitochondrial membrane potential. In fact, the respiratory flux was elevated per amount of ETC, possibly through enhanced ETC coupling. Furthermore, overexpression of AKIP1 reduced and silencing of AKIP1 increased mitochondrial superoxide production, suggesting that AKIP1 modulates the efficiency of electron flux through the ETC. Together, this suggests that AKIP1 overexpression improves mitochondrial function to enhance respiration without excess superoxide generation, thereby implicating a role for AKIP1 in mitochondrial stress adaptation. Upregulation of AKIP1 during different forms of cardiac stress may therefore be an adaptive mechanism to protect the heart.

  12. α,β-Unsaturated aldehyde pollutant acrolein suppresses cardiomyocyte contractile function: Role of TRPV1 and oxidative stress.

    Wu, Zhenbiao; He, Emily Y; Scott, Glenda I; Ren, Jun

    2015-01-01

    Air pollution is associated with an increased prevalence of heart disease and is known to trigger a proinflammatory response via stimulation of transient receptor potential vanilloid cation channels (TRPV1, also known as the capsaicin receptor). This study was designed to examine the effect of acrolein, an essential α,β-unsaturated aldehyde pollutant, on myocardial contractile function and the underlying mechanism involved with a focus on TRPV1 and oxidative stress. Cardiomyocyte mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix MyoCam® system including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90 ), fura-2 fluorescence intensity (FFI) and intracellular Ca(2+) decay. Changes in apoptosis and TRPV1 were evaluated using Western blot analysis. The degree of oxidative stress was assessed using the ratio between reduced and oxidized glutathione. Results obtained revealed that exposure of cardiomyocytes to acrolein acutely compromised contractile and intracellular Ca(2+) properties including depressed PS, ± dL/dt and ΔFFI, as well as prolonged TR90 and intracellular Ca(2+) decay. In addition, acrolein exposure upregulated TRPV1 associated with an increase in both apoptosis and oxidative stress. However, the acrolein-induced cardiomyocyte contractile and intracellular Ca(2+) anomalies, as well as apoptosis (as evidenced by Bcl-2, Bax, FasL, Caspase-3 and -8), were negated by the reactive oxygen species (ROS) scavenger glutathione or the TRPV1 antagonist capsazepine. Collectively these data suggest that the α,β-unsaturated aldehyde pollutant acrolein may play a role in the pathogenesis and sequelae of air pollution-induced heart disease via a TRPV1- and oxidative stress-dependent mechanism. © 2013 Wiley Periodicals, Inc.

  13. Manipulation-free cultures of human iPSC-derived cardiomyocytes offer a novel screening method for cardiotoxicity.

    Rajasingh, Sheeja; Isai, Dona Greta; Samanta, Saheli; Zhou, Zhi-Gang; Dawn, Buddhadeb; Kinsey, William H; Czirok, Andras; Rajasingh, Johnson

    2018-04-05

    Induced pluripotent stem cell (iPSC)-based cardiac regenerative medicine requires the efficient generation, structural soundness and proper functioning of mature cardiomyocytes, derived from the patient's somatic cells. The most important functional property of cardiomyocytes is the ability to contract. Currently available methods routinely used to test and quantify cardiomyocyte function involve techniques that are labor-intensive, invasive, require sophisticated instruments or can adversely affect cell vitality. We recently developed optical flow imaging method analyses and quantified cardiomyocyte contractile kinetics from video microscopic recordings without compromising cell quality. Specifically, our automated particle image velocimetry (PIV) analysis of phase-contrast video images captured at a high frame rate yields statistical measures characterizing the beating frequency, amplitude, average waveform and beat-to-beat variations. Thus, it can be a powerful assessment tool to monitor cardiomyocyte quality and maturity. Here we demonstrate the ability of our analysis to characterize the chronotropic responses of human iPSC-derived cardiomyocytes to a panel of ion channel modulators and also to doxorubicin, a chemotherapy agent with known cardiotoxic side effects. We conclude that the PIV-derived beat patterns can identify the elongation or shortening of specific phases in the contractility cycle, and the obtained chronotropic responses are in accord with known clinical outcomes. Hence, this system can serve as a powerful tool to screen the new and currently available pharmacological compounds for cardiotoxic effects.

  14. Investigation on plasmonic responses in multilayered nanospheres including asymmetry and spatial nonlocal effects

    Dong, Tianyu; Shi, Yi; Liu, Hui; Chen, Feng; Ma, Xikui; Mittra, Raj

    2017-12-01

    In this work, we present a rigorous approach for analyzing the optical response of multilayered spherical nano-particles comprised of either plasmonic metal or dielectric, when there is no longer radial symmetry and when nonlocality is included. The Lorenz-Mie theory is applied, and a linearized hydrodynamic Drude model as well as the general nonlocal optical response model for the metals are employed. Additional boundary conditions, viz., the continuity of normal components of polarization current density and the continuity of first-order pressure of free electron density, respectively, are incorporated when handling interfaces involving metals. The application of spherical addition theorems, enables us to express a spherical harmonic about one origin to spherical harmonics about a different origin, and leads to a linear system of equations for the inward- and outward-field modal coefficients for all the layers in the nanoparticle. Scattering matrices at interfaces are obtained and cascaded to obtain the expansion coefficients, to yield the final solution. Through extensive modelling of stratified concentric and eccentric metal-involved spherical nanoshells illuminating by a plane wave, we show that, within a nonlocal description, significant modifications of plasmonic response appear, e.g. a blue-shift in the extinction / scattering spectrum and a broadening spectrum of the resonance. In addition, it has been demonstrated that core-shell nanostructures provide an option for tunable Fano-resonance generators. The proposed method shows its capability and flexibility to analyze the nonlocal response of eccentric hybrid metal-dielectric multilayer structures as well as adjoined metal-involved nanoparticles, even when the number of layers is large.

  15. CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart.

    Melisa Gomez-Velazquez

    2017-08-01

    Full Text Available Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development.

  16. Early Administration of Glutamine Protects Cardiomyocytes from Post-Cardiac Arrest Acidosis

    Yan-Ren Lin

    2016-01-01

    Full Text Available Postcardiac arrest acidosis can decrease survival. Effective medications without adverse side effects are still not well characterized. We aimed to analyze whether early administration of glutamine could improve survival and protect cardiomyocytes from postcardiac arrest acidosis using animal and cell models. Forty Wistar rats with postcardiac arrest acidosis (blood pH < 7.2 were included. They were divided into study (500 mg/kg L-alanyl-L-glutamine, n=20 and control (normal saline, n=20 groups. Each of the rats received resuscitation. The outcomes were compared between the two groups. In addition, cardiomyocytes derived from human induced pluripotent stem cells were exposed to HBSS with different pH levels (7.3 or 6.5 or to culture medium (control. Apoptosis-related markers and beating function were analyzed. We found that the duration of survival was significantly longer in the study group (p<0.05. In addition, in pH 6.5 or pH 7.3 HBSS buffer, the expression levels of cell stress (p53 and apoptosis (caspase-3, Bcl-xL markers were significantly lower in cardiomyocytes treated with 50 mM L-glutamine than those without L-glutamine (RT-PCR. L-glutamine also increased the beating function of cardiomyocytes, especially at the lower pH level (6.5. More importantly, glutamine decreased cardiomyocyte apoptosis and increased these cells’ beating function at a low pH level.

  17. CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart

    Gomez-Velazquez, Melisa; Badia-Careaga, Claudio; Lechuga-Vieco, Ana Victoria; Nieto-Arellano, Rocio; Rollan, Isabel; Alvarez, Alba; Torroja, Carlos; Caceres, Eva F.; Roy, Anna R.; Galjart, Niels; Sanchez-Cabo, Fatima; Enriquez, Jose Antonio; Gomez-Skarmeta, Jose Luis

    2017-01-01

    Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development. PMID:28846746

  18. CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart.

    Gomez-Velazquez, Melisa; Badia-Careaga, Claudio; Lechuga-Vieco, Ana Victoria; Nieto-Arellano, Rocio; Tena, Juan J; Rollan, Isabel; Alvarez, Alba; Torroja, Carlos; Caceres, Eva F; Roy, Anna R; Galjart, Niels; Delgado-Olguin, Paul; Sanchez-Cabo, Fatima; Enriquez, Jose Antonio; Gomez-Skarmeta, Jose Luis; Manzanares, Miguel

    2017-08-01

    Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development.

  19. SU-F-J-86: Method to Include Tissue Dose Response Effect in Deformable Image Registration

    Zhu, J; Liang, J; Chen, S; Qin, A; Yan, D [Beaumont Health Systeml, Royal Oak, MI (United States)

    2016-06-15

    Purpose: Organ changes shape and size during radiation treatment due to both mechanical stress and radiation dose response. However, the dose response induced deformation has not been considered in conventional deformable image registration (DIR). A novel DIR approach is proposed to include both tissue elasticity and radiation dose induced organ deformation. Methods: Assuming that organ sub-volume shrinkage was proportional to the radiation dose induced cell killing/absorption, the dose induced organ volume change was simulated applying virtual temperature on each sub-volume. Hence, both stress and heterogeneity temperature induced organ deformation. Thermal stress finite element method with organ surface boundary condition was used to solve deformation. Initial boundary correspondence on organ surface was created from conventional DIR. Boundary condition was updated by an iterative optimization scheme to minimize elastic deformation energy. The registration was validated on a numerical phantom. Treatment dose was constructed applying both the conventional DIR and the proposed method using daily CBCT image obtained from HN treatment. Results: Phantom study showed 2.7% maximal discrepancy with respect to the actual displacement. Compared with conventional DIR, subvolume displacement difference in a right parotid had the mean±SD (Min, Max) to be 1.1±0.9(−0.4∼4.8), −0.1±0.9(−2.9∼2.4) and −0.1±0.9(−3.4∼1.9)mm in RL/PA/SI directions respectively. Mean parotid dose and V30 constructed including the dose response induced shrinkage were 6.3% and 12.0% higher than those from the conventional DIR. Conclusion: Heterogeneous dose distribution in normal organ causes non-uniform sub-volume shrinkage. Sub-volume in high dose region has a larger shrinkage than the one in low dose region, therefore causing more sub-volumes to move into the high dose area during the treatment course. This leads to an unfavorable dose-volume relationship for the normal organ

  20. Neomysin inhibits Ca2+-stimulated phosphatidylinositol hydrolysis and protects cultured rat cardiomyocytes from Ca2+-dependent cell injury

    Babson, J.R.; Dougherty, J.M.

    1991-01-01

    Exposure of cultured rat cardiomyocytes to ionomycin and extracellular Ca 2+ leads to a rapid, sustained increase in intracellular free Ca 2+ as monitored by Ca 2+ -dependent phosphorylase a activation and to a subsequent loss of cardiomyocyte viability as determined by lactate dehydrogenase (LDH) leakage. The intracellular free Ca 2+ increase coincided with a rapid hydrolysis of phosphatidylinositol that preceded cell death. Phosphatidylinositol hydrolysis was monitored by the release of radiolabeled phosphoinositides from cardiomyocytes prelabeled with [2- 3 H]-myo-inositol. Neomycin, a known inhibitor of phospholipase C, inhibited the phosphatidylinositol hydrolysis and markedly reduced the extent of cell injury. Inhibitors of other Ca 2+ -activated processes, including intracellular proteases and phospholipase A 2 , had no effect on ionomycin-mediated cell injury. These data suggest that ionomycin-induced Ca 2+ -dependent cell injury in cultured cardiomyocytes may be due in part to the stimulation of phosphatidylinositol hydrolysis, presumably catalyzed by a Ca 2+ -dependent phospholipase C

  1. Identification and purification of human induced pluripotent stem cell-derived atrial-like cardiomyocytes based on sarcolipin expression.

    Rebecca Josowitz

    Full Text Available The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN. When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.

  2. Optimization of Gluten-Free Tulumba Dessert Formulation Including Corn Flour: Response Surface Methodology Approach

    Yildiz Önder

    2017-03-01

    Full Text Available Tulumba dessert is widely preferred in Turkey; however, it cannot be consumed by celiac patients because it includes gluten. The diversity of gluten-free products should be expanded so that celiac patients may meet their daily needs regularly. In this study, corn flour (CF / potato starch (PS blend to be used in the gluten-free tulumba dessert formulation was optimized using the Response Surface Methodology (RSM. Increasing ratio of PS in the CF-PS led to a decrease in hardness of the dessert and to an increase in expansion, viscosity, adhesiveness, yield of dessert both with and without syrup (P0.05, additionally these desserts had a much higher sensory score compared to the control sample in terms of the overall quality and pore structure (P<0.05.

  3. Cardiomyocyte apoptosis vs autophagy with prolonged doxorubicin treatment: comparison with osteosarcoma cells.

    Tacar, Oktay; Indumathy, Sivanjah; Tan, Mei Lin; Baindur-Hudson, Swati; Friedhuber, Anna M; Dass, Crispin R

    2015-02-01

    Doxorubicin (Dox) is a frontline chemotherapeutic against osteosarcoma (OS) that is plagued by side effects, particularly in the heart. The specific objective of this article is to investigate whether low-dose Dox treatment had pro-autophagic effects in cardiomyocytes as well as osteosarcoma cells. This study characterises apoptotic (Bax) and autophagic (Beclin-1) biomarker levels in human OS and cardiomyocyte cell lines as well as in various tissues when mice are exposed to low (1 mg/kg, thrice weekly) and high (3 mg/kg thrice weekly) dose Dox for a month. There was a decrease in Bax and increase in Beclin-1 in cardiac tissue in the high-dose group. Dox decreased Beclin-1 in the skin and liver, with no clear indication in the stomach, small intestine and testis. At low Dox doses of 10 and 100 nm in cardiomyocytes and OS cells, there is a pro-apoptotic effect, with a quicker response in the 100-nm condition, and a slower but steady increase of a pro-apoptotic response at the lower 10-nm dose. However, electron microscopy images revealed changes to human OS cells that resembled autophagy. Human prostate, breast and colorectal cells treated with 10-nm Dox showed ∼ 40% reduction in cell viability after 24 h. In culture, cells of both cardiomyocytes and OS revealed a predominant pro-apoptotic response at the expense of autophagy, although both seemed to be occurring in vivo. © 2014 Royal Pharmaceutical Society.

  4. Emergency response facilities including primary and secondary prevention strategies across 79 professional football clubs in England.

    Malhotra, Aneil; Dhutia, Harshil; Gati, Sabiha; Yeo, Tee-Joo; Finocchiaro, Gherardo; Keteepe-Arachi, Tracey; Richards, Thomas; Walker, Mike; Birt, Robin; Stuckey, David; Robinson, Laurence; Tome, Maite; Beasley, Ian; Papadakis, Michael; Sharma, Sanjay

    2017-06-14

    To assess the emergency response planning and prevention strategies for sudden cardiac arrest (SCA) across a wide range of professional football clubs in England. A written survey was sent to all professional clubs in the English football league, namely the Premiership, Championship, League 1 and League 2. Outcomes included: (1) number of clubs performing cardiac screening and frequency of screening; (2) emergency planning and documentation; (3) automated external defibrillator (AED) training and availability; and (4) provision of emergency services at sporting venues. 79 clubs (86%) responded to the survey. 100% clubs participated in cardiac screening. All clubs had AEDs available on match days and during training sessions. 100% Premiership clubs provided AED training to designated staff. In contrast, 30% of lower division clubs with AEDs available did not provide formal training. Most clubs (n=66; 83%) reported the existence of an emergency action plan for SCA but formal documentation was variable. All clubs in the Premiership and League 1 provided an ambulance equipped for medical emergencies on match days compared with 75% of clubs in the Championship and 66% in League 2. The majority of football clubs in England have satisfactory prevention strategies and emergency response planning in line with European recommendations. Additional improvements such as increasing awareness of European guidelines for emergency planning, AED training and mentorship with financial support to lower division clubs are necessary to further enhance cardiovascular safety of athletes and spectators and close the gap between the highest and lower divisions. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  5. Analysis of cardiomyocyte movement in the developing murine heart

    Hashimoto, Hisayuki [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Yuasa, Shinsuke, E-mail: yuasa@a8.keio.jp [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Tabata, Hidenori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Tohyama, Shugo; Seki, Tomohisa; Egashira, Toru; Hayashiji, Nozomi; Hattori, Fumiyuki; Kusumoto, Dai; Kunitomi, Akira; Takei, Makoto; Kashimura, Shin; Yozu, Gakuto; Shimojima, Masaya; Motoda, Chikaaki; Muraoka, Naoto [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Nakajima, Kazunori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Sakaue-Sawano, Asako; Miyawaki, Atsushi [Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Fukuda, Keiichi [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan)

    2015-09-04

    The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle. - Highlights: • We directly visualized cardiomyocyte movement inside the developing murine heart. • Cell cycle related genes were upregulated in the proliferating cardiomyocytes. • Time-lapse imaging revealed that proliferating murine cardiomyocytes stayed in place. • Murine ventricular cardiomyocytes proliferate on site during development.

  6. How to include the variability of TMS responses in simulations: a speech mapping case study

    De Geeter, N.; Lioumis, P.; Laakso, A.; Crevecoeur, G.; Dupré, L.

    2016-11-01

    When delivered over a specific cortical site, TMS can temporarily disrupt the ongoing process in that area. This allows mapping of speech-related areas for preoperative evaluation purposes. We numerically explore the observed variability of TMS responses during a speech mapping experiment performed with a neuronavigation system. We selected four cases with very small perturbations in coil position and orientation. In one case (E) a naming error occurred, while in the other cases (NEA, B, C) the subject appointed the images as smoothly as without TMS. A realistic anisotropic head model was constructed of the subject from T1-weighted and diffusion-weighted MRI. The induced electric field distributions were computed, associated to the coil parameters retrieved from the neuronavigation system. Finally, the membrane potentials along relevant white matter fibre tracts, extracted from DTI-based tractography, were computed using a compartmental cable equation. While only minor differences could be noticed between the induced electric field distributions of the four cases, computing the corresponding membrane potentials revealed different subsets of tracts were activated. A single tract was activated for all coil positions. Another tract was only triggered for case E. NEA induced action potentials in 13 tracts, while NEB stimulated 11 tracts and NEC one. The calculated results are certainly sensitive to the coil specifications, demonstrating the observed variability in this study. However, even though a tract connecting Broca’s with Wernicke’s area is only triggered for the error case, further research is needed on other study cases and on refining the neural model with synapses and network connections. Case- and subject-specific modelling that includes both electromagnetic fields and neuronal activity enables demonstration of the variability in TMS experiments and can capture the interaction with complex neural networks.

  7. Facilitated ethanol metabolism promotes cardiomyocyte contractile dysfunction through autophagy in murine hearts.

    Guo, Rui; Hu, Nan; Kandadi, Machender R; Ren, Jun

    2012-04-01

    Chronic drinking leads to myocardial contractile dysfunction where ethanol metabolism plays an essential role. Acetaldehyde, the main ethanol metabolite, mediates alcohol-induced cell injury although the underlying mechanism is still elusive. This study was designed to examine the mechanism involved in accelerated ethanol metabolism-induced cardiac defect with a focus on autophagy. Wild-type FVB and cardiac-specific overexpression of alcohol dehydrogenase mice were placed on a 4% nutrition-balanced alcohol diet for 8 weeks. Myocardial histology, immunohistochemistry, autophagy markers and signal molecules were examined. Expression of micro RNA miR-30a, a potential target of Beclin 1, was evaluated by real-time PCR. Chronic alcohol intake led to cardiac acetaldehyde accumulation, hypertrophy and overt autophagosome accumulation (LC3-II and Atg7), the effect of which was accentuated by ADH. Signaling molecules governing autophagy initiation including class III PtdIns3K, phosphorylation of mTOR and p70S6K were enhanced and dampened, respectively, following alcohol intake. These alcohol-induced signaling responses were augmented by ADH. ADH accentuated or unmasked alcohol-induced downregulation of Bcl-2, Bcl-xL and MiR-30a. Interestingly, ADH aggravated alcohol-induced p62 accumulation. Autophagy inhibition using 3-MA abolished alcohol-induced cardiomyocyte contractile anomalies. Moreover, acetaldehyde led to cardiomyocyte contractile dysfunction and autophagy induction, which was ablated by 3-MA. Ethanol or acetaldehyde increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by 3-MA but unaffected by lysosomal inhibition using bafilomycin A(1), E64D and pepstatin A. In summary, these data suggested that facilitated acetaldehyde production via ADH following alcohol intake triggered cardiac autophagosome formation along with impaired lysosomal degradation, en route to myocardial defect.

  8. Directed Differentiation of Zebrafish Pluripotent Embryonic Cells to Functional Cardiomyocytes

    Yao Xiao

    2016-09-01

    Full Text Available A cardiomyocyte differentiation in vitro system from zebrafish embryos remains to be established. Here, we have determined pluripotency window of zebrafish embryos by analyzing their gene-expression patterns of pluripotency factors together with markers of three germ layers, and have found that zebrafish undergoes a very narrow period of pluripotency maintenance from zygotic genome activation to a brief moment after oblong stage. Based on the pluripotency and a combination of appropriate conditions, we established a rapid and efficient method for cardiomyocyte generation in vitro from primary embryonic cells. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes. Notably, these in vitro generated cardiomyocytes exhibited typical contractile kinetics and electrophysiological features. The system provides a new paradigm of cardiomyocyte differentiation from primary embryonic cells in zebrafish. The technology provides a new platform for the study of heart development and regeneration, in addition to drug discovery, disease modeling, and assessment of cardiotoxic agents.

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  1. File list: Pol.CDV.50.AllAg.Cardiomyocytes [Chip-atlas[Archive

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    Full Text Available NoD.CDV.50.AllAg.Cardiomyocytes mm9 No description Cardiovascular Cardiomyocytes ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.CDV.50.AllAg.Cardiomyocytes.bed ...

  4. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes.

    Helen P McWilliams-Koeppen

    Full Text Available Light chain (AL amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(PH-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

  5. Cardiomyocyte Overexpression of FABP4 Aggravates Pressure Overload-Induced Heart Hypertrophy.

    Ji Zhang

    Full Text Available Fatty acid binding protein 4 (FABP4 is a member of the intracellular lipid-binding protein family, responsible for the transportation of fatty acids. It is considered to express mainly in adipose tissues, and be strongly associated with inflammation, obesity, diabetes and cardiovasculardiseases. Here we report that FABP4 is also expressed in cardiomyocytes and plays an important role in regulating heart function under pressure overload. We generated heart-specific transgenic FABP4 (FABP4-TG mice using α myosin-heavy chain (α-MHC promoter and human FABP4 sequence, resulting in over-expression of FABP4 in cardiomyocytes. The FABP4-TG mice displayed normal cardiac morphology and contractile function. When they were subjected to the transverse aorta constriction (TAC procedure, the FABP4-TG mice developed more cardiac hypertrophy correlated with significantly increased ERK phosphorylation, compared with wild type controls. FABP4 over-expression in cardiomyocytes activated phosphor-ERK signal and up-regulate the expression of cardiac hypertrophic marker genes. Conversely, FABP4 induced phosphor-ERK signal and hypertrophic gene expressions can be markedly inhibited by an ERK inhibitor PD098059 as well as the FABP4 inhibitor BMS309403. These results suggest that FABP4 over-expression in cardiomyocytes can aggravate the development of cardiac hypertrophy through the activation of ERK signal pathway.

  6. Inflammatory and mitochondrial gene expression data in GPER-deficient cardiomyocytes from male and female mice

    Hao Wang

    2017-02-01

    Full Text Available We previously showed that cardiomyocyte-specific G protein-coupled estrogen receptor (GPER gene deletion leads to sex-specific adverse effects on cardiac structure and function; alterations which may be due to distinct differences in mitochondrial and inflammatory processes between sexes. Here, we provide the results of Gene Set Enrichment Analysis (GSEA based on the DNA microarray data from GPER-knockout versus GPER-intact (intact cardiomyocytes. This article contains complete data on the mitochondrial and inflammatory response-related gene expression changes that were significant in GPER knockout versus intact cardiomyocytes from adult male and female mice. The data are supplemental to our original research article “Cardiomyocyte-specific deletion of the G protein-coupled estrogen receptor (GPER leads to left ventricular dysfunction and adverse remodeling: a sex-specific gene profiling” (Wang et al., 2016 [1]. Data have been deposited to the Gene Expression Omnibus (GEO database repository with the dataset identifier GSE86843.

  7. Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes

    Susann Björk

    2017-11-01

    paced beating rates (1, 2 Hz. Cumulating doses of E-4031 produced prolonged APDs, followed by EADs and drug-induced quiescence. These observations were corroborated by patch clamp and contractility measurements. Similar responses, although more modest were seen with the IKs potassium channel blocker JNJ-303. In conclusion, optogenetic measurements of AP waveforms combined with optical pacing compare well with the patch clamp gold standard. Combined with video motion contractile measurements, optogenetic imaging provides an appealing alternative for electrophysiological screening of human cardiomyocyte responses in pharmacological efficacy and safety testings.

  8. Airfoil wake and linear theory gust response including sub and superresonant flow conditions

    Henderson, Gregory H.; Fleeter, Sanford

    1992-01-01

    The unsteady aerodynamic gust response of a high solidity stator vane row is examined in terms of the fundamental gust modeling assumptions with particular attention given to the effects near an acoustic resonance. A series of experiments was performed with gusts generated by rotors comprised of perforated plates and airfoils. It is concluded that, for both the perforated plate and airfoil wake generated gusts, the unsteady pressure responses do not agree with the linear-theory gust predictions near an acoustic resonance. The effects of the acoustic resonance phenomena are clearly evident on the airfoil surface unsteady pressure responses. The transition of the measured lift coefficients across the acoustic resonance from the subresonant regime to the superresonant regime occurs in a simple linear fashion.

  9. Seismic response of nuclear reactors in layered liquefiable soil deposits including nonlinear soil-structure interaction

    Zaman, M.; Mamoon, S.M.

    1989-01-01

    Analysis of seismic response of structures located at a site with potential for soil liquefaction has drawn attention of many researchers. The topic is particularly important in the design of critical facilities like nuclear reactors and defense installations. This paper presents the results of a study involving evaluation of coupled seismic response of structures (model nuclear reactors) and characteristics of soil liquefaction at a site. The analysis procedure employed is based on the nonlinear finite element (FE) technique and accounts for the interaction effects due to a neighboring structure. Emphasis is given to the following features: prediction of spatial and temporal variation of pore water pressure; identification of the on-set of liquefaction based on the effective stress approach, and tracing the propagation of the liquefied zones with time and resulting response of the structures

  10. Mitochondria Play a Central Role in Nonischemic Cardiomyocyte Necrosis: Common to Acute and Chronic Stressor States

    Khan, M. Usman; Cheema, Yaser; Shahbaz, Atta U.; Ahokas, Robert A.; Sun, Yao; Gerling, Ivan C.; Bhattacharya, Syamal K.; Weber, Karl T.

    2012-01-01

    The survival of cardiomyocytes must be ensured as the myocardium adjusts to a myriad of competing physiologic and pathophysiologic demands. A significant loss of these contractile cells, together with their replacement by stiff fibrillar collagen in the form of fibrous tissue accounts for a transition from a usually efficient muscular pump into one that is failing. Cellular and subcellular mechanisms involved in the pathogenic origins of cardiomyocyte cell death have long been of interest. This includes programmed molecular pathways to either necrosis or apoptosis which are initiated from ischemic or nonischemic origins. Herein we focus on the central role played by a mitochondriocentric signal-transducer-effector pathway to nonischemic cardiomyocyte necrosis which is common to acute and chronic stressor states. We begin by building upon the hypothesis advanced by Albrecht Fleckenstein and coworkers some 40 years ago based on the importance of calcitropic hormone- mediated intracellular Ca2+ overloading which predominantly involves subsarcolemmal mitochondria and is the signal to pathway activation. Other pathway components, which came to be recognized in subsequent years, include the induction of oxidative stress and opening of the mitochondrial inner membrane permeability transition pore. The ensuing loss of cardiomyocytes and consequent replacement fibrosis, or scarring, represents a disease of adaptation and a classic example of when homeostasis begets dyshomeostasis. PMID:22328074

  11. The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold, and heat

    Nakashima, Kazuo; Yamaguchi-Shinozaki, Kazuko; Shinozaki, Kazuo

    2014-01-01

    Drought negatively impacts plant growth and the productivity of crops around the world. Understanding the molecular mechanisms in the drought response is important for improvement of drought tolerance using molecular techniques. In plants, abscisic acid (ABA) is accumulated under osmotic stress conditions caused by drought, and has a key role in stress responses and tolerance. Comprehensive molecular analyses have shown that ABA regulates the expression of many genes under osmotic stress cond...

  12. Subjective Response to Foot-Fall Noise, Including Localization of the Source Position

    Brunskog, Jonas; Hwang, Ha Dong; Jeong, Cheol-Ho

    2011-01-01

    annoyance, using simulated binaural room impulse responses, with sources being a moving point source or a nonmoving surface source, and rooms being a room with a reverberation time of 0.5 s or an anechoic room. The paper concludes that no strong effect of the source localization on the annoyance can...

  13. Simulated selection responses for breeding programs including resistance and resilience to parasites in Creole goats

    Gunia, M.; Phocas, F.; Gourdine, J.L.; Bijma, P.; Mandonnet, N.

    2013-01-01

    The Creole goat is a local breed used for meat production in Guadeloupe (French West Indies). As in other tropical countries, improvement of parasite resistance is needed. In this study, we compared predicted selection responses for alternative breeding programs with or without parasites resistance

  14. Hybridising Sport Education and Teaching for Personal and Social Responsibility to Include Students with Disabilities

    Menendez, Jose Ignacio; Fernandez-Rio, Javier

    2017-01-01

    The present study aimed to explore the impact of the combination of two pedagogical models, Sport Education and Teaching for Personal and Social Responsibility, for learners with disabilities experiencing a contactless kickboxing learning unit. Twelve secondary education students agreed to participate. Five had disabilities (intellectual and…

  15. A two-dimensional simulation model of phosphorus uptake including crop growth and P-response

    Mollier, A.; Willigen, de P.; Heinen, M.; Morel, C.; Schneider, A.; Pellerin, S.

    2008-01-01

    Modelling nutrient uptake by crops implies considering and integrating the processes controlling the soil nutrient supply, the uptake by the root system and relationships between the crop growth response and the amount of nutrient absorbed. We developed a model that integrates both dynamics of maize

  16. Habituation of the cold shock response may include a significant perceptual component.

    Barwood, Martin J; Corbett, Jo; Wagstaff, Christopher R D

    2014-02-01

    Accidental immersion in cold water is a risk factor for many occupations. Habituation to cold-water immersion (CWI) is one practical means of reducing the cold shock response (CSR) on immersion. We investigated whether repeated thermoneutral water immersion (TWI) induced a perceptual habituation (i.e., could lessen perceived threat and anxiety) and consequently reduce the CSR on subsequent CWI. There were 12 subjects who completed seven 7-min head-out immersions. Immersions one and seven were CWls [15.0 (0.1) degrees C], and immersions two to six were TWI [34.9 (0.10) degrees C]. Anxiety 120-cm visual analogue scale) and the cardiorespiratory responses [heart rate (f(C)), respiratory frequency (f(R)), tidal volume (V(T)), and minute ventilation (V(E))] to immersion were measured throughout. Data were compared within subject between conditions using ANOVA to an alpha level of 0.05. Acute anxiety was significantly reduced after repeated exposure to the immersion scenario (i.e., TWI): CWI-1: 6.3 (4.4) cm; and CWI-2: 4.5 (4.0) cm [condition mean (SD)]. These differences did not influence the peak in the CSR. The f(C), f(R), and V(E) responses were similar between CWI-1 and CWI-2. V(T) response was significantly lower in CWI-2; mean (SD) across the immersion: CWI-1 1.27 (0.17) vs. CWI-2 1.11 0.21 L. Repeated TWI lessened the anxiety associated with CWI (perceptual habituation). This had a negligible effect on the primary components of the CSR, but did lower VT, which may reduce the volume of any aspirated water in an emergency situation. Reducing the threat appraisal of an environmental stressor may be a useful biproduct of survival training, thereby minimizing psychophysiological strain.

  17. CORPORATE SOCIAL RESPONSIBILITY AS CONDITION OF INCLUDING UKRAINE IN EUROPE AND WORLD ECONOMIC SPACE

    T. Lytvynenko

    2013-09-01

    Full Text Available The thesis that companies’ activities for introduction of corporate social responsibility stimulates the speed of to the processes of the technical upgrade, modernization of company’s activity and increase of its profitability is proved within the article. Those Ukrainian companies, which have high index of activities transparency, are also the most profitable. However, we can’t observe any significant increment of number of companies joining the Global agreement. One of the explanations we could name is the unproved idea supported by some politicians and economists about a shadow (‘black’ market that allegedly allows creating workplaces and taking off social tension in society on the certain stage. Insignificant values of index of citizens’ trust to activity of industries holds on the development socially of responsible business. Trust considered as a part of the general social capital. The Government of Ukraine must support initiative of companies to introduce social responsibility of business, as many European governments do it. It is also important to inform society of advantages of CSR.

  18. The soluble guanylyl cyclase activator bay 58-2667 selectively limits cardiomyocyte hypertrophy.

    Jennifer C Irvine

    Full Text Available Although evidence now suggests cGMP is a negative regulator of cardiac hypertrophy, the direct consequences of the soluble guanylyl cyclase (sGC activator BAY 58-2667 on cardiac remodeling, independent of changes in hemodynamic load, has not been investigated. In the present study, we tested the hypothesis that the NO(•-independent sGC activator BAY 58-2667 inhibits cardiomyocyte hypertrophy in vitro. Concomitant impact of BAY 58-2667 on cardiac fibroblast proliferation, and insights into potential mechanisms of action, were also sought. Results were compared to the sGC stimulator BAY 41-2272.Neonatal rat cardiomyocytes were incubated with endothelin-1 (ET(1, 60nmol/L in the presence and absence of BAY 41-2272 and BAY 58-2667 (0.01-0.3 µmol/L. Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. The impact of both sGC ligands on basal and stimulated cardiac fibroblast proliferation in vitro was also determined.We now demonstrate that BAY 58-2667 (0.01-0.3 µmol/L elicited concentration-dependent antihypertrophic actions, inhibiting ET(1-mediated increases in cardiomyocyte 2D area and de novo protein synthesis, as well as suppressing ET(1-induced cardiomyocyte superoxide generation. This was accompanied by potent increases in cardiomyocyte cGMP accumulation and activity of its downstream signal, vasodilator-stimulated phosphoprotein (VASP, without elevating cardiomyocyte cAMP. In contrast, submicromolar concentrations of BAY 58-2667 had no effect on basal or stimulated cardiac fibroblast proliferation. Indeed, only at concentrations ≥10 µmol/L was inhibition of cardiac fibrosis seen in vitro. The effects of BAY 58-2667 in both cell types were mimicked by BAY 41-2272.Our results demonstrate that BAY 58-2667 elicits protective, cardiomyocyte-selective effects in vitro. These actions are associated with sGC activation and are evident in the absence of confounding hemodynamic factors, at low (submicromolar

  19. Effects of embedment including slip and separation on seismic SSI response of a nuclear reactor building

    Saxena, Navjeev; Paul, D.K.

    2012-01-01

    Highlights: ► Both the slip and separation of reactor base reduce with increase in embedment. ► The slip and separation become insignificant beyond 1/4 and 1/2 embedment respectively. ► The stresses in reactor reduce significantly upto 1/4 embedment. ► The stress reduction with embedment is more pronounced in case of tensile stresses. ► The modeling of interface is important beyond 1/8 embedment as stresses are underestimated otherwise. - Abstract: The seismic response of nuclear reactor containment building considering the effects of embedment, slip and separation at soil–structure interface requires modeling of the soil, structure and interface altogether. Slip and separation at the interface causes stress redistribution in the soil and the structure around the interface. The embedment changes the dynamic characteristics of the soil–structure system. Consideration of these aspects allows capturing the realistic response of the structure, which has been a research gap and presented here individually as well as taken together. Finite element analysis has been carried out in time domain to attempt the highly nonlinear problem. The study draws important conclusions useful for design of nuclear reactor containment building.

  20. Seismic response and fragility evaluation for an Eastern US NPP including soil-structure interaction effects

    Ghiocel, Dan M.; Wilson, Paul R.; Thomas, Gary G.; Stevenson, John D.

    1998-01-01

    The paper discusses methodological aspects involved in a probabilistic seismic soil-structure interaction (SSI) analysis for a Seismic Probabilistic Risk Assessment (SPRA) review. An example of an Eastern US nuclear power plant (NPP) is presented. The approach presented herein follows the current practice of the Individual Plant Examination for External Events (IPEEE) program in the US. The NPP is founded on a relatively soft soil deposit, and thus the SSI effects on seismic responses are significant. Probabilistic models used for the idealization of the seismic excitation and the surrounding soil deposit are described. Using a lognormal format, computed random variability effects were combined with those proposed in the SPRA methodology guidelines. Probabilistic floor response spectra and structural fragilities for different NPP buildings were computed. Structural capacities were determined following the current practice which assumes independent median safety factors for strength and inelastic absorption. Limitations of the IPEEE practice for performing SPRA are discussed and alternate procedures, more rigorous and simple to implement, are suggested

  1. Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis.

    Qiu, Xiao-Xu; Liu, Yang; Zhang, Yi-Fan; Guan, Ya-Na; Jia, Qian-Qian; Wang, Chen; Liang, He; Li, Yong-Qin; Yang, Huang-Tian; Qin, Yong-Wen; Huang, Shuang; Zhao, Xian-Xian; Jing, Qing

    2017-10-02

    Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional

  2. Integrated outage management: Leveraging utility system assets including GIS and AMR for optimum outage response

    Finamore, E. P.

    2004-02-01

    The control of electrical system outages is discussed. The principal argument advanced is that traditional stand-alone methods of outage response will no longer get the job done without utility companies integrating their outage management systems with other system assets such as GIS (geographic information system) and AMR (advanced metering systems). Many meter reading systems, while primarily supporting customer billing, can also provide outage alarm and some are also capable of service restoration notification, which is an invaluable benefit to service operators since it obviates the need for verifying system restoration by labour-intensive on-site visits or customer call-backs. If successfully leveraged, optimization of all utility assets and improvements in labour productivity can results in improved outage management performance gains without affecting performance in other areas.

  3. SOX6 and PDCD4 enhance cardiomyocyte apoptosis through LPS-induced miR-499 inhibition.

    Jia, Zhuqing; Wang, Jiaji; Shi, Qiong; Liu, Siyu; Wang, Weiping; Tian, Yuyao; Lu, Qin; Chen, Ping; Ma, Kangtao; Zhou, Chunyan

    2016-02-01

    Sepsis-induced cardiac apoptosis is one of the major pathogenic factors in myocardial dysfunction. As it enhances numerous proinflammatory factors, lipopolysaccharide (LPS) is considered the principal mediator in this pathological process. However, the detailed mechanisms involved are unclear. In this study, we attempted to explore the mechanisms involved in LPS-induced cardiomyocyte apoptosis. We found that LPS stimulation inhibited microRNA (miR)-499 expression and thereby upregulated the expression of SOX6 and PDCD4 in neonatal rat cardiomyocytes. We demonstrate that SOX6 and PDCD4 are target genes of miR-499, and they enhance LPS-induced cardiomyocyte apoptosis by activating the BCL-2 family pathway. The apoptosis process enhanced by overexpression of SOX6 or PDCD4, was rescued by the cardiac-abundant miR-499. Overexpression of miR-499 protected the cardiomyocytes against LPS-induced apoptosis. In brief, our results demonstrate the existence of a miR-499-SOX6/PDCD4-BCL-2 family pathway in cardiomyocytes in response to LPS stimulation.

  4. Inhibition of MMP-2 Expression with siRNA Increases Baseline Cardiomyocyte Contractility and Protects against Simulated Ischemic Reperfusion Injury

    Han-Bin Lin

    2014-01-01

    Full Text Available Matrix metalloproteinases (MMPs significantly contribute to ischemia reperfusion (I/R injury, namely, by the degradation of contractile proteins. However, due to the experimental models adopted and lack of isoform specificity of MMP inhibitors, the cellular source and identity of the MMP(s involved in I/R injury remain to be elucidated. Using isolated adult rat cardiomyocytes, subjected to chemically induced I/R-like injury, we show that specific inhibition of MMP-2 expression and activity using MMP-2 siRNA significantly protected cardiomyocyte contractility from I/R-like injury. This was also associated with increased expression of myosin light chains 1 and 2 (MLC1/2 in comparison to scramble siRNA transfection. Moreover, the positive effect of MMP-2 siRNA transfection on cardiomyocyte contractility and MLC1/2 expression levels was also observed under control conditions, suggesting an important additional role for MMP-2 in physiological sarcomeric protein turnover. This study clearly demonstrates that intracellular expression of MMP-2 plays a significant role in sarcomeric protein turnover, such as MLC1 and MLC2, under aerobic (physiological conditions. In addition, this study identifies intracellular/autocrine, cardiomyocyte-produced MMP-2, rather than paracrine/extracellular, as responsible for the degradation of MLC1/2 and consequent contractile dysfunction in cardiomyocytes subjected to I/R injury.

  5. Differential Gene Expression Patterns in Chicken Cardiomyocytes during Hydrogen Peroxide-Induced Apoptosis.

    Wan, Chunyun; Xiang, Jinmei; Li, Youwen; Guo, Dingzong

    2016-01-01

    Hydrogen peroxide (H2O2) is both an exogenous and endogenous cytotoxic agent that can reliably induce apoptosis in numerous cell types for studies on apoptosis signaling pathways. However, little is known of these apoptotic processes in myocardial cells of chicken, a species prone to progressive heart failure. Sequencing of mRNA transcripts (RNA-Seq) allows for the identification of differentially expressed genes under various physiological and pathological conditions to elucidate the molecular pathways involved, including cellular responses to exogenous and endogenous toxins. We used RNA-seq to examine genes differentially expressed during H2O2-induced apoptosis in primary cultures of embryonic chicken cardiomyocytes. Following control or H2O2 treatment, RNA was extracted and sequencing performed to identify novel transcripts up- or downregulated in the H2O2 treatment group and construct protein-protein interaction networks. Of the 19,268 known and 2,160 novel transcripts identified in both control and H2O2 treatment groups, 4,650 showed significant differential expression. Among them, 55.63% were upregulated and 44.37% downregulated. Initiation of apoptosis by H2O2 was associated with upregulation of caspase-8, caspase-9, and caspase-3, and downregulation of anti-apoptotic genes API5 and TRIA1. Many other differentially expressed genes were associated with metabolic pathways (including 'Fatty acid metabolism', 'Alanine, aspartate, and glutamate metabolism', and 'Biosynthesis of unsaturated fatty acids') and cell signaling pathways (including 'PPAR signaling pathway', 'Adipocytokine signaling pathway', 'TGF-beta signaling pathway', 'MAPK signaling pathway', and 'p53 signaling pathway'). In chicken cardiomyocytes, H2O2 alters the expression of numerous genes linked to cell signaling and metabolism as well as genes directly associated with apoptosis. In particular, H2O2 also affects the biosynthesis and processing of proteins and unsaturated fatty acids. These

  6. Acoustical sensing of cardiomyocyte cluster beating

    Tymchenko, Nina; Kunze, Angelika [Dept. of Applied Physics, Chalmers University of Technology, 412 96 Göteborg (Sweden); Dahlenborg, Kerstin [Cellectis, 413 46 Göteborg (Sweden); Svedhem, Sofia, E-mail: sofia.svedhem@chalmers.se [Dept. of Applied Physics, Chalmers University of Technology, 412 96 Göteborg (Sweden); Steel, Daniella [Cellectis, 413 46 Göteborg (Sweden)

    2013-06-14

    Highlights: •An example of the application of QCM-D to live cell studies. •Detection of human pluripotent stem cell-derived cardiomyocyte cluster beating. •Clusters were studied in a thin liquid film and in a large liquid volume. •The QCM-D beating profile provides an individual fingerprint of the hPS-CMCs. -- Abstract: Spontaneously beating human pluripotent stem cell-derived cardiomyocytes clusters (CMCs) represent an excellent in vitro tool for studies of human cardiomyocyte function and for pharmacological cardiac safety assessment. Such testing typically requires highly trained operators, precision plating, or large cell quantities, and there is a demand for real-time, label-free monitoring of small cell quantities, especially rare cells and tissue-like structures. Array formats based on sensing of electrical or optical properties of cells are being developed and in use by the pharmaceutical industry. A potential alternative to these techniques is represented by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, which is an acoustic surface sensitive technique that measures changes in mass and viscoelastic properties close to the sensor surface (from nm to μm). There is an increasing number of studies where QCM-D has successfully been applied to monitor properties of cells and cellular processes. In the present study, we show that spontaneous beating of CMCs on QCM-D sensors can be clearly detected, both in the frequency and the dissipation signals. Beating rates in the range of 66–168 bpm for CMCs were detected and confirmed by simultaneous light microscopy. The QCM-D beating profile was found to provide individual fingerprints of the hPS-CMCs. The presented results point towards acoustical assays for evaluation cardiotoxicity.

  7. A predator-prey model with a holling type I functional response including a predator mutual interference

    Seo, G.; DeAngelis, D.L.

    2011-01-01

    The most widely used functional response in describing predator-prey relationships is the Holling type II functional response, where per capita predation is a smooth, increasing, and saturating function of prey density. Beddington and DeAngelis modified the Holling type II response to include interference of predators that increases with predator density. Here we introduce a predator-interference term into a Holling type I functional response. We explain the ecological rationale for the response and note that the phase plane configuration of the predator and prey isoclines differs greatly from that of the Beddington-DeAngelis response; for example, in having three possible interior equilibria rather than one. In fact, this new functional response seems to be quite unique. We used analytical and numerical methods to show that the resulting system shows a much richer dynamical behavior than the Beddington-DeAngelis response, or other typically used functional responses. For example, cyclic-fold, saddle-fold, homoclinic saddle connection, and multiple crossing bifurcations can all occur. We then use a smooth approximation to the Holling type I functional response with predator mutual interference to show that these dynamical properties do not result from the lack of smoothness, but rather from subtle differences in the functional responses. ?? 2011 Springer Science+Business Media, LLC.

  8. Polycystin-2-dependent control of cardiomyocyte autophagy.

    Criollo, Alfredo; Altamirano, Francisco; Pedrozo, Zully; Schiattarella, Gabriele G; Li, Dan L; Rivera-Mejías, Pablo; Sotomayor-Flores, Cristian; Parra, Valentina; Villalobos, Elisa; Battiprolu, Pavan K; Jiang, Nan; May, Herman I; Morselli, Eugenia; Somlo, Stefan; de Smedt, Humbert; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

    2018-05-01

    Considerable evidence points to critical roles of intracellular Ca 2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca 2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca 2+ homeostasis and autophagy. Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2 F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca 2+ chelation using BAPTA-AM, whereas removal of extracellular Ca 2+ had no effect, pointing to a role of intracellular Ca 2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca 2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca 2+ -channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca 2+ . Furthermore, PC2 ablation was associated with impaired Ca 2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca 2+ stores. Finally, we provide evidence that Ca 2+ -mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. Together, this study unveils PC2 as a novel regulator of autophagy acting

  9. The Response of Tigriopus californicusto Chlorophytic Macroalgae, Including Cladophora trichotomaKützing

    Powlik, J. J.; Lewis, A. G.; Verma, N.

    1997-03-01

    The harpacticoid copepod Tigriopus californicusis not observed to maintain populations in supralittoral pools containing the alga Cladophora trichotoma. Laboratory microcosms and field treatments in Barkley Sound, British Columbia, Canada were inoculated with T. californicusand monitored seasonally (winter, spring, summer). After 5 days, C. trichotomamicrocosms held fewer survivors (18·6±7·3%) compared to treatments without vegetation (95·6±0·1%) or those containing the alga Enteromorpha compressa(93·8±5·4%), mean±SE. Though more difficult to determine in situ, results from inoculated field sites were similar. Copepodite and adult T. californicuswere the most adversely affected, while the survival of nauplii was generally maintained over 5-day intervals of observation. Other invertebrates, including littorines, barnacles and mussels were more abundant in C. trichotomapools at lower tidal elevations. Although several factors (exposure, nutrient supply, predation) are likely to contribute to the observed ' exclusion ' of T. californicusfrom pools containing C. trichotoma, the present results additionally suggest the deleterious influence of an unidentified chemical exudate from C. trichotoma. The particular susceptibility of mature T. californicusto this exudate precludes the establishment of viable copepod populations in these pools. 1997 Academic Press Limited

  10. Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

    Birkedal Rikke

    2009-12-01

    Full Text Available Abstract Background Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss, which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine. Results Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. Conclusions The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that

  11. The location of energetic compartments affects energetic communication in cardiomyocytes

    Rikke eBirkedal

    2014-09-01

    Full Text Available The heart relies on accurate regulation of mitochondrial energy supply to match energy demand. The main regulators are Ca2+ and feedback of ADP and Pi. Regulation via feedback has intrigued for decades. First, the heart exhibits a remarkable metabolic stability. Second, diffusion of ADP and other molecules is restricted specifically in heart and red muscle, where a fast feedback is needed the most. To explain the regulation by feedback, compartmentalization must be taken into account. Experiments and theoretical approaches suggest that cardiomyocyte energetic compartmentalization is elaborate with barriers obstructing diffusion in the cytosol and at the level of the mitochondrial outer membrane (MOM. A recent study suggests the barriers are organized in a lattice with dimensions in agreement with those of intracellular structures. Here, we discuss the possible location of these barriers. The more plausible scenario includes a barrier at the level of MOM. Much research has focused on how the permeability of MOM itself is regulated, and the importance of the creatine kinase system to facilitate energetic communication. We hypothesize that at least part of the diffusion restriction at the MOM level is not by MOM itself, but due to the close physical association between the sarcoplasmic reticulum (SR and mitochondria. This will explain why animals with a disabled creatine kinase system exhibit rather mild phenotype modifications. Mitochondria are hubs of energetics, but also ROS production and signaling. The close association between SR and mitochondria may form a diffusion barrier to ADP added outside a permeabilised cardiomyocyte. But in vivo, it is the structural basis for the mitochondrial-SR coupling that is crucial for the regulation of mitochondrial Ca2+-transients to regulate energetics, and for avoiding Ca2+-overload and irreversible opening of the mitochondrial permeability transition pore.

  12. A Non-Destructive Culturing and Cell Sorting Method for Cardiomyocytes and Neurons Using a Double Alginate Layer

    Terazono, Hideyuki; Kim, Hyonchol; Hayashi, Masahito; Hattori, Akihiro; Nomura, Fumimasa; Kaneko, Tomoyuki; Yasuda, Kenji

    2012-01-01

    A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES) cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture. PMID:22870332

  13. A non-destructive culturing and cell sorting method for cardiomyocytes and neurons using a double alginate layer.

    Hideyuki Terazono

    Full Text Available A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.

  14. A piezoelectric electrospun platform for in situ cardiomyocyte contraction analysis

    Beringer, Laura Toth

    Flexible, self-powered materials are in demand for a multitude of applications such as energy harvesting, robotic devices, and lab-on-a chip medical diagnostics. Lab-on-a-chip materials or cell-based biosensors can provide new diagnostic or therapeutic tools for numerous diseases. This dissertation explores the fabrication and characterization of a cell-based sensor termed a nanogenerator with three major aims. The first aim of this research was to fabricate a piezoelectric material that could act as both a cell scaffold and sensor and characterize the response to cell-scale deformation. Electrospinning piezoelectric fluoropolymers into nanofibers can provide both of these functionalities in a facile method. PVDF-TrFe was electrospun in an aligned format and interfaced with a flexible plastic substrate in order to create a platform for voltage response characterization after small force cantilever deformations. Voltage peak signals were an average of +/- 0.4 V, and this response did not change after platform sterilization. However, when placed in cell culture media, piezoelectric response was dampened, which was taken into consideration for the next two aims. An aligned electrospun coaxial fiber system of PVDF-TrFe and collagen was created and interfaced with the nanogenerator for the second aim in order to provide a more biologically favorable surface for cells to adhere to. These nanogenerators were successfully characterized for their piezoelectric response, which was an average of +/- 0.1 V. Additionally, the aligned coaxial collagen/PVDF-TrFe fibers supported both neuron and HeLa cell attachment and growth, demonstrating that they were not cytotoxic. To assess the potential for the nanogenerators to be used as a contractile analysis lab-on-a-chip based device, HeLa cell contraction was induced with potassium chloride and signal response was analyzed. The nanogenerator system was able to detect both the resting state of HeLa cells, a contraction state, and a

  15. DPP4 deficiency exerts protective effect against H2O2 induced oxidative stress in isolated cardiomyocytes.

    Hui-Chun Ku

    Full Text Available Apart from the antihyperglycemic effects, DPP4 inhibitors and GLP-1 molecules are involved in the preservation of cardiac functions. We have demonstrated that DPP4-deficient rats possess resistance to endotoxemia and ischemia/reperfusion stress. However, whether the decrease of DPP4 activity simply augmented the GLP-1 signaling or that such decrease resulted in a change of cellular function remain unclear. Accordingly, we investigated the responses of H(2O(2-induced oxidative stress in adult wild-type and DPP4-deficient rats isolated cardiomyocytes. The coadministration of GLP-1 or DPP4 inhibitor was also performed to define the mechanisms. Cell viability, ROS concentration, catalase activity, glucose uptake, prosurvival, proapoptotic signaling, and contractile function were examined after cells exposed to H(2O(2. DPP4-deficient cardiomyocytes were found to be resistant to H(2O(2-induced cell death via activating AKT signaling, enhancing glucose uptake, preserving catalase activity, diminishing ROS level and proapoptotic signaling. GLP-1 concentration-dependently improved cell viability in wild-type cardiomyocyte against ROS stress, and the ceiling response concentration (200 nM was chosen for studies. GLP-1 was shown to decrease H(2O(2-induced cell death by its receptor-dependent AKT pathway in wild-type cardiomyocytes, but failed to cause further activation of AKT in DPP4-deficient cardiomyocytes. Acute treatment of DPP4 inhibitor only augmented the protective effect of low dose GLP-1, but failed to alter fuel utilization or ameliorate cell viability in wild-type cardiomyocytes after H(2O(2 exposure. The improvement of cell viability after H(2O(2 exposure was correlated with the alleviation of cellular contractile dysfunction in both DPP4-deficient and GLP-1 treated wild-type cardiomyocytes. These findings demonstrated that GLP-1 receptor-dependent pathway is important and exert protective effect in wild-type cardiomyocyte. Long term loss of

  16. Differentiation and characterization of rhesus monkey atrial and ventricular cardiomyocytes from induced pluripotent stem cells.

    Zhang, Xiaoqian; Cao, Henghua; Bai, Shuyun; Huo, Weibang; Ma, Yue

    2017-04-01

    The combination of non-human primate animals and their induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) provides not only transplantation models for cell-based therapy of heart diseases, but also opportunities for heart-related drug research on both cellular and animal levels. However, the subtypes and electrophysiology properties of non-human primate iPSC-CMs hadn't been detailed characterized. In this study, we generated rhesus monkey induced pluripotent stem cells (riPSCs), and efficiently differentiated them into ventricular or atrial cardiomyocytes by modulating retinoic acid (RA) pathways. Our results revealed that the electrophysiological characteristics and response to canonical drugs of riPSC-CMs were similar with those of human pluripotent stem cell derived CMs. Therefore, rhesus monkeys and their iPSC-CMs provide a powerful and practicable system for heart related biomedical research. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Effects of 5-fluorouracil on morphology, cell cycle, proliferation, apoptosis, autophagy and ROS production in endothelial cells and cardiomyocytes.

    Chiara Focaccetti

    Full Text Available Antimetabolites are a class of effective anticancer drugs interfering in essential biochemical processes. 5-Fluorouracil (5-FU and its prodrug Capecitabine are widely used in the treatment of several solid tumors (gastro-intestinal, gynecological, head and neck, breast carcinomas. Therapy with fluoropyrimidines is associated with a wide range of adverse effects, including diarrhea, dehydration, abdominal pain, nausea, stomatitis, and hand-foot syndrome. Among the 5-FU side effects, increasing attention is given to cardiovascular toxicities induced at different levels and intensities. Since the mechanisms related to 5-FU-induced cardiotoxicity are still unclear, we examined the effects of 5-FU on primary cell cultures of human cardiomyocytes and endothelial cells, which represent two key components of the cardiovascular system. We analyzed at the cellular and molecular level 5-FU effects on cell proliferation, cell cycle, survival and induction of apoptosis, in an experimental cardioncology approach. We observed autophagic features at the ultrastructural and molecular levels, in particular in 5-FU exposed cardiomyocytes. Reactive oxygen species (ROS elevation characterized the endothelial response. These responses were prevented by a ROS scavenger. We found induction of a senescent phenotype on both cell types treated with 5-FU. In vivo, in a xenograft model of colon cancer, we showed that 5-FU treatment induced ultrastructural changes in the endothelium of various organs. Taken together, our data suggest that 5-FU can affect, both at the cellular and molecular levels, two key cell types of the cardiovascular system, potentially explaining some manifestations of 5-FU-induced cardiovascular toxicity.

  18. Analysis of mitochondrial 3D-deformation in cardiomyocytes during active contraction reveals passive structural anisotropy of orthogonal short axes.

    Yael Yaniv

    Full Text Available The cardiomyocyte cytoskeleton, composed of rigid and elastic elements, maintains the isolated cell in an elongated cylindrical shape with an elliptical cross-section, even during contraction-relaxation cycles. Cardiomyocyte mitochondria are micron-sized, fluid-filled passive spheres distributed throughout the cell in a crystal-like lattice, arranged in pairs sandwiched between the sarcomere contractile machinery, both longitudinally and radially. Their shape represents the extant 3-dimensional (3D force-balance. We developed a novel method to examine mitochondrial 3D-deformation in response to contraction and relaxation to understand how dynamic forces are balanced inside cardiomyocytes. The variation in transmitted light intensity induced by the periodic lattice of myofilaments alternating with mitochondrial rows can be analyzed by Fourier transformation along a given cardiomyocyte axis to measure mitochondrial deformation along that axis. This technique enables precise detection of changes in dimension of ∼1% in ∼1 µm (long-axis structures with 8 ms time-resolution. During active contraction (1 Hz stimulation, mitochondria deform along the length- and width-axes of the cell with similar deformation kinetics in both sarcomere and mitochondrial structures. However, significant deformation anisotropy (without hysteresis was observed between the orthogonal short-axes (i.e., width and depth of mitochondria during electrical stimulation. The same degree of deformation anisotropy was also found between the myocyte orthogonal short-axes during electrical stimulation. Therefore, the deformation of the mitochondria reflects the overall deformation of the cell, and the apparent stiffness and stress/strain characteristics of the cytoskeleton differ appreciably between the two cardiomyocyte orthogonal short-axes. This method may be applied to obtaining a better understanding of the dynamic force-balance inside cardiomyocytes and of changes in the

  19. Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy

    Kiso, Hironori; Ohba, Takayoshi; Iino, Kenji; Sato, Kazuhiro; Terata, Yutaka; Murakami, Manabu; Ono, Kyoichi; Watanabe, Hiroyuki; Ito, Hiroshi

    2013-01-01

    Highlights: •Transient receptor potential canonical (TRPC1, 3 and 6) are up-regulated by ET-1. •Sildenafil inhibited hypertrophic responses (BNP, Ca entry, NFAT activation). •Sildenafil suppressed TRPC1, 3 and 6 expression. -- Abstract: Background: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. Methods and results: In cultured neonatal rat cardiomyocytes, a 48 h treatment with 10 nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. Conclusions: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression

  20. Both cardiomyocyte and endothelial cell Nox4 mediate protection against hemodynamic overload-induced remodelling.

    Zhang, Min; Mongue-Din, Heloise; Martin, Daniel; Catibog, Norman; Smyrnias, Ioannis; Zhang, Xiaohong; Yu, Bin; Wang, Minshu; Brandes, Ralf P; Schröder, Katrin; Shah, Ajay M

    2018-03-01

    NADPH oxidase-4 (Nox4) is an important reactive oxygen species (ROS) source that is upregulated in the haemodynamically overloaded heart. Our previous studies using global Nox4 knockout (Nox4KO) mice demonstrated a protective role of Nox4 during chronic abdominal aortic banding, involving a paracrine enhancement of myocardial capillary density. However, other authors who studied cardiac-specific Nox4KO mice reported detrimental effects of Nox4 in response to transverse aortic constriction (TAC). It has been speculated that these divergent results are due to cell-specific actions of Nox4 (i.e. cardiomyocyte Nox4 detrimental but endothelial Nox4 beneficial) and/or differences in the model of pressure overload (i.e. abdominal banding vs. TAC). This study aimed to (i) investigate whether the effects of Nox4 on pressure overload-induced cardiac remodelling vary according to the pressure overload model and (ii) compare the roles of cardiomyocyte vs. endothelial cell Nox4. Global Nox4KO mice subjected to TAC developed worse cardiac remodelling and contractile dysfunction than wild-type littermates, consistent with our previous results with abdominal aortic banding. Next, we generated inducible cardiomyocyte-specific Nox4 KO mice (Cardio-Nox4KO) and endothelial-specific Nox4 KO mice (Endo-Nox4KO) and studied their responses to pressure overload. Both Cardio-Nox4KO and Endo-Nox4KO developed worse pressure overload-induced cardiac remodelling and dysfunction than wild-type littermates, associated with significant decrease in protein levels of HIF1α and VEGF and impairment of myocardial capillarization. Cardiomyocyte as well as endothelial cell Nox4 contributes to protection against chronic hemodynamic overload-induced cardiac remodelling, at least in part through common effects on myocardial capillary density. © The Author 2017 Published by Oxford University Press on behalf of the European Society of Cardiology.

  1. 30 CFR 254.23 - What information must I include in the “Emergency response action plan” section?

    2010-07-01

    ... response operations on your behalf. You must describe the team's organizational structure as well as the... include the number and types of personnel available from each identified labor source. (d) A planned... primary and secondary radio frequencies that will be used. (e) A listing of the types and characteristics...

  2. Posintro™-HBsAg, a modified ISCOM including HBsAg, induces strong cellular and humoral responses

    Schiött, Asa; Larsson, Kristina; Manniche, Søren

    2011-01-01

    HBsAg vaccine formulation, Posintro™-HBsAg, was compared to two commercial hepatitis B vaccines including aluminium or monophosphoryl lipid A (MPL) and the two adjuvant systems MF59 and QS21 in their efficiency to prime both cellular and humoral immune responses. The Posintro™-HBsAg induced...

  3. An essential role of Nrf2 in American ginseng-mediated anti-oxidative actions in cardiomyocytes.

    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

  4. Generation and purification of human stem cell-derived cardiomyocytes

    Schwach, Verena; Passier, Robert

    2016-01-01

    © 2016 International Society of Differentiation Efficient and reproducible generation and purification of human stem cell-derived cardiomyocytes (CMs) is crucial for regenerative medicine, disease modeling, drug screening and study of developmental events during cardiac specification. Established

  5. Adrenaline in pro-oxidant conditions elicits intracellular survival pathways in isolated rat cardiomyocytes

    Costa, Vera Marisa; Silva, Renata; Ferreira, Rita; Amado, Francisco; Carvalho, Felix; Bastos, Maria Lourdes de; Albuquerque Carvalho, Rui; Carvalho, Marcia; Remiao, Fernando

    2009-01-01

    In several pathologic conditions, like cardiac ischemia/reperfusion, the sustained elevation of plasma and interstitial catecholamine levels, namely adrenaline (ADR), and the generation of reactive oxygen species (ROS) are hallmarks. The present work aimed to investigate in cardiomyocytes which intracellular signalling pathways are altered by ADR redox ability. To mimic pathologic conditions, freshly isolated calcium tolerant cardiomyocytes from adult rat were incubated with ADR alone or in the presence of a system capable of generating ROS [(xanthine with xanthine oxidase) (X/XO)]. ADR elicited a pro-oxidant signal with generation of reactive species, which was largely magnified by the ROS generating system. However, no change in cardiomyocytes viability was observed. The pro-oxidant signal promoted the translocation to the nucleus of the transcription factors, Heat shock factor-1 (HSF-1) and Nuclear factor-κB (NF-κB). In addition, proteasome activity was compromised in the experimental groups where the generation of reactive species occurred. The decrease in the proteasome activity of the ADR group resulted from its redox sensitivity, since the activity was recovered by adding the ROS scavenger, tiron. Proteasome inhibition seemed to elicit an increase in HSP70 levels. Furthermore, retention of mitochondrial cytochrome c and inhibition of caspase 3 activity were observed by X/XO incubation in presence or absence of ADR. In conclusion, in spite of all the insults inflicted to the cardiomyocytes, they were capable to activate intracellular responses that enabled their survival. These mechanisms, namely the pathways altered by catecholamine proteasome inhibition, should be further characterized, as they could be of relevance in the ischemia preconditioning and the reperfusion injury

  6. The Role of the Pulmonary Embolism Response Team: How to Build One, Who to Include, Scenarios, Organization, and Algorithms.

    Galmer, Andrew; Weinberg, Ido; Giri, Jay; Jaff, Michael; Weinberg, Mitchell

    2017-09-01

    Pulmonary embolism response teams (PERTs) are multidisciplinary response teams aimed at delivering a range of diagnostic and therapeutic modalities to patients with pulmonary embolism. These teams have gained traction on a national scale. However, despite sharing a common goal, individual PERT programs are quite individualized-varying in their methods of operation, team structures, and practice patterns. The tendency of such response teams is to become intensely structured, algorithmic, and inflexible. However, in their current form, PERT programs are quite the opposite. They are being creatively customized to meet the needs of the individual institution based on available resources, skills, personnel, and institutional goals. After a review of the essential core elements needed to create and operate a PERT team in any form, this article will discuss the more flexible feature development of the nascent PERT team. These include team planning, member composition, operational structure, benchmarking, market analysis, and rudimentary financial operations. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells.

    Anders Waldenström

    Full Text Available BACKGROUND: Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions. METHODOLOGY/PRINCIPAL FINDINGS: Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls. CONCLUSIONS/SIGNIFICANCE: Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted "cardiosomes", can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes.

  8. The Role of Reactive Oxygen Species in β-Adrenergic Signaling in Cardiomyocytes from Mice with the Metabolic Syndrome.

    Monica Llano-Diez

    Full Text Available The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute β-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS. Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice. We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: β-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to β-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after β-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute β-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.

  9. In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells.

    Szaraz, Peter; Gratch, Yarden S; Iqbal, Farwah; Librach, Clifford L

    2017-08-09

    Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells. Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein α, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. Our results demonstrate that this

  10. Renal hypertension prevents run training modification of cardiomyocyte diastolic Ca2+ regulation in male rats.

    Palmer, B M; Lynch, J M; Snyder, S M; Moore, R L

    2001-06-01

    The combined effects of endurance run training and renal hypertension on cytosolic Ca2+ concentration ([Ca2+]c) dynamics and Na+-dependent Ca2+ regulation in rat left ventricular cardiomyocytes were examined. Male Fischer 344 rats underwent stenosis of the left renal artery [hypertensive (Ht), n = 18] or a sham operation [normotensive (Nt), n = 20]. One-half of the rats from each group were treadmill trained for >16 wk. Cardiomyocyte fura 2 fluorescence ratio transients were recorded for 7 min during electrical pacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29 degrees C. The rate of [Ca2+]c decline was not changed by run training in the Nt group but was reduced in the Ht group. At 7 min, cardiomyocytes were exposed to 10 mM caffeine in the absence of Na+ and Ca2+, which triggered sarcoplasmic reticular Ca2+ release and suppressed Ca2+ efflux via Na+/Ca2+ exchanger. External Na+ was then added, and Na+-dependent Ca2+ efflux rate was recorded. Treadmill training significantly enhanced Na+-dependent Ca2+ efflux rate under these conditions in the Nt group but not in the Ht group. These data provide evidence that renal hypertension prevents the normal run training-induced modifications in diastolic [Ca2+]c regulation mechanisms, including Na+/Ca2+ exchanger.

  11. Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes.

    Christophe M Raynaud

    Full Text Available Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context, we explored the growth and differentiation potential of mesenchymal progenitors (MPs derived in vitro from human embryonic stem cells (hESCs. Similar to MPs isolated from bone marrow, hESC derived MPs (hESC-MPs efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1, hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as NKX2-5, MEF2C, HAND2 and MYOCD. Nevertheless, NKX2-5+ hESC-MP derivatives did not form contractile cardiomyocytes, raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that, although hESC-MP derived cells expressed a suite of cardiac related genes, they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes, but treated cells retain a mesenchymal like phenotype.

  12. Mechanisms of greater cardiomyocyte functions on conductive nanoengineered composites for cardiovascular applications

    Stout DA

    2012-11-01

    Full Text Available David A Stout,1,2 Jennie Yoo,2 Adriana Noemi Santiago-Miranda,3 Thomas J Webster1,41School of Engineering, 2Division of Biology and Medicine, Brown University, Providence, RI, 3Department of Chemical Engineering, University of Puerto Rico, Mayagües, PR, 4Department of Orthopedics, Brown University, Providence, RI, USABackground: Recent advances in nanotechnology (materials with at least one dimension between 1 nm and 100 nm have led to the use of nanomaterials in numerous medical device applications. Recently, nanomaterials have been used to create innovative biomaterials for cardiovascular applications. Specifically, carbon nanofibers (CNF embedded in poly(lactic-co-glycolic-acid (PLGA have been shown to promote cardiomyocyte growth compared with conventional polymer substrates, but the mechanisms involved in such events remain unknown. The aim of this study was to determine the basic mechanism of cell growth on these novel nanocomposites.Methods: CNF were added to biodegradable PLGA (50:50 PGA:PLA weight ratio to increase the conductivity, mechanical and cytocompatibility properties of pure PLGA. For this reason, different PLGA to CNF ratios (100:0, 75:25, 50:50, 25:75, and 0:100 wt% with different PLGA densities (0.1, 0.05, 0.025, and 0.0125 g/mL were used, and their compatibility with cardiomyocytes was assessed.Results: Throughout all the cytocompatibility experiments, cardiomyocytes were viable and expressed important biomarkers, including cardiac troponin T, connexin-43, and alpha-sarcomeric actin (α-SCA. Adhesion and proliferation experiments indicated that a PLGA density of 0.025 g/mL with a PLGA to CNF ratio of 75:25 and 50:50 (wt% promoted the best overall cell growth, ie, a 55% increase in cardiomyocyte density after 120 hours compared with pure PLGA and a 75% increase compared with the control at the same time point for 50:50 (wt%. The PLGA:CNF materials were conductive, and their conductivity increased as greater amounts of CNF

  13. Study on the seismic response of reactor vessel of pool type LMFBR including fluid-structure interaction

    Tanimoto, K.; Ito, T.; Fujita, K.; Kurihara, C.; Sawada, Y.; Sakurai, A.

    1988-01-01

    The paper presents the seismic response of reactor vessel of pool type LMFBR with fluid-structure interaction. The reactor vessel has bottom support arrangement, the same core support system as Super-Phenix in France. Due to the bottom support arrangement, the level of core support is lower than that of the side support arrangement. So, in this reactor vessel, the displacement of the core top tends to increase because of the core's rocking. In this study, we investigated the vibration and seismic response characteristics of the reactor vessel. Therefore, the seismic experiments were carried out using one-eighth scale model and the seismic response including FSI and sloshing were investigated. From this study, the effect of liquid on the vibration characteristics and the seismic response characteristics of reactor vessel were clarified and sloshing characteristics were also clarified. It was confirmed that FEM analysis with FSI can reproduce the seismic behavior of the reactor vessel and is applicable to seismic design of the pool type LMFBR with bottom support arrangement. (author). 5 refs, 14 figs, 2 tabs

  14. Transcriptome analysis of the spalax hypoxia survival response includes suppression of apoptosis and tight control of angiogenesis

    Malik Assaf

    2012-11-01

    Full Text Available Abstract Background The development of complex responses to hypoxia has played a key role in the evolution of mammals, as inadequate response to this condition is frequently associated with cardiovascular diseases, developmental disorders, and cancers. Though numerous studies have used mice and rats in order to explore mechanisms that contribute to hypoxia tolerance, these studies are limited due to the high sensitivity of most rodents to severe hypoxia. The blind subterranean mole rat Spalax is a hypoxia tolerant rodent, which exhibits unique longevity and therefore has invaluable potential in hypoxia and cancer research. Results Using microarrays, transcript abundance was measured in brain and muscle tissues from Spalax and rat individuals exposed to acute and chronic hypoxia for varying durations. We found that Spalax global gene expression response to hypoxia differs from that of rat and is characterized by the activation of functional groups of genes that have not been strongly associated with the response to hypoxia in hypoxia sensitive mammals. Using functional enrichment analysis of Spalax hypoxia induced genes we found highly significant overrepresentation of groups of genes involved in anti apoptosis, cancer, embryonic/sexual development, epidermal growth factor receptor binding, coordinated suppression and activation of distinct groups of transcription factors and membrane receptors, in addition to angiogenic related processes. We also detected hypoxia induced increases of different critical Spalax hub gene transcripts, including antiangiogenic genes associated with cancer tolerance in Down syndrome human individuals. Conclusions This is the most comprehensive study of Spalax large scale gene expression response to hypoxia to date, and the first to use custom Spalax microarrays. Our work presents novel patterns that may underlie mechanisms with critical importance to the evolution of hypoxia tolerance, with special relevance to

  15. A review of sex differences in sexual jealousy, including self-report data, psychophysiological responses, interpersonal violence, and morbid jealousy.

    Harris, Christine R

    2003-01-01

    The specific innate modular theory of jealousy hypothesizes that natural selection shaped sexual jealousy as a mechanism to prevent cuckoldry, and emotional jealousy as a mechanism to prevent resource loss. Therefore, men should be primarily jealous over a mate's sexual infidelity and women over a mate's emotional infidelity. Five lines of evidence have been offered as support: self-report responses, psychophysiological data, domestic violence (including spousal abuse and homicide), and morbid jealousy cases. This article reviews each line of evidence and finds only one hypothetical measure consistent with the hypothesis. This, however, is contradicted by a variety of other measures (including reported reactions to real infidelity). A meta-analysis of jealousy-inspired homicides, taking into account base rates for murder, found no evidence that jealousy disproportionately motivates men to kill. The findings are discussed from a social-cognitive theoretical perspective.

  16. Human Stem Cell Derived Cardiomyocytes: An Alternative ...

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally, a WHO report on the global composite impact of chemicals on health reported that 16% of the total burden of cardiovascular disease was attributed to environmental chemical exposure with 2.5 million deaths per year. Clearly, the cardiovascular system, at all its various developmental and life stages, represents a critical target organ system that can be adversely affected by existing and emerging chemicals (e.g., engineered nanomaterials) in a variety of environmental media. The ability to assess chemical cardiac risk and safety is critically needed but extremely challenging due to the number and categories of chemicals in commerce, as indicated. This presentation\\session will evaluate the use of adult human stem cell derived cardiomyocytes, and existing platforms, as an alternative model to evaluate environmental chemical cardiac toxicity as well as provide key information for the development of predictive adverse outcomes pathways associated with environmental chemical exposures. (This abstract does not represent EPA policy) Rapid and translatable chemical safety screening models for cardiotoxicity current status for informing regulatory decisions, a workshop sponsored by the Society

  17. Wind-induced response analysis of a wind turbine tower including the blade-tower coupling effect

    Xiao-bo CHEN; Jing LI; Jian-yun CHEN

    2009-01-01

    To analyze wind-induced response characteristics of a wind turbine tower more accurately, the blade-tower coupling effect was investigated. The mean wind velocity of the rotating blades and tower was simulated according to wind shear effects,and the fluctuating wind velocity time series of the wind turbine were simulated by a harmony superposition method. A dynamic finite element method (FEM) was used to calculate the wind-induced response of the blades and tower. Wind-induced responses of the tower were calculated in two cases (one included the blade-tower coupling effect, and the other only added the mass of blades and the hub at the top of the tower), and then the maximal displacements at the top of the tower of the tow cases were compared with each other. As a result of the influence of the blade-tower coupling effect and the total base shear of the blades, the maximal displacement of the first case increased nearly by 300% compared to the second case. To obtain more precise analysis, the blade-tower coupling effect and the total base shear of the blades should be considered simultaneously in the design of wind turbine towers.

  18. High Glucose-Induced Cardiomyocyte Death May Be Linked to Unbalanced Branched-Chain Amino Acids and Energy Metabolism

    Xi Zhang

    2018-04-01

    Full Text Available High glucose-induced cardiomyocyte death is a common symptom in advanced-stage diabetic patients, while its metabolic mechanism is still poorly understood. The aim of this study was to explore metabolic changes in high glucose-induced cardiomyocytes and the heart of streptozotocin-induced diabetic rats by 1H-NMR-based metabolomics. We found that high glucose can promote cardiomyocyte death both in vitro and in vivo studies. Metabolomic results show that several metabolites exhibited inconsistent variations in vitro and in vivo. However, we also identified a series of common metabolic changes, including increases in branched-chain amino acids (BCAAs: leucine, isoleucine and valine as well as decreases in aspartate and creatine under high glucose condition. Moreover, a reduced energy metabolism could also be a common metabolic characteristic, as indicated by decreases in ATP in vitro as well as AMP, fumarate and succinate in vivo. Therefore, this study reveals that a decrease in energy metabolism and an increase in BCAAs metabolism could be implicated in high glucose-induced cardiomyocyte death.

  19. Developing an explicit strategy towards social responsibility in the NHS: a case for including NHS managers in this strategy.

    Merali, Faruk

    2006-01-01

    To explore the concept of corporate social responsibility (CSR) within the UK National Health Service (NHS) and to examine how it may be developed to positively influence the psyche, behaviour and performance of NHS managers. Primary research based upon semi-structured individual face to face interviews with 20 NHS managers. Theoretical frameworks and concepts relating to organisational culture and CSR are drawn upon to discuss the findings. The NHS managers see themselves as being driven by altruistic core values. However, they feel that the public does not believe that they share the altruistic NHS value system. The study is based on a relatively small sample of NHS managers working exclusively in London and may not necessarily represent the views of managers either London-wide or nation-wide. It is suggested that an explicit recognition by the NHS of the socially responsible commitment of its managers within its CSR strategy would help challenge the existing negative public image of NHS managers and in turn improve the managers' self esteem and morale. This paper addresses the relative lacunae in research relating to public sector organisations (such as the NHS) explicitly including the role and commitment of its staff within the way it publicises its CSR strategy. This paper would be of interest to a wide readership including public sector and NHS policy formulators, NHS practitioners, academics and students.

  20. Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

    Aguilar, David; Strom, Joshua; Chen, Qin M.

    2014-01-01

    Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes. - Highlights: • Corticosteroids act as a cytoprotective agent in cardiomyocytes • Corticosteroids induce GILZ expression in cardiomyocytes • Elevated GILZ results in resistance against apoptosis induced by doxorubicin • GILZ induces Bcl-xL protein without inducing Bcl-xL mRNA

  1. Factors that contribute to biomarker responses in humans including a study in individuals taking Vitamin C supplementation.

    Anderson, D

    2001-09-01

    It is possible in many situations to identify humans exposed to potentially toxic materials in the workplace and in the environment. As in most human studies, there tends to be a high degree of interindividual variability in response to chemical insults. Some non-exposed control individuals exhibit as high a level of damage as some exposed individuals and some of these have levels of damage as low as many of the controls. Thus, it is only the mean values of the groups that can substantiate an exposure-related problem; the data on an individual basis are still of limited use. While human lymphocytes remain the most popular cell type for monitoring purposes, sperm, buccal, nasal, epithelial and placental cells are also used. However, for interpretation of responses, the issue of confounding factors must be addressed. There are endogenous confounding factors, such as age, gender, and genetic make-up and exogenous ones, including lifestyle habits (smoking, drinking, etc.) There are biomarkers of exposure, effect/response and susceptibility and the last may be influenced by the genotype and polymorphism genes existing in a population. From our own studies, confounding effects on cytogenetic damage and ras oncoproteins will be considered in relation to workers exposed to vinyl chloride and petroleum emissions and to volunteers taking Vitamin C supplementation. Smoking history, exposure and duration of employment affected the worker studies. For petroleum emissions, so did gender and season of exposure. For the non-smoking volunteer Vitamin C supplementation study, cholesterol levels, plasma Vitamin C levels, lipid peroxidation products and DNA damage in the Comet assay were also measured. Gender affected differences in Vitamin C levels, antioxidant capacity and the number of chromosome aberrations induced by bleomycin challenge in vitro. The results were the same for both high and low cholesterol subjects. The relationship between biomarkers and the various factors which

  2. Probe train including a flaw detector and a radiation responsive recording means with alignment means having a natural curved cast

    Stone, R.M.

    1975-01-01

    An inspection system for a multitube steam generator comprising a probe train for insertion in a tube to be inspected is described. The probe train includes, in series, directional probe means, such as an eddy current probe, for indicating the longitudinal and angular location of an irregularity at or in the wall of the tube, and radiation responsive recording means nonrotatable relative to the eddy current probe during operation and in substantially close longitudinal relationship thereto for receiving an image of the irregularity when laterally adjacent thereto; elongated alignment means joined to at least one end of the probe train against rotation relative thereto and insertable in the tube for controlling or determining the angular orientation of the probe train within the tube; means for propelling the probe train longitudinally within the tube; and a source of radiation insertable in another tube of the steam generator to a position therealong laterally adjacent the indicated irregularity for irradiation of the irregularity to project said image on the recording means. The directional probe means may preferably be an eddy current probe and the radiation responsive recording means may preferably be a film bearing cassette probe. The alignment means may be provided by a resilient naturally curved plastic cable, which cable might also be used to propel the probe train. (auth)

  3. A meta-analysis including dose-response relationship between night shift work and the risk of colorectal cancer.

    Wang, Xiao; Ji, Alin; Zhu, Yi; Liang, Zhen; Wu, Jian; Li, Shiqi; Meng, Shuai; Zheng, Xiangyi; Xie, Liping

    2015-09-22

    A meta-analysis was conducted to quantitatively evaluate the correlation between night shift work and the risk of colorectal cancer. We searched for publications up to March 2015 using PubMed, Web of Science, Cochrane Library, EMBASE and the Chinese National Knowledge Infrastructure databases, and the references of the retrieved articles and relevant reviews were also checked. OR and 95% CI were used to assess the degree of the correlation between night shift work and risk of colorectal cancer via fixed- or random-effect models. A dose-response meta-analysis was performed as well. The pooled OR estimates of the included studies illustrated that night shift work was correlated with an increased risk of colorectal cancer (OR = 1.318, 95% CI 1.121-1.551). No evidence of publication bias was detected. In the dose-response analysis, the rate of colorectal cancer increased by 11% for every 5 years increased in night shift work (OR = 1.11, 95% CI 1.03-1.20). In conclusion, this meta-analysis indicated that night shift work was associated with an increased risk of colorectal cancer. Further researches should be conducted to confirm our findings and clarify the potential biological mechanisms.

  4. Mouse preimplantation embryo responses to culture medium osmolarity include increased expression of CCM2 and p38 MAPK activation

    Watson Andrew J

    2007-01-01

    Full Text Available Abstract Background Mechanisms that confer an ability to respond positively to environmental osmolarity are fundamental to ensuring embryo survival during the preimplantation period. Activation of p38 mitogen-activated protein kinase (MAPK occurs following exposure to hyperosmotic treatment. Recently, a novel scaffolding protein called Osmosensing Scaffold for MEKK3 (OSM was linked to p38 MAPK activation in response to sorbitol-induced hypertonicity. The human ortholog of OSM is cerebral cavernous malformation 2 (CCM2. The present study was conducted to investigate whether CCM2 is expressed during mouse preimplantation development and to determine whether this scaffolding protein is associated with p38 MAPK activation following exposure of preimplantation embryos to hyperosmotic environments. Results Our results indicate that Ccm2 along with upstream p38 MAPK pathway constituents (Map3k3, Map2k3, Map2k6, and Map2k4 are expressed throughout mouse preimplantation development. CCM2, MAP3K3 and the phosphorylated forms of MAP2K3/MAP2K6 and MAP2K4 were also detected throughout preimplantation development. Embryo culture in hyperosmotic media increased p38 MAPK activity in conjunction with elevated CCM2 levels. Conclusion These results define the expression of upstream activators of p38 MAPK during preimplantation development and indicate that embryo responses to hyperosmotic environments include elevation of CCM2 and activation of p38 MAPK.

  5. Finite Element Modeling and Analysis of Nonlinear Impact and Frictional Motion Responses Including Fluid—Structure Coupling Effects

    Yong Zhao

    1997-01-01

    Full Text Available A nonlinear three dimensional (3D single rack model and a nonlinear 3D whole pool multi-rack model are developed for the spent fuel storage racks of a nuclear power plant (NPP to determine impacts and frictional motion responses when subjected to 3D excitations from the supporting building floor. The submerged free standing rack system and surrounding water are coupled due to hydrodynamic fluid-structure interaction (FSI using potential theory. The models developed have features that allow consideration of geometric and material nonlinearities including (1 the impacts of fuel assemblies to rack cells, a rack to adjacent racks or pool walls, and rack support legs to the pool floor; (2 the hydrodynamic coupling of fuel assemblies with their storing racks, and of a rack with adjacent racks, pool walls, and the pool floor; and (3 the dynamic motion behavior of rocking, twisting, and frictional sliding of rack modules. Using these models 3D nonlinear time history dynamic analyses are performed per the U.S. Nuclear Regulatory Commission (USNRC criteria. Since few such modeling, analyses, and results using both the 3D single and whole pool multiple rack models are available in the literature, this paper emphasizes description of modeling and analysis techniques using the SOLVIA general purpose nonlinear finite element code. Typical response results with different Coulomb friction coefficients are presented and discussed.

  6. Functional Differences in Engineered Myocardium from Embryonic Stem Cell-Derived versus Neonatal Cardiomyocytes

    Feinberg, Adam W.; Ripplinger, Crystal M.; van der Meer, Peter; Sheehy, Sean P.; Domian, Ibrahim; Chien, Kenneth R.; Parker, Kevin Kit

    2013-01-01

    Stem cell-derived cardiomyocytes represent unique tools for cell-and tissue-based regenerative therapies, drug discovery and safety, and studies of fundamental heart-failure mechanisms. However, the degree to which stem cell-derived cardiomyocytes compare to mature cardiomyocytes is often debated.

  7. File list: ALL.CDV.20.AllAg.Cardiomyocytes [Chip-atlas[Archive

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  8. File list: His.CDV.05.AllAg.Cardiomyocytes [Chip-atlas[Archive

    Full Text Available His.CDV.05.AllAg.Cardiomyocytes mm9 Histone Cardiovascular Cardiomyocytes SRX305918...,SRX305920,SRX1121699,SRX305919 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.CDV.05.AllAg.Cardiomyocytes.bed ...

  9. File list: ALL.CDV.50.AllAg.Cardiomyocytes [Chip-atlas[Archive

    Full Text Available ALL.CDV.50.AllAg.Cardiomyocytes mm9 All antigens Cardiovascular Cardiomyocytes SRX3...05918,SRX305920,SRX305919,SRX1121699,SRX1121694 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.CDV.50.AllAg.Cardiomyocytes.bed ...

  10. File list: ALL.CDV.05.AllAg.Cardiomyocytes [Chip-atlas[Archive

    Full Text Available ALL.CDV.05.AllAg.Cardiomyocytes mm9 All antigens Cardiovascular Cardiomyocytes SRX3...05918,SRX305920,SRX1121699,SRX305919,SRX1121694 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.CDV.05.AllAg.Cardiomyocytes.bed ...

  11. File list: His.CDV.20.AllAg.Cardiomyocytes [Chip-atlas[Archive

    Full Text Available His.CDV.20.AllAg.Cardiomyocytes mm9 Histone Cardiovascular Cardiomyocytes SRX112169...9,SRX305918,SRX305920,SRX305919 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.CDV.20.AllAg.Cardiomyocytes.bed ...

  12. File list: His.CDV.50.AllAg.Cardiomyocytes [Chip-atlas[Archive

    Full Text Available His.CDV.50.AllAg.Cardiomyocytes mm9 Histone Cardiovascular Cardiomyocytes SRX305918...,SRX305920,SRX305919,SRX1121699 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.CDV.50.AllAg.Cardiomyocytes.bed ...

  13. File list: His.CDV.10.AllAg.Cardiomyocytes [Chip-atlas[Archive

    Full Text Available His.CDV.10.AllAg.Cardiomyocytes mm9 Histone Cardiovascular Cardiomyocytes SRX112169...9,SRX305919,SRX305918,SRX305920 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.CDV.10.AllAg.Cardiomyocytes.bed ...

  14. Alamandine acts via MrgD to induce AMPK/NO activation against Ang II hypertrophy in cardiomyocytes.

    de Jesus, Itamar Couto Guedes; Scalzo, Sergio; Alves, Fabiana; Marques, Kariny; Rocha-Resende, Cibele; Bader, Michael; Santos, Robson A Souza; Guatimosim, Silvia

    2018-02-14

    The renin-angiotensin system (RAS) plays a pivotal role in the pathogenesis of cardiovascular diseases. New members of this system have been characterized and shown to have biologically relevant actions. Alamandine and its receptor MrgD are recently identified components of RAS. In the cardiovascular system alamandine actions included vasodilation, antihypertensive and anti-fibrosis effects. Currently, the actions of alamandine on cardiomyocytes are unknown. Here our goal was twofold: (1) to unravel the signaling molecules activated by the alamandine/MrgD axis in cardiomyocytes; (2) to evaluate the ability of this axis to prevent against Angiotensin II (Ang II)-induced hypertrophy. In cardiomyocytes from C57BL/6 mice, alamandine treatment induced an increase in nitric oxide (NO) production, which was blocked by D-Pro 7 -Ang-(1-7), a MrgD antagonist. This NO rise correlated with increased phosphorylation of AMPK. Alamandine induced NO production was preserved in Mas -/- myocytes, and lost in MrgD -/- cells. Binding of fluorescent-labeled alamandine was observed in wild-type cells, but it was dramatically reduced in MrgD -/- myocytes. We also assessed the consequences of prolonged alamandine exposure to cultured neonatal rat cardiomyocytes (NRCMs) treated with Ang II. Treatment of NRCMs with alamandine prevented Ang II-induced hypertrophy. Moreover, antihypertrophic actions of alamandine were mediated via MrgD and NO, since they could be prevented by D-Pro 7 -Ang-(1-7) or inhibitors of NO synthase or AMPK. β-alanine, a MrgD agonist, recapitulated alamandine's cardioprotective effects in cardiomyocytes. Our data show that alamandine via MrgD induces AMPK/NO signaling to counterregulate Ang II induced hypertrophy. These findings highlight the therapeutic potential of the alamandine/MrgD axis in the heart.

  15. Troglitazone stimulates β-arrestin-dependent cardiomyocyte contractility via the angiotensin II type 1A receptor

    Tilley, Douglas G.; Nguyen, Anny D.; Rockman, Howard A.

    2010-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) agonists are commonly used to treat cardiovascular diseases, and are reported to have several effects on cardiovascular function that may be due to PPARγ-independent signaling events. Select angiotensin receptor blockers (ARBs) interact with and modulate PPARγ activity, thus we hypothesized that a PPARγ agonist may exert physiologic effects via the angiotensin II type 1 A receptor (AT1 A R). In AT1 A R-overexpressing HEK 293 cells, both angiotensin II (Ang II) and the PPARγ agonist troglitazone (Trog) enhanced AT1 A R internalization and recruitment of endogenous β-arrestin1/2 (βarr1/2) to the AT1 A R. A fluorescence assay to measure diacylglycerol (DAG) accumulation showed that although Ang II induced AT1 A R-G q protein-mediated DAG accumulation, Trog had no impact on DAG generation. Trog-mediated recruitment of βarr1/2 was selective to AT1 A R as the response was prevented by an ARB- and Trog-mediated βarr1/2 recruitment to β1-adrenergic receptor (β1AR) was not observed. In isolated mouse cardiomyocytes, Trog increased both % and rate of cell shortening to a similar extent as Ang II, effects which were blocked with an ARB. Additionally, these effects were found to be βarr2-dependent, as cardiomyocytes isolated from βarr2-KO mice showed blunted contractile responses to Trog. These findings show for the first time that the PPARγ agonist Trog acts at the AT1 A R to simultaneously block G q protein activation and induce the recruitment of βarr1/2, which leads to an increase in cardiomyocyte contractility.

  16. Structural phenotyping of stem cell-derived cardiomyocytes.

    Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2015-03-10

    Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes.

    Tan, Xiaobing; Dai, Qingli; Guo, Tao; Xu, Jingshu; Dai, Qingyuan

    2018-01-22

    Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy. Copyright © 2017. Published by Elsevier Inc.

  18. Vegetation response to invasive Tamarix control in southwestern U.S. rivers: a collaborative study including 416 sites.

    González, Eduardo; Sher, Anna A; Anderson, Robert M; Bay, Robin F; Bean, Daniel W; Bissonnete, Gabriel J; Bourgeois, Bérenger; Cooper, David J; Dohrenwend, Kara; Eichhorst, Kim D; El Waer, Hisham; Kennard, Deborah K; Harms-Weissinger, Rebecca; Henry, Annie L; Makarick, Lori J; Ostoja, Steven M; Reynolds, Lindsay V; Robinson, W Wright; Shafroth, Patrick B

    2017-09-01

    Most studies assessing vegetation response following control of invasive Tamarix trees along southwestern U.S. rivers have been small in scale (e.g., river reach), or at a regional scale but with poor spatial-temporal replication, and most have not included testing the effects of a now widely used biological control. We monitored plant composition following Tamarix control along hydrologic, soil, and climatic gradients in 244 treated and 172 reference sites across six U.S. states. This represents the largest comprehensive assessment to date on the vegetation response to the four most common Tamarix control treatments. Biocontrol by a defoliating beetle (treatment 1) reduced the abundance of Tamarix less than active removal by mechanically using hand and chain-saws (2), heavy machinery (3) or burning (4). Tamarix abundance also decreased with lower temperatures, higher precipitation, and follow-up treatments for Tamarix resprouting. Native cover generally increased over time in active Tamarix removal sites, however, the increases observed were small and was not consistently increased by active revegetation. Overall, native cover was correlated to permanent stream flow, lower grazing pressure, lower soil salinity and temperatures, and higher precipitation. Species diversity also increased where Tamarix was removed. However, Tamarix treatments, especially those generating the highest disturbance (burning and heavy machinery), also often promoted secondary invasions of exotic forbs. The abundance of hydrophytic species was much lower in treated than in reference sites, suggesting that management of southwestern U.S. rivers has focused too much on weed control, overlooking restoration of fluvial processes that provide habitat for hydrophytic and floodplain vegetation. These results can help inform future management of Tamarix-infested rivers to restore hydrogeomorphic processes, increase native biodiversity and reduce abundance of noxious species. © 2017 by the

  19. Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection.

    Fornaro, Michele; Sharthiya, Harsh; Tiwari, Vaibhav

    2018-03-09

    This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

  20. Gene transfer of heterologous G protein-coupled receptors to cardiomyocytes: differential effects on contractility.

    Laugwitz, K L; Weig, H J; Moretti, A; Hoffmann, E; Ueblacker, P; Pragst, I; Rosport, K; Schömig, A; Ungerer, M

    2001-04-13

    In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to beta-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective beta-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V(2) vasopressin receptors (rV(2)-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant beta(2)-adrenergic receptors (rbeta(2)-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V(2)-Rs are uniquely coupled to Gs, PTH1-Rs and beta(2)-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rbeta(2)-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV(2)-R-expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of beta(2)-ARS: In the absence of receptor agonists, rPTH1-Rs and rbeta(2)-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV(2)-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V(2)-Rs might be more suited to test the effects of c

  1. Proarrhythmia risk prediction using human induced pluripotent stem cell-derived cardiomyocytes.

    Yamazaki, Daiju; Kitaguchi, Takashi; Ishimura, Masakazu; Taniguchi, Tomohiko; Yamanishi, Atsuhiro; Saji, Daisuke; Takahashi, Etsushi; Oguchi, Masao; Moriyama, Yuta; Maeda, Sanae; Miyamoto, Kaori; Morimura, Kaoru; Ohnaka, Hiroki; Tashibu, Hiroyuki; Sekino, Yuko; Miyamoto, Norimasa; Kanda, Yasunari

    2018-04-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to become a useful tool for proarrhythmia risk prediction in the non-clinical drug development phase. Several features including electrophysiological properties, ion channel expression profile and drug responses were investigated using commercially available hiPSC-CMs, such as iCell-CMs and Cor.4U-CMs. Although drug-induced arrhythmia has been extensively examined by microelectrode array (MEA) assays in iCell-CMs, it has not been fully understood an availability of Cor.4U-CMs for proarrhythmia risk. Here, we evaluated the predictivity of proarrhythmia risk using Cor.4U-CMs. MEA assay revealed linear regression between inter-spike interval and field potential duration (FPD). The hERG inhibitor E-4031 induced reverse-use dependent FPD prolongation. We next evaluated the proarrhythmia risk prediction by a two-dimensional map, which we have previously proposed. We determined the relative torsade de pointes risk score, based on the extent of FPD with Fridericia's correction (FPDcF) change and early afterdepolarization occurrence, and calculated the margins normalized to free effective therapeutic plasma concentrations. The drugs were classified into three risk groups using the two-dimensional map. This risk-categorization system showed high concordance with the torsadogenic information obtained by a public database CredibleMeds. Taken together, these results indicate that Cor.4U-CMs can be used for drug-induced proarrhythmia risk prediction. Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  2. Alterations in cardiomyocyte function after pulmonary treatment with stainless steel welding fume in rats.

    Popstojanov, Risto; Antonini, James M; Salmen, Rebecca; Ye, Morgan; Zheng, Wen; Castranova, Vincent; Fekedulegn, Desta B; Kan, Hong

    2014-01-01

    Welding fume is composed of a complex of different metal particulates. Pulmonary exposure to different welding fumes may exert a negative impact on cardiac function, although the underlying mechanisms remain unclear. To explore the effect of welding fumes on cardiac function, Sprague-Dawley rats were exposed by intratracheal instillation to 2 mg/rat of manual metal arc hard surfacing welding fume (MMA-HS) once per week for 7 wk. Control rats received saline. Cardiomyocytes were isolated enzymatically at d 1 and 7 postexposure. Intracellular calcium ([Ca(2+)]i) transients (fluorescence ratio) were measured on the stage of an inverted phase-contrast microscope using a myocyte calcium imaging/cell length system. Phosphorylation levels of cardiac troponin I (cTnI) were determined by Western blot. The levels of nonspecific inflammatory marker C-reactive protein (CRP) and proinflammatory cytokine interleukin-6 (IL-6) in serum were measured by enzyme-linked immunosorbent assay (ELISA). Contraction of isolated cardiomyocytes was significantly reduced at d 1 and d 7 postexposure. Intracellular calcium levels were decreased in response to extracellular calcium stimulation at d 7 postexposure. Changes of intracellular calcium levels after isoprenaline hydrochloride (ISO) stimulation were not markedly different between groups at either time point. Phosphorylation levels of cTnI in the left ventricle were significantly lower at d 1 postexposure. The serum levels of CRP were not markedly different between groups at either time point. Serum levels of IL-6 were not detectable in both groups. Cardiomyocyte alterations observed after welding fume treatment were mainly due to alterations in intracellular calcium handling and phosphorylation levels of cTnI.

  3. Polycystin-1 Is a Cardiomyocyte Mechanosensor That Governs L-Type Ca2+ Channel Protein Stability.

    Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K; Morales, Cyndi R; Contreras-Ferrat, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J; Somlo, Stefan; Rothermel, Beverly A; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

    2015-06-16

    L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress-induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction-induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. © 2015 American Heart Association, Inc.

  4. Common marmoset embryonic stem cell can differentiate into cardiomyocytes

    Chen Hao; Hattori, Fumiyuki; Murata, Mitsushige; Li Weizhen; Yuasa, Shinsuke; Onizuka, Takeshi; Shimoji, Kenichiro; Ohno, Yohei; Sasaki, Erika; Kimura, Kensuke; Hakuno, Daihiko

    2008-01-01

    Common marmoset monkeys have recently attracted much attention as a primate research model, and are preferred to rhesus and cynomolgus monkeys due to their small bodies, easy handling and efficient breeding. We recently reported the establishment of common marmoset embryonic stem cell (CMESC) lines that could differentiate into three germ layers. Here, we report that our CMESC can also differentiate into cardiomyocytes and investigated their characteristics. After induction, FOG-2 was expressed, followed by GATA4 and Tbx20, then Nkx2.5 and Tbx5. Spontaneous beating could be detected at days 12-15. Immunofluorescent staining and ultrastructural analyses revealed that they possessed characteristics typical of functional cardiomyocytes. They showed sinus node-like action potentials, and the beating rate was augmented by isoproterenol stimulation. The BrdU incorporation assay revealed that CMESC-derived cardiomyocytes retained a high proliferative potential for up to 24 weeks. We believe that CMESC-derived cardiomyocytes will advance preclinical studies in cardiovascular regenerative medicine

  5. A non-cardiomyocyte autonomous mechanism of cardioprotection involving the SLO1 BK channel

    Andrew P. Wojtovich

    2013-03-01

    Full Text Available Opening of BK-type Ca2+ activated K+ channels protects the heart against ischemia-reperfusion (IR injury. However, the location of BK channels responsible for cardioprotection is debated. Herein we confirmed that openers of the SLO1 BK channel, NS1619 and NS11021, were protective in a mouse perfused heart model of IR injury. As anticipated, deletion of the Slo1 gene blocked this protection. However, in an isolated cardiomyocyte model of IR injury, protection by NS1619 and NS11021 was insensitive to Slo1 deletion. These data suggest that protection in intact hearts occurs by a non-cardiomyocyte autonomous, SLO1-dependent, mechanism. In this regard, an in-situ assay of intrinsic cardiac neuronal function (tachycardic response to nicotine revealed that NS1619 preserved cardiac neurons following IR injury. Furthermore, blockade of synaptic transmission by hexamethonium suppressed cardioprotection by NS1619 in intact hearts. These results suggest that opening SLO1 protects the heart during IR injury, via a mechanism that involves intrinsic cardiac neurons. Cardiac neuronal ion channels may be useful therapeutic targets for eliciting cardioprotection.

  6. Spatiotemporal stability of neonatal rat cardiomyocyte monolayers spontaneous activity is dependent on the culture substrate.

    Jonathan Boudreau-Béland

    Full Text Available In native conditions, cardiac cells must continuously comply with diverse stimuli necessitating a perpetual adaptation. Polydimethylsiloxane (PDMS is commonly used in cell culture to study cellular response to changes in the mechanical environment. The aim of this study was to evaluate the impact of using PDMS substrates on the properties of spontaneous activity of cardiomyocyte monolayer cultures. We compared PDMS to the gold standard normally used in culture: a glass substrate. Although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 and increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol and acetylcholine. These results reveal the importance of carefully selecting the culture substrate for studies involving mechanical stimulation, especially for tissue engineering or pharmacological high-throughput screening of cardiac tissue analog.

  7. Cardiomyocyte-specific deletion of the G protein-coupled estrogen receptor (GPER) leads to left ventricular dysfunction and adverse remodeling: A sex-specific gene profiling analysis.

    Wang, Hao; Sun, Xuming; Chou, Jeff; Lin, Marina; Ferrario, Carlos M; Zapata-Sudo, Gisele; Groban, Leanne

    2017-08-01

    Activation of G protein-coupled estrogen receptor (GPER) by its agonist, G1, protects the heart from stressors such as pressure-overload, ischemia, a high-salt diet, estrogen loss, and aging, in various male and female animal models. Due to nonspecific effects of G1, the exact functions of cardiac GPER cannot be concluded from studies using systemic G1 administration. Moreover, global knockdown of GPER affects glucose homeostasis, blood pressure, and many other cardiovascular-related systems, thereby confounding interpretation of its direct cardiac actions. We generated a cardiomyocyte-specific GPER knockout (KO) mouse model to specifically investigate the functions of GPER in cardiomyocytes. Compared to wild type mice, cardiomyocyte-specific GPER KO mice exhibited adverse alterations in cardiac structure and impaired systolic and diastolic function, as measured by echocardiography. Gene deletion effects on left ventricular dimensions were more profound in male KO mice compared to female KO mice. Analysis of DNA microarray data from isolated cardiomyocytes of wild type and KO mice revealed sex-based differences in gene expression profiles affecting multiple transcriptional networks. Gene Set Enrichment Analysis (GSEA) revealed that mitochondrial genes are enriched in GPER KO females, whereas inflammatory response genes are enriched in GPER KO males, compared to their wild type counterparts of the same sex. The cardiomyocyte-specific GPER KO mouse model provides us with a powerful tool to study the functions of GPER in cardiomyocytes. The gene expression profiles of the GPER KO mice provide foundational information for further study of the mechanisms underlying sex-specific cardioprotection by GPER. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Comparison of the IKr blockers moxifloxacin, dofetilide and E-4031 in five screening models of pro-arrhythmia reveals lack of specificity of isolated cardiomyocytes

    Nalos, L; Varkevisser, R; Jonsson, Mkb

    2012-01-01

    Background and purpose Drug discovery and development require testing of new chemical entities for possible adverse effects. For cardiac safety screening, improved assays are urgently needed and isolated adult cardiomyocytes (CM) and human embryonic stem cell-derived cardiomyocytes (hESC-CM) may......-4031 (unsafe compounds). Experimental approach The assays included: 1. The anesthetized remodeled chronic complete AV-block (CAVB) dog, 2. The anesthetized methoxamine sensitized unremodeled rabbit, 3. Multi-cellular hESC-CM clusters, 4. Isolated CM obtained from the CAVB dog and 5. Isolated CM...

  9. MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

    Sala, Luca; van Meer, Berend J; Tertoolen, Leon G J; Bakkers, Jeroen; Bellin, Milena; Davis, Richard P; Denning, Chris; Dieben, Michel A E; Eschenhagen, Thomas; Giacomelli, Elisa; Grandela, Catarina; Hansen, Arne; Holman, Eduard R; Jongbloed, Monique R M; Kamel, Sarah M; Koopman, Charlotte D; Lachaud, Quentin; Mannhardt, Ingra; Mol, Mervyn P H; Mosqueira, Diogo; Orlova, Valeria V; Passier, Robert; Ribeiro, Marcelo C; Saleem, Umber; Smith, Godfrey L; Burton, Francis L; Mummery, Christine L

    2018-02-02

    There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac "organoids," engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models. © 2017 The Authors.

  10. Similar to spironolactone, oxymatrine is protective in aldosterone-induced cardiomyocyte injury via inhibition of calpain and apoptosis-inducing factor signaling.

    Ting-Ting Xiao

    Full Text Available Accumulating evidence indicates that oxymatrine (OMT possesses variously pharmacological properties, especially on the cardiovascular system. We previously demonstrated that activated calpain/apoptosis-inducing factor (AIF-mediated pathway was the key molecular mechanism in aldosterone (ALD induces cardiomyocytes apoptosis. In the present study, we extended the experimentation by investigating the effect of OMT on cardiomyocytes exposed to ALD, as compared to spironolactone (Spiro, a classical ALD receptor antagonist. Cardiomyocytes were pre-incubated with OMT, Spiro or vehicle for 1 h, and then, cardiomyocytes were exposed to ALD 24 h. The cell injury was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and lactate dehydrogenase (LDH leakage ratio. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay, annexin V/PI staining, and relative caspase-3 activity assay. Furthermore, expression of pro-apoptotic proteins including truncated Bid (tBid, calpain and AIF were evaluated by western blot analysis. ALD stimulation increased cardiomyocytes apoptosis, caspase-3 activity and protein expression of calpain, tBid and AIF in the cytosol (p<0.05. Pre-incubated with cardiomyocytes injury and increased caspase-3 activity were significantly attenuated (p<0.05. Furthermore, OMT suppressed ALD-induced high expression of calpain and AIF. And these effects of OMT could be comparable to Spiro. These findings indicated that OMT might be a potential cardioprotective-agent against excessive ALD-induced cardiotoxicity, at least in part, mediated through inhibition of calpain/AIF signaling.

  11. Postnatal ablation of Foxm1 from cardiomyocytes causes late onset cardiac hypertrophy and fibrosis without exacerbating pressure overload-induced cardiac remodeling.

    Craig Bolte

    Full Text Available Heart disease remains a leading cause of morbidity and mortality in the industrialized world. Hypertrophic cardiomyopathy is the most common genetic cardiovascular disorder and the most common cause of sudden cardiac death. Foxm1 transcription factor (also known as HFH-11B, Trident, Win or MPP2 plays an important role in the pathogenesis of various cancers and is a critical mediator of post-injury repair in multiple organs. Foxm1 has been previously shown to be essential for heart development and proliferation of embryonic cardiomyocytes. However, the role of Foxm1 in postnatal heart development and in cardiac injury has not been evaluated. To delete Foxm1 in postnatal cardiomyocytes, αMHC-Cre/Foxm1(fl/fl mice were generated. Surprisingly, αMHC-Cre/Foxm1(fl/fl mice exhibited normal cardiomyocyte proliferation at postnatal day seven and had no defects in cardiac structure or function but developed cardiac hypertrophy and fibrosis late in life. The development of cardiomyocyte hypertrophy and cardiac fibrosis in aged Foxm1-deficient mice was associated with reduced expression of Hey2, an important regulator of cardiac homeostasis, and increased expression of genes critical for cardiac remodeling, including MMP9, αSMA, fibronectin and vimentin. We also found that following aortic constriction Foxm1 mRNA and protein were induced in cardiomyocytes. However, Foxm1 deletion did not exacerbate cardiac hypertrophy or fibrosis following chronic pressure overload. Our results demonstrate that Foxm1 regulates genes critical for age-induced cardiomyocyte hypertrophy and cardiac fibrosis.

  12. Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker.

    Yukihiro Saito

    Full Text Available Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (If flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN channels and attenuation of the inward rectifier K+ current (IK1 flowing through inward rectifier potassium (Kir channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs and induced cardiomyocytes that originally show poor IK1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs function as a biological pacemaker in vitro.The rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of IK1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger If currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded

  13. Effect of hepatocyte growth factor and angiotensin II on rat cardiomyocyte hypertrophy

    Chen, Ai-Lan [Department of Cardiology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Ou, Cai-Wen [The Fourth Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); He, Zhao-Chu [Department of Cardiology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Liu, Qi-Cai [Experimental Medical Research Center, Guangzhou Medical University, Guangzhou (China); Dong, Qi [Department of Physiology, Guangzhou Medical University, Guangzhou (China); Chen, Min-Sheng [Guangzhou Key Laboratory of Cardiovascular Disease, Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China)

    2012-10-15

    Angiotensin II (Ang II) plays an important role in cardiomyocyte hypertrophy. The combined effect of hepatocyte growth factor (HGF) and Ang II on cardiomyocytes is unknown. The present study was designed to determine the effect of HGF on cardiomyocyte hypertrophy and to explore the combined effect of HGF and Ang II on cardiomyocyte hypertrophy. Primary cardiomyocytes were isolated from neonatal rat hearts and cultured in vitro. Cells were treated with Ang II (1 µM) alone, HGF (10 ng/mL) alone, and Ang II (1 µM) plus HGF (10 ng/mL) for 24, 48, and 72 h. The amount of [{sup 3}H]-leucine incorporation was then measured to evaluate protein synthesis. The mRNA levels of β-myosin heavy chain and atrial natriuretic factor were determined by real-time PCR to evaluate the presence of fetal phenotypes of gene expression. The cell size of cardiomyocytes was also studied. Ang II (1 µM) increased cardiomyocyte hypertrophy. Similar to Ang II, treatment with 1 µM HGF promoted cardiomyocyte hypertrophy. Moreover, the combination of 1 µM Ang II and 10 ng/mL HGF clearly induced a combined pro-hypertrophy effect on cardiomyocytes. The present study demonstrates for the first time a novel, combined effect of HGF and Ang II in promoting cardiomyocyte hypertrophy.

  14. Particulate matter exposure exacerbates high glucose-induced cardiomyocyte dysfunction through ROS generation.

    Li Zuo

    Full Text Available Diabetes mellitus and fine particulate matter from diesel exhaust (DEP are both important contributors to the development of cardiovascular disease (CVD. Diabetes mellitus is a progressive disease with a high mortality rate in patients suffering from CVD, resulting in diabetic cardiomyopathy. Elevated DEP levels in the air are attributed to the development of various CVDs, presumably since fine DEP (<2.5 µm in diameter can be inhaled and gain access to the circulatory system. However, mechanisms defining how DEP affects diabetic or control cardiomyocyte function remain poorly understood. The purpose of the present study was to evaluate cardiomyocyte function and reactive oxygen species (ROS generation in isolated rat ventricular myocytes exposed overnight to fine DEP (0.1 µg/ml, and/or high glucose (HG, 25.5 mM. Our hypothesis was that DEP exposure exacerbates contractile dysfunction via ROS generation in cardiomyocytes exposed to HG. Ventricular myocytes were isolated from male adult Sprague-Dawley rats cultured overnight and sarcomeric contractile properties were evaluated, including: peak shortening normalized to baseline (PS, time-to-90% shortening (TPS(90, time-to-90% relengthening (TR(90 and maximal velocities of shortening/relengthening (±dL/dt, using an IonOptix field-stimulator system. ROS generation was determined using hydroethidine/ethidium confocal microscopy. We found that DEP exposure significantly increased TR(90, decreased PS and ±dL/dt, and enhanced intracellular ROS generation in myocytes exposed to HG. Further studies indicated that co-culture with antioxidants (0.25 mM Tiron and 0.5 mM N-Acetyl-L-cysteine completely restored contractile function in DEP, HG and HG+DEP-treated myocytes. ROS generation was blocked in HG-treated cells with mitochondrial inhibition, while ROS generation was blocked in DEP-treated cells with NADPH oxidase inhibition. Our results suggest that DEP exacerbates myocardial dysfunction in isolated

  15. Cardioprotective Effects of Quercetin in Cardiomyocyte under Ischemia/Reperfusion Injury

    Yi-Wen Chen

    2013-01-01

    Full Text Available Quercetin, a polyphenolic compound existing in many vegetables, fruits, has antiinflammatory, antiproliferation, and antioxidant effect on mammalian cells. Quercetin was evaluated for protecting cardiomyocytes from ischemia/reperfusion injury, but its protective mechanism remains unclear in the current study. The cardioprotective effects of quercetin are achieved by reducing the activity of Src kinase, signal transducer and activator of transcription 3 (STAT3, caspase 9, Bax, intracellular reactive oxygen species production, and inflammatory factor and inducible MnSOD expression. Fluorescence two-dimensional differential gel electrophoresis (2D-DIGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS can reveal the differentially expressed proteins of H9C2 cells treated with H2O2 or quercetin. Although 17 identified proteins were altered in H2O2-induced cells, these proteins such as alpha-soluble NSF attachment protein (α-SNAP, Ena/VASP-like protein (Evl, and isopentenyl-diphosphate delta-isomerase 1 (Idi-1 were reverted by pretreatment with quercetin, which correlates with kinase activation, DNA repair, lipid, and protein metabolism. Quercetin dephosphorylates Src kinase in H2O2-induced H9C2 cells and likely blocks the H2O2-induced inflammatory response through STAT3 kinase modulation. This probably contributes to prevent ischemia/reperfusion injury in cardiomyocytes.

  16. Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes.

    Sheyda R Frolova

    Full Text Available The ability of azobenzene trimethylammonium bromide (azoTAB to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav, calcium (ICav, and potassium (IKv currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na+ and calcium (Ca2+ currents and potentiation of net potassium (K+ currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.

  17. Effects of Mechanical Coupling Between Cardiomyocytes and Cardiac Fibroblasts on Myocardium

    Zorlutuna, Pinar; Nguyen, Trung Dung; Nagarajan, Neerajha

    Cardiomyocytes show excitatory responses to stimulation solely by mechanical forces through their stretch-activated ion channels, and can fire action potentials upon mechanical stimulation through a pathway known as mechano-electric feedback. Furthermore, cardiomyocyte (CM) - cardiac fibroblasts (CF) can couple mechanically through cell-cell junctions. Here we investigated the effects of CM and CF mechanical coupling on myocardial physiology and pathology using a bio-nanoindentered coupled with fast calcium imaging and microelectrode arrays. In order to study mechanical signal transmission, we measured the contractile forces generated by CMs, as well as by CFs that were coupled to the CMs. We observed that CFs were beating with the same frequency but at smaller magnitude compared to CMs, and their contractility was dependent on the substrate stiffness. Our results showed that beating CMs actively stretched neighbouring CFs through the deformation of the substrate the cells were seeded on, which promoted the myocardial contractility through mechanical coupling. The results also revealed that CM contractility was propagated greater on soft substrates than stiff ones. Results of this study could help identify the role of the infarcted tissue stiffness and size on heart failure. This study is supported by NSF Grant No: 1530884.

  18. Dissociation between cardiomyocyte function and remodeling with beta-adrenergic receptor blockade in isolated canine mitral regurgitation.

    Pat, Betty; Killingsworth, Cheryl; Denney, Thomas; Zheng, Junying; Powell, Pamela; Tillson, Michael; Dillon, A Ray; Dell'Italia, Louis J

    2008-12-01

    The low-pressure volume overload of isolated mitral regurgitation (MR) is associated with increased adrenergic drive, left ventricular (LV) dilatation, and loss of interstitial collagen. We tested the hypothesis that beta1-adrenergic receptor blockade (beta1-RB) would attenuate LV remodeling after 4 mo of MR in the dog. beta1-RB did not attenuate collagen loss or the increase in LV mass in MR dogs. Using MRI and three-dimensional (3-D) analysis, there was a 70% increase in the LV end-diastolic (LVED) volume-to-LV mass ratio, a 23% decrease in LVED midwall circumferential curvature, and a >50% increase in LVED 3-D radius/wall thickness in MR dogs that was not attenuated by beta1-RB. However, beta1-RB caused a significant increase in LVED length from the base to apex compared with untreated MR dogs. This was associated with an increase in isolated cardiomyocyte length (171+/-5 microm, P<0.05) compared with normal (156+/-3 microm) and MR (165+/-4 microm) dogs. Isolated cardiomyocyte fractional shortening was significantly depressed in MR dogs compared with normal dogs (3.73+/-0.31 vs. 5.02+/-0.26%, P<0.05) and normalized with beta1-RB (4.73+/-0.48%). In addition, stimulation with the beta-adrenergic receptor agonist isoproterenol (25 nM) increased cardiomyocyte fractional shortening by 215% (P<0.05) in beta1-RB dogs compared with normal (56%) and MR (50%) dogs. In summary, beta1-RB improved LV cardiomyocyte function and beta-adrenergic receptor responsiveness despite further cell elongation. The failure to attenuate LV remodeling associated with MR could be due to a failure to improve ultrastructural changes in extracellular matrix organization.

  19. Rcan1-1L overexpression induces mitochondrial autophagy and improves cell survival in angiotensin II-exposed cardiomyocytes

    Duan, Hongyan; Li, Yongqiang; Yan, Lijie; Yang, Haitao; Wu, Jintao; Qian, Peng; Li, Bing; Wang, Shanling

    2015-01-01

    Mitochondrial autophagy is an important adaptive stress response and can be modulated by various key molecules. A previous study found that the regulator of calcineurin 1-1L (Rcan1-1L) may regulate mitochondrial autophagy and cause mitochondria degradation in neurocytes. However, the effect of Rcan1-1L on cardiomyocytes has not been determined. In the present study, we aimed to investigate the role of Rcan1-1L in angiotensin II (Ang II)-exposed human cardiomyocytes. Above all, Human adult cardiac myocytes (HACMs) were exposed to 200 nmol/L Ang II for 4 days. Enhanced H 2 O 2 production, cytochrome C release and mitochondrial permeability were observed in these cells, which were blocked by valsartan. Consistently, Ang II exposure significantly reduced cardiomyocyte viability. However, transfection of Rcan1-1L vector promoted cell viability and ameliorated the apoptosis caused by Ang II. Rcan1-1L clearly promoted mitochondrial autophagy in HACMs, with elevated autophagy protein (ATG) 5 and light chain 3 (LC3) expression. Transient mitochondrial biogenesis and reduced cytochrome C release was also induced by Rcan1-1L. Additionally, Rcan1-1L significantly inhibited calcineurin/nuclear factor of activated T cells (NFAT) signaling. We thus conclude that Rcan1-1L may play a protective role in Ang II-treated cardiomyocytes through the induction of mitochondrial autophagy, and may be an alternative method of cardiac protection. - Highlights: • Transfection of Rcan1-1L into HACMs promoted cell viability and reduced apoptosis. • Transfection of Rcan1-1L promoted mitochondrial autophagy in HACMs. • Rcan1-1L inhibited the calcineurin/nuclear factor of activated T cells signaling

  20. Rcan1-1L overexpression induces mitochondrial autophagy and improves cell survival in angiotensin II-exposed cardiomyocytes

    Duan, Hongyan; Li, Yongqiang; Yan, Lijie; Yang, Haitao; Wu, Jintao; Qian, Peng; Li, Bing; Wang, Shanling, E-mail: shanglingwang@126.com

    2015-07-01

    Mitochondrial autophagy is an important adaptive stress response and can be modulated by various key molecules. A previous study found that the regulator of calcineurin 1-1L (Rcan1-1L) may regulate mitochondrial autophagy and cause mitochondria degradation in neurocytes. However, the effect of Rcan1-1L on cardiomyocytes has not been determined. In the present study, we aimed to investigate the role of Rcan1-1L in angiotensin II (Ang II)-exposed human cardiomyocytes. Above all, Human adult cardiac myocytes (HACMs) were exposed to 200 nmol/L Ang II for 4 days. Enhanced H{sub 2}O{sub 2} production, cytochrome C release and mitochondrial permeability were observed in these cells, which were blocked by valsartan. Consistently, Ang II exposure significantly reduced cardiomyocyte viability. However, transfection of Rcan1-1L vector promoted cell viability and ameliorated the apoptosis caused by Ang II. Rcan1-1L clearly promoted mitochondrial autophagy in HACMs, with elevated autophagy protein (ATG) 5 and light chain 3 (LC3) expression. Transient mitochondrial biogenesis and reduced cytochrome C release was also induced by Rcan1-1L. Additionally, Rcan1-1L significantly inhibited calcineurin/nuclear factor of activated T cells (NFAT) signaling. We thus conclude that Rcan1-1L may play a protective role in Ang II-treated cardiomyocytes through the induction of mitochondrial autophagy, and may be an alternative method of cardiac protection. - Highlights: • Transfection of Rcan1-1L into HACMs promoted cell viability and reduced apoptosis. • Transfection of Rcan1-1L promoted mitochondrial autophagy in HACMs. • Rcan1-1L inhibited the calcineurin/nuclear factor of activated T cells signaling.

  1. Evaluation of Seismic Response Trends from Long-Term Monitoring of Two Instrumented RC Buildings Including Soil-Structure Interaction

    Faheem Butt

    2012-01-01

    Full Text Available This paper presents analyses of the seismic responses of two reinforced concrete buildings monitored for a period of more than two years. One of the structures was a three-storey reinforced concrete (RC frame building with a shear core, while the other was a three-storey RC frame building without a core. Both buildings are part of the same large complex but are seismically separated from the rest of it. Statistical analysis of the relationships between maximum free field accelerations and responses at different points on the buildings was conducted and demonstrated strong correlation between those. System identification studies using recorded accelerations were undertaken and revealed that natural frequencies and damping ratios of the building structures vary during different earthquake excitations. This variation was statistically examined and relationships between identified natural frequencies and damping ratios, and the peak response acceleration at the roof level were developed. A general trend of decreasing modal frequencies and increasing damping ratios was observed with increased level of shaking and response. Moreover, the influence of soil structure interaction (SSI on the modal characteristics was evaluated. SSI effects decreased the modal frequencies and increased some of the damping ratios.

  2. Simulation of spatially varying ground motions including incoherence, wave‐passage and differential site‐response effects

    Konakli, Katerina; Der Kiureghian, Armen

    2012-01-01

    A method is presented for simulating arrays of spatially varying ground motions, incorporating the effects of incoherence, wave passage, and differential site response. Non‐stationarity is accounted for by considering the motions as consisting of stationary segments. Two approaches are developed....

  3. Future arrangements for Great Britain's gas quality specifications. Government Response (including Summary of Responses) to consultation on future arrangements for Great Britain's gas quality specifications

    2007-01-01

    Last year the Government consulted on GB's future gas quality specifications. The background is that some streams of imported gases have an energy content that exceeds the upper limit specified in GB's current gas quality regulations. The policy issue was whether to stick with the current regulated specifications, but at the cost of having to process imported gas to bring it within those specifications; or, at some time after 2020, to adjust those specifications, but at the cost of having to check (and potentially change) approximately 45m domestic gas appliances in 22m households, to ensure that they are capable of burning the high energy gas safely (and with a residual safety risk that some appliances are missed). The Consultation Document sought views on the proposal (backed up by the Partial Regulatory Impact Assessment) to adopt the 'no change' option. In the light of the responses, this is what the Government now proposes. The associated Impact Assessment estimates the total net benefit of the 'no change' option at UK Pounds 1.5bn - 14bn, with a best estimate of UK Pounds 8bn (NPV, 2005). The risks for gas prices and security of supply (included in the Impact Assessment) are judged to be small. The Government proposes a 'forward plan', consisting of support for Ofgem's work on maximising the commercial flexibilities for the GB gas market to handle gases of different specifications, whilst continuing to engage in EU discussions on the gas quality issue. (Author)

  4. Effectiveness of Tuned Mass Dampers in Seismic Response Control of Isolated Bridges Including Soil-Structure Interaction

    Said Elias

    Full Text Available Abstract The effect of soil-structure interaction (SSI on the dynamic responses of seismically isolated three-span continuous reinforced concrete (RC bridge is investigated. Also, tuned mass damper(s (TMD/s is/are installed to control undesirable bearing displacement, even under the SSI effect. The TMDs are placed at the mid-span of the bridge and each tuned with a modal frequency, while controlling up to first few modes as desirable. The soil surrounding the foundation of pier is modeled by frequency independent coefficients. Dynamic analysis is carried out in time domain using direct integration method. In order to specify the effects of the SSI, the responses of the non-isolated, isolated, and controlled isolated bridge are compared. It is observed that the soil surrounding the pier has significant effects on the bearing displacement of the isolated RC bridges. In addition, it is observed that the seismic responses of isolated RC bridge reduced significantly with installation of the TMDs.

  5. Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology.

    Rajagopal, Vijay; Bass, Gregory; Ghosh, Shouryadipta; Hunt, Hilary; Walker, Cameron; Hanssen, Eric; Crampin, Edmund; Soeller, Christian

    2018-04-18

    With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the

  6. Cardiac injury of the newborn mammalian heart accelerates cardiomyocyte terminal differentiation

    Zebrowski, David C.; Jensen, Charlotte H.; Becker, Robert

    2017-01-01

    exhibited midbody formation consistent with successful abscission, whereas those from 3 day-old cardiomyocytes after apical resection exhibited midbody formation consistent with abscission failure. Lastly, injured hearts failed to fully regenerate as evidenced by persistent scarring and reduced wall motion......After birth cardiomyocytes undergo terminal differentiation, characterized by binucleation and centrosome disassembly, rendering the heart unable to regenerate. Yet, it has been suggested that newborn mammals regenerate their hearts after apical resection by cardiomyocyte proliferation. Thus, we...... increased rate of binucleation there was a nearly 2-fold increase in the number of cardiomyocytes in mitosis indicating that the majority of injury-induced cardiomyocyte cell cycle activity results in binucleation, not proliferation. Concurrently, cardiomyocytes undergoing cytokinesis from embryonic hearts...

  7. Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration.

    Patterson, Michaela; Barske, Lindsey; Van Handel, Ben; Rau, Christoph D; Gan, Peiheng; Sharma, Avneesh; Parikh, Shan; Denholtz, Matt; Huang, Ying; Yamaguchi, Yukiko; Shen, Hua; Allayee, Hooman; Crump, J Gage; Force, Thomas I; Lien, Ching-Ling; Makita, Takako; Lusis, Aldons J; Kumar, S Ram; Sucov, Henry M

    2017-09-01

    Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes.

  8. Psychometric properties including reliability, validity and responsiveness of the Majeed pelvic score in patients with chronic sacroiliac joint pain.

    Bajada, Stefan; Mohanty, Khitish

    2016-06-01

    The Majeed scoring system is a disease-specific outcome measure that was originally designed to assess pelvic injuries. The aim of this study was to determine the psychometric properties of the Majeed scoring system for chronic sacroiliac joint pain. Internal consistency, content validity, criterion validity, construct validity and responsiveness to change was assessed prospectively for the Majeed scoring system in a cohort of 60 patients diagnosed with sacroiliac joint pain. This diagnosis was confirmed with CT-guided sacroiliac joint anaesthetic block. The overall Majeed score showed acceptable internal consistency (Cronbach alpha = 0.63). Similarly, it showed acceptable floor (0 %) and ceiling (0 %) effects. On the other hand, the domains of pain, work, sitting and sexual intercourse had high (>30 %) floor effects. Significant correlation with the physical component of the Short Form-36 (p = 0.005) and Oswestry disability index (p ≤ 0.001) was found indicating acceptable criterion validity. The overall Majeed score showed acceptable construct validity with all five developed hypotheses showing significance (p ≤ 0.05). The overall Majeed score showed acceptable responsiveness to change with a large (≥0.80) effect size and standardized response mean. Overall the Majeed scoring system demonstrated acceptable psychometric properties for outcome assessment in chronic sacroiliac joint pain. Thus, its use in this condition is adequate. However, some domains demonstrated suboptimal performance indicating that improvement might be achieved with the development of an outcome measure specific for sacroiliac joint dysfunction and degeneration.

  9. Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

    Banzet, N.; Richaud, C.; Deveaux, Y.; Kazmaier, M.; Gagnon, J.; Triantaphylides, C.

    1998-01-01

    Changes in gene expression, by application of H2O2, O2.- generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H2O2 and gamma irradiation, but not to O2.- generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I-IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H2O2 and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H2O2 action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H2O2 pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential

  10. Rac1 modulates cardiomyocyte adhesion during mouse embryonic development

    Abu-Issa, Radwan, E-mail: rabuissa@umich.edu

    2015-01-24

    Highlights: • Conditional knockout of Rac1 using Nkx2.5 Cre line is lethal at E13.5. • The myocardium of the mutant is thin and disorganized. • The phenotype is not due to cardiomyocyte low proliferation or apoptosis. • The phenotype is due to specific defect in cardiomyocyte adhesion. - Abstract: Rac1, a member of the Rho subfamily of small GTPases, is involved in morphogenesis and differentiation of many cell types. Here we define a role of Rac1 in cardiac development by specifically deleting Rac1 in the pre-cardiac mesoderm using the Nkx2.5-Cre transgenic driver line. Rac1-conditional knockout embryos initiate heart development normally until embryonic day 11.5 (E11.5); their cardiac mesoderm is specified, and the heart tube is formed and looped. However, by E12.5-E13.5 the mutant hearts start failing and embryos develop edema and hemorrhage which is probably the cause for the lethality observed soon after. The hearts of Rac1-cKO embryos exhibit disorganized and thin myocardial walls and defects in outflow tract alignment. No significant differences of cardiomyocyte death or proliferation were found between developing control and mutant embryos. To uncover the role of Rac1 in the heart, E11.5 primary heart cells were cultured and analyzed in vitro. Rac1-deficient cardiomyocytes were less spread, round and loosely attached to the substrate and to each other implying that Rac1-mediated signaling is required for appropriate cell–cell and/or cellmatrix adhesion during cardiac development.

  11. Rac1 modulates cardiomyocyte adhesion during mouse embryonic development

    Abu-Issa, Radwan

    2015-01-01

    Highlights: • Conditional knockout of Rac1 using Nkx2.5 Cre line is lethal at E13.5. • The myocardium of the mutant is thin and disorganized. • The phenotype is not due to cardiomyocyte low proliferation or apoptosis. • The phenotype is due to specific defect in cardiomyocyte adhesion. - Abstract: Rac1, a member of the Rho subfamily of small GTPases, is involved in morphogenesis and differentiation of many cell types. Here we define a role of Rac1 in cardiac development by specifically deleting Rac1 in the pre-cardiac mesoderm using the Nkx2.5-Cre transgenic driver line. Rac1-conditional knockout embryos initiate heart development normally until embryonic day 11.5 (E11.5); their cardiac mesoderm is specified, and the heart tube is formed and looped. However, by E12.5-E13.5 the mutant hearts start failing and embryos develop edema and hemorrhage which is probably the cause for the lethality observed soon after. The hearts of Rac1-cKO embryos exhibit disorganized and thin myocardial walls and defects in outflow tract alignment. No significant differences of cardiomyocyte death or proliferation were found between developing control and mutant embryos. To uncover the role of Rac1 in the heart, E11.5 primary heart cells were cultured and analyzed in vitro. Rac1-deficient cardiomyocytes were less spread, round and loosely attached to the substrate and to each other implying that Rac1-mediated signaling is required for appropriate cell–cell and/or cellmatrix adhesion during cardiac development

  12. Shock Wave Therapy Promotes Cardiomyocyte Autophagy and Survival during Hypoxia

    Ling Du

    2017-06-01

    Full Text Available Background: Autophagy plays an important role in cardiovascular disease. Controversy still exists regarding the effect of autophagy on ischemic/hypoxic myocardium. Cardiac shock wave therapy (CSWT is an effective alternative treatment for refractory ischemic heart disease. Whether CSWT can regulate cardiomyocyte autophagy under hypoxic conditions is not clear. We established a myocardial hypoxia model using the H9c2 cell line and performed shock waves (SWs treatment to evaluate the effect of SW on autophagy. Methods: The H9c2 cells were incubated under hypoxic conditions, and SW treatment was then performed at energies of 0.02, 0.05, or 0.10 mJ/mm2. The cell viability and intracellular ATP level were examined. Western blot analysis was used to assess the expression of LC3B, AMPK, mTOR, Beclin-1, Sirt1, and HIF-1α. Autophagic vacuoles were visualized by monodansylcadaverine staining. Results: After the 24-hour hypoxic period, cardiomyocyte viability and ATP levels were decreased and autophagy was significantly increased in H9c2 cells. SW treatment with an energy of 0.05 mJ/mm2 significantly increased the cellular viability, ATP level, LC3B-II/I, and number of autophagic vacuoles. In addition, phosphorylated AMPK and Sirt1 were increased and phosphorylated mTOR and HIF-1α were decreased after SW treatment. Conclusion: SW treatment can potentially promote cardiomyocyte autophagy during hypoxia and protect cardiomyocyte function by regulating the AMPK/mTOR pathway.

  13. Predicting species distribution and abundance responses to climate change: why it is essential to include biotic interactions across trophic levels.

    Van der Putten, Wim H; Macel, Mirka; Visser, Marcel E

    2010-07-12

    Current predictions on species responses to climate change strongly rely on projecting altered environmental conditions on species distributions. However, it is increasingly acknowledged that climate change also influences species interactions. We review and synthesize literature information on biotic interactions and use it to argue that the abundance of species and the direction of selection during climate change vary depending on how their trophic interactions become disrupted. Plant abundance can be controlled by aboveground and belowground multitrophic level interactions with herbivores, pathogens, symbionts and their enemies. We discuss how these interactions may alter during climate change and the resulting species range shifts. We suggest conceptual analogies between species responses to climate warming and exotic species introduced in new ranges. There are also important differences: the herbivores, pathogens and mutualistic symbionts of range-expanding species and their enemies may co-migrate, and the continuous gene flow under climate warming can make adaptation in the expansion zone of range expanders different from that of cross-continental exotic species. We conclude that under climate change, results of altered species interactions may vary, ranging from species becoming rare to disproportionately abundant. Taking these possibilities into account will provide a new perspective on predicting species distribution under climate change.

  14. KChIP2 genotype dependence of transient outward current (Ito) properties in cardiomyocytes isolated from male and female mice.

    Waldschmidt, Lara; Junkereit, Vera; Bähring, Robert

    2017-01-01

    The transient outward current (Ito) in cardiomyocytes is largely mediated by Kv4 channels associated with Kv Channel Interacting Protein 2 (KChIP2). A knockout model has documented the critical role of KChIP2 in Ito expression. The present study was conducted to characterize in both sexes the dependence of Ito properties, including current magnitude, inactivation kinetics, recovery from inactivation and voltage dependence of inactivation, on the number of functional KChIP2 alleles. For this purpose we performed whole-cell patch-clamp experiments on isolated left ventricular cardiomyocytes from male and female mice which had different KChIP2 genotypes; i.e., wild-type (KChIP2+/+), heterozygous knockout (KChIP2+/-) or complete knockout of KChIP2 (KChIP2-/-). We found in both sexes a KChIP2 gene dosage effect (i.e., a proportionality between number of alleles and phenotype) on Ito magnitude, however, concerning other Ito properties, KChIP2+/- resembled KChIP2+/+. Only in the total absence of KChIP2 (KChIP2-/-) we observed a slowing of Ito kinetics, a slowing of recovery from inactivation and a negative shift of a portion of the voltage dependence of inactivation. In a minor fraction of KChIP2-/- myocytes Ito was completely lost. The distinct KChIP2 genotype dependences of Ito magnitude and inactivation kinetics, respectively, seen in cardiomyocytes were reproduced with two-electrode voltage-clamp experiments on Xenopus oocytes expressing Kv4.2 and different amounts of KChIP2. Our results corroborate the critical role of KChIP2 in controlling Ito properties. They demonstrate that the Kv4.2/KChIP2 interaction in cardiomyocytes is highly dynamic, with a clear KChIP2 gene dosage effect on Kv4 channel surface expression but not on inactivation gating.

  15. Novel distribution of calreticulin to cardiomyocyte mitochondria and its increase in a rat model of dilated cardiomyopathy

    Zhang, Ming [Department of Cardiology, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Department of Respiratory Medicine, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Wei, Jin, E-mail: weijindr@163.com [Department of Cardiology, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Li, Yali [Department of Respiratory Medicine, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Shan, Hu; Yan, Rui; Lin, Lin [Department of Cardiology, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Zhang, Qiuhong [Department of Respiratory Medicine, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Xue, Jiahong [Department of Cardiology, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China)

    2014-06-20

    Highlights: • Calreticulin can also be found in cardiomyocyte mitochondria. • The mitochondrial content of calreticulin is increased in DCM hearts. • Increased expression of mitochondrial CRT may induce mitochondrial damage. • Mitochondrial CRT may inhibit the phosphorylation of mitochondrial STAT3. - Abstract: Background: Calreticulin (CRT), a Ca{sup 2+}-binding chaperone of the endoplasmic reticulum, can also be found in several other locations including the cytosol, nucleus, secretory granules, the outer side of the plasma membrane, and the extracellular matrix. Whether CRT is localized at mitochondria of cardiomyocytes and whether such localization is affected under DCM are still unclear. Methods and results: The DCM model was generated in rats by the daily oral administration of furazolidone for thirty weeks. Echocardiographic and hemodynamic studies demonstrated enlarged left ventricular dimensions and reduced systolic and diastolic function in DCM rats. Immuno-electron microscopy and Western blot showed that CRT was present in cardiomyocyte mitochondria and the mitochondrial content of CRT was increased in DCM hearts (P < 0.05). Morphometric analysis showed notable myocardial apoptosis and mitochondrial swelling with fractured or dissolved cristae in the DCM hearts. Compared with the control group, the mitochondrial membrane potential level of the freshly isolated cardiac mitochondria and the enzyme activities of cytochrome c oxidase and succinate dehydrogenase in the model group were significantly decreased (P < 0.05), and the myocardial apoptosis index and the caspase activities of caspase-9 and caspase-3 were significantly increased (P < 0.05). Pearson linear correlation analysis showed that the mitochondrial content of CRT had negative correlations with the mitochondrial function, and a positive correlation with myocardial apoptosis index (P < 0.001). The protein expression level of cytochrome c and the phosphorylation activity of STAT3 in the

  16. Rapid Induction of Aldosterone Synthesis in Cultured Neonatal Rat Cardiomyocytes under High Glucose Conditions

    Masami Fujisaki

    2013-01-01

    Full Text Available In addition to classical adrenal cortical biosynthetic pathway, there is increasing evidence that aldosterone is produced in extra-adrenal tissues. Although we previously reported aldosterone production in the heart, the concept of cardiac aldosterone synthesis remains controversial. This is partly due to lack of established experimental models representing aldosterone synthase (CYP11B2 expression in robustly reproducible fashion. We herein investigated suitable conditions in neonatal rat cardiomyocytes (NRCMs culture system producing CYP11B2 with considerable efficacy. NRCMs were cultured with various glucose doses for 2–24 hours. CYP11B2 mRNA expression and aldosterone concentrations secreted from NRCMs were determined using real-time PCR and enzyme immunoassay, respectively. We found that suitable conditions for CYP11B2 induction included four-hour incubation with high glucose conditions. Under these particular conditions, CYP11B2 expression, in accordance with aldosterone secretion, was significantly increased compared to those observed in the cells cultured under standard-glucose condition. Angiotensin II receptor blocker partially inhibited this CYP11B2 induction, suggesting that there is local renin-angiotensin-aldosterone system activation under high glucose conditions. The suitable conditions for CYP11B2 induction in NRCMs culture system are now clarified: high-glucose conditions with relatively brief period of culture promote CYP11B2 expression in cardiomyocytes. The current system will help to accelerate further progress in research on cardiac tissue aldosterone synthesis.

  17. Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform.

    Denning, Chris; Borgdorff, Viola; Crutchley, James; Firth, Karl S A; George, Vinoj; Kalra, Spandan; Kondrashov, Alexander; Hoang, Minh Duc; Mosqueira, Diogo; Patel, Asha; Prodanov, Ljupcho; Rajamohan, Divya; Skarnes, William C; Smith, James G W; Young, Lorraine E

    2016-07-01

    Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  18. A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

    Judge, Luke M; Perez-Bermejo, Juan A; Truong, Annie; Ribeiro, Alexandre Js; Yoo, Jennie C; Jensen, Christina L; Mandegar, Mohammad A; Huebsch, Nathaniel; Kaake, Robyn M; So, Po-Lin; Srivastava, Deepak; Pruitt, Beth L; Krogan, Nevan J; Conklin, Bruce R

    2017-07-20

    Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.

  19. Mutations in Alström Protein Impair Terminal Differentiation of Cardiomyocytes

    Shenje, Lincoln T.; Andersen, Peter; Halushka, Marc K.; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B.; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A. S.; Chen, Yan; Chelko, Stephen; Crosson, Jane E.

    2014-01-01

    Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inher...

  20. Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

    Shenje, Lincoln T; Andersen, Peter; Halushka, Marc K; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A S; Chen, Yan; Chelko, Stephen; Crosson, Jane E; Scheel, Janet; Vricella, Luca; Craig, Brian D; Marosy, Beth A; Mohr, David W; Hetrick, Kurt N; Romm, Jane M; Scott, Alan F; Valle, David; Naggert, Jürgen K; Kwon, Chulan; Doheny, Kimberly F; Judge, Daniel P

    2014-03-04

    Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

  1. Solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation: piece by piece.

    Lundy, David J; Lee, Desy S; Hsieh, Patrick C H

    2017-03-01

    There is a growing need for in vitro models which can serve as platforms for drug screening and basic research. Human adult cardiomyocytes cannot be readily obtained or cultured, and so pluripotent stem cell-derived cardiomyocytes appear to be an attractive option. Unfortunately, these cells are structurally and functionally immature-more comparable to foetal cardiomyocytes than adult. A recent study by Ruan et al ., provides new insights into accelerating the maturation process and takes us a step closer to solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation.

  2. A constitutive model for the compressive response of metallic closed-cell foams including micro-inertia effects

    Barthélémy Romain

    2015-01-01

    Full Text Available Metallic foams have known a keen interest in the last decades. Their ability to undergo very large deformations while transmitting low stress levels make them capable of performing functions of protective layers against intense loadings and of energy absorbers, for instance. The behaviour of metal foams varies considerably between quasi-static and dynamic regimes. Those differences can be linked to the strain-rate sensitivity of the skeleton material and to micro-inertial effects (induced by the crushing of the foam cells. In the present work, a micromechanical model has been developed to take into account micro-inertia effects on the macroscopic behaviour of closed-cell foams under dynamic loading conditions. The proposed modelling is based on the dynamic homogenisation procedure introduced by Molinari and Mercier (J. Mech. Phys. Solids 49 (2001 1497–1516. Within this framework, the macrostress is the sum of two terms. The first one is a static stress, that can be described with any existing model of metal foam. The second contribution is a dynamic stress related to micro-inertia effects. Considering an initially spherical shell as a Representative Volume Element (RVE of the foam material, a closed-form expression of the dynamic stress was obtained. The proposed modelling was applied to shock propagation in aluminium foams (it should however be noted that the present theory is not restricted to uniaxial deformation but can be applied to arbitrary loadings. From experimental data of the literature, it is observed that incorporating micro-inertia effects allows one to achieve a better description of the foam shock response. This indicates that micro-inertia may have a significant influence on the dynamic behaviour of metallic foams.

  3. Recombinant adeno-associated virus-delivered hypoxia-inducible stanniocalcin-1 expression effectively inhibits hypoxia-induced cell apoptosis in cardiomyocytes.

    Shi, Xin; Wang, Jianzhong; Qin, Yan

    2014-12-01

    Ischemia/hypoxia-induced oxidative stress is detrimental for the survival of cardiomyocytes and cardiac function. Stanniocalcin-1 (STC-1), a glycoprotein, has been found to play an inhibitory role in the production of reactive oxygen species (ROS). Here, we speculated that the overexpression of STC-1 might alleviate oxidative damage in cardiomyocytes under conditions of hypoxia. To control the expression of STC-1 in hypoxia, we constructed a recombinant adeno-associated virus (AAV) carrying the hypoxia-responsive element (HRE) to mediate hypoxia induction. Cardiomyocytes were infected with AAV-HRE-STC-1 and cultured in normoxic or hypoxic conditions, and STC-1 overexpression was only detected in hypoxic cultured cardiomyocytes by using quantitative real-time polymerase chain reaction and Western blot analysis. Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, AAV-HRE-STC-1 infection was shown to significantly enhance cell survival under hypoxia. Hypoxia-induced cell apoptosis was inhibited by AAV-HRE-STC-1 infection by using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide apoptosis assay. Moreover, the proapoptotic protein Caspase-3 and anti-apoptotic protein Bcl-2, which were dysregulated by hypoxia, were reversed by AAV-HRE-STC-1 infection. AAV-HRE-STC-1-mediated STC-1 overexpression markedly inhibited ROS production in cardiomyocytes cultured under hypoxic conditions. AAV-HRE-STC-1 infection significantly upregulated uncoupled protein 3 (UCP3), whereas silencing of UCP3 blocked the inhibitory effect of AAV-HRE-STC-1 on ROS production. In contrast, AAV-HRE-STC-1 infection had no effect on UCP2, and knockdown of UCP2 did not block the inhibitory effect of AAV-HRE-STC-1 on ROS production in the cardiomyocytes cultured under hypoxic conditions. Taken together, STC1 activates antioxidant pathway in cardiomyocytes through the induction of UCP3, implying that AAV-HRE-STC-1 has potential in the treatment of ischemic

  4. Mutation of KREMEN1, a modulator of Wnt signaling, is responsible for ectodermal dysplasia including oligodontia in Palestinian families.

    Issa, Yasmin A; Kamal, Lara; Rayyan, Amal Abu; Dweik, Dima; Pierce, Sarah; Lee, Ming K; King, Mary-Claire; Walsh, Tom; Kanaan, Moien

    2016-10-01

    Tooth development is controlled by the same processes that regulate formation of other ectodermal structures. Mutations in the genes underlying these processes may cause ectodermal dysplasia, including severe absence of primary or permanent teeth. Four consanguineous Palestinian families presented with oligodontia and hair and skin features of ectodermal dysplasia. Appearance of ectodermal dysplasia was consistent with autosomal recessive inheritance. Exome sequencing followed by genotyping of 56 informative relatives in the 4 families suggests that the phenotype is due to homozygosity for KREMEN1 p.F209S (c.626 T>C) on chromosome 22 at g.29,521,399 (hg19). The variant occurs in the highly conserved extracellular WSC domain of KREMEN1, which is known to be a high affinity receptor of Dickkopf-1, a component of the Dickkopf-Kremen-LRP6 complex, and a potent regulator of Wnt signaling. The Wnt signaling pathway is critical to development of ectodermal structures. Mutations in WNT10A, LRP6, EDA, and other genes in this pathway lead to tooth agenesis with or without other ectodermal anomalies. Our results implicate KREMEN1 for the first time in a human disorder and provide additional details on the role of the Wnt signaling in ectodermal and dental development.

  5. Doxorubicin impairs the insulin-like growth factor-1 system and causes insulin-like growth factor-1 resistance in cardiomyocytes.

    Patrizia Fabbi

    Full Text Available Insulin-like growth factor-1 (IGF-1 promotes the survival of cardiomyocytes by activating type 1 IGF receptor (IGF-1R. Within the myocardium, IGF-1 action is modulated by IGF binding protein-3 (IGFBP-3, which sequesters IGF-1 away from IGF-1R. Since cardiomyocyte apoptosis is implicated in anthracycline cardiotoxicity, we investigated the effects of the anthracycline, doxorubicin, on the IGF-1 system in H9c2 cardiomyocytes.Besides inducing apoptosis, concentrations of doxorubicin comparable to those observed in patients after bolus infusion (0.1-1 µM caused a progressive decrease in IGF-1R and increase in IGFBP-3 expression. Exogenous IGF-1 was capable to rescue cardiomyocytes from apoptosis triggered by 0.1 and 0.5 µM, but not 1 µM doxorubicin. The loss of response to IGF-1 was paralleled by a significant reduction in IGF-1 availability and signaling, as assessed by free hormone levels in conditioned media and Akt phosphorylation in cell lysates, respectively. Doxorubicin also dose-dependently induced p53, which is known to repress the transcription of IGF1R and induce that of IGFBP3. Pre-treatment with the p53 inhibitor, pifithrin-α, prevented apoptosis and the changes in IGF-1R and IGFBP-3 elicited by doxorubicin. The decrease in IGF-1R and increase in IGFBP-3, as well as apoptosis, were also antagonized by pre-treatment with the antioxidant agents, N-acetylcysteine, dexrazoxane, and carvedilol.Doxorubicin down-regulates IGF-1R and up-regulates IGFBP-3 via p53 and oxidative stress in H9c2 cells. This leads to resistance to IGF-1 that may contribute to doxorubicin-initiated apoptosis. Further studies are needed to confirm these findings in human cardiomyocytes and explore the possibility of manipulating the IGF-1 axis to protect against anthracycline cardiotoxicity.

  6. Apelin and APJ orchestrate complex tissue-specific control of cardiomyocyte hypertrophy and contractility in the hypertrophy-heart failure transition.

    Parikh, Victoria Nicole; Liu, Jing; Shang, Ching; Woods, Christopher; Chang, Alex Chia Yu; Zhao, Mingming; Charo, David N; Grunwald, Zachary; Huang, Yong; Seo, Kinya; Tsao, Philip S; Bernstein, Daniel; Ruiz-Lozano, Pilar; Quertermous, Thomas; Ashley, Euan A

    2018-05-18

    The G protein coupled receptor APJ is a promising therapeutic target for heart failure. Constitutive deletion of APJ in the mouse is protective against the hypertrophy-heart failure transition via elimination of ligand-independent, β-arrestin dependent stretch transduction. However, the cellular origin of this stretch transduction and the details of its interaction with apelin signaling remain unknown. We generated mice with conditional elimination of APJ in the endothelium (APJ endo-/- ) and myocardium (APJ myo-/- ). No baseline difference was observed in LV function in APJ endo-/- , APJ myo-/- or controls (APJ endo+/+ , APJ myo+/+ ). After exposure to transaortic constriction (TAC), APJ endo-/- animals developed left ventricular failure while APJ myo-/- were protected. At the cellular level, carbon fiber stretch of freshly isolated single cardiomyocytes demonstrated decreased contractile response to stretch in APJ -/- cardiomyocytes compared to APJ +/+ cardiomyocytes. Calcium transient did not change with stretch in either APJ -/- or APJ +/+ cardiomyocytes. Application of apelin to APJ +/+ cardiomyocytes resulted in decreased calcium transient. Further, hearts of mice treated with apelin exhibited decreased phosphorylation at Troponin I (cTnI) N-terminal residues (Ser 22,23), consistent with increased calcium sensitivity. These data establish that APJ stretch transduction is mediated specifically by myocardial APJ, that APJ is necessary for stretch-induced increases in contractility, and that apelin opposes APJ's stretch-mediated hypertrophy signaling by lowering calcium transient while maintaining contractility through myofilament calcium sensitization. These findings underscore apelin's unique potential as a therapeutic agent that can simultaneously support cardiac function and protect against the hypertrophy-heart failure transition.

  7. [Over-expression of BDNF inhibits angiotensin II-induced apoptosis of cardiomyocytes in SD rats].

    Cao, Jingli; Wu, Yingfeng; Liu, Geming; Li, Zhenlong

    2018-03-01

    Objective To investigate the role and molecular mechanism of brain-derived neurotrophic factor (BDNF) against the process of cardiomyocyte hypertrophy and apoptosis. Methods Cardiomyocyte hypertrophy were estabolished by angiotensin II (Ang II) in neonatal cardiomyocytes in vitro and incomplete ligature of abdominal aorta of SD rats in vivo. BDNF over-expressing recombinant vector pcDNA5-BDNF was transfected into cardiomyocytes by liposomes. Immunofluorescence staining was used to detect the effect of BDNF transfection on the surface area of myocardial cells. The effect of BDNF transfection on the apoptosis of cardiomyocytes was assayed by flow cytometry. Real-time fluorescent quantitative PCR was performed to detect the effect of over-expression of BDNF on the expressions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNAs in cardiomyocytes. Western blot assay was used to observe the changes of BDNF, ANP and BNP, calmodulin kinase 2 (CaMK2) and phosphorylated calmodulin kinase 2 (p-CaMK2), calcineurin (CaN), p-CaN, nuclear factor of activated T cells 3 (NFATC3) and p-NFATC3 protein expressions in the myocardial tissues and cardiomyocytes. Results The expression of BDNF protein increased significantly in cardiac hypertrophy animal and cell models in a time-dependent manner. Compared with the untransfected control cardiomyocytes, the surface area of cardiomyocytes, the rate of apoptosis, the levels of ANP and BNP mRNA and protein expression, the levels of p-CaMK2 and CaN protein in the BDNF over-expressed cardiomyocytes were remarkably reduced, while the level of p-NFATC3 protein rose significantly. Conclusion BDNF inhibits the apoptosis of cardiomyocytes induced by Ang II, and it plays the role by inhibiting CaMK2 and CaN signaling pathways.

  8. Pulmonary response to surface‐coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: A toxicogenomic study

    Halappanavar, Sabina; Jackson, Petra; Williams, Andrew

    2011-01-01

    with acute phase, inflammation and immune response 5 days post exposure with concomitant changes in several miRNAs. The role of these miRNAs in pulmonary response to inhaled particles is unknown and warrants further research. Environ. Mol. Mutagen., 2011. © 2011 Wiley‐Liss, Inc....... in increased levels of mRNA for acute phase markers serum amyloid A‐1 (Saa1) and serum amyloid A‐3 (Saa3), several C‐X‐C and C‐C motif chemokines, and cytokine tumor necrosis factor genes. Protein analysis of Saa1 and 3 showed selective upregulation of Saa3 in lung tissues. Sixteen miRNAs were induced by more...... than 1.2‐fold (adjusted P‐value changes in the expression of genes associated...

  9. High-dose benfotiamine rescues cardiomyocyte contractile dysfunction in streptozotocin-induced diabetes mellitus.

    Ceylan-Isik, Asli F; Wu, Shan; Li, Qun; Li, Shi-Yan; Ren, Jun

    2006-01-01

    Diabetic cardiomyopathy is characterized by cardiac dysfunction. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse cardiomyocytes. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg ip). Fourteen days later, control and diabetic (fasting plasma glucose > 13.9 mM) mice were put on benfotiamine therapy (100 mg.kg(-1).day(-1) ip) for another 14 days. Mechanical and intracellular Ca2+ properties were evaluated in left ventricular myocytes using an IonOptix MyoCam system. The following indexes were evaluated: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening, resting and rise of intracellular Ca2+ in response to electrical stimulus, sarcoplasmic reticulum (SR) Ca2+ load, and intracellular Ca2+ decay rate (tau). Two- or four-week STZ treatment led to hyperglycemia, prolonged TPS and TR90, reduced SR Ca2+ load, elevated resting intracellular Ca2+ level and prolonged tau associated with normal PS, maximal velocity of shortening/relengthening, and intracellular Ca2+ rise in response to electrical stimulus. Benfotiamine treatment abolished prolongation in TPS, TR90, and tau, as well as reduction in SR Ca2+ load without affecting hyperglycemia and elevated resting intracellular Ca2+. Diabetes triggered oxidative stress, measured by GSH-to-GSSG ratio and formation of advanced glycation end product (AGE) in the hearts. Benfotiamine treatment alleviated oxidative stress without affecting AGE or protein carbonyl formation. Collectively, our results indicated that benfotiamine may rescue STZ-induced cardiomyocyte dysfunction but not AGE formation in short-term diabetes.

  10. Basal and β-Adrenergic Cardiomyocytes Contractility Dysfunction Induced by Dietary Protein Restriction is Associated with Downregulation of SERCA2a Expression and Disturbance of Endoplasmic Reticulum Ca2+ Regulation in Rats

    Arlete R. Penitente

    2014-07-01

    Full Text Available Background: The mechanisms responsible for the cardiac dysfunction associated with dietary protein restriction (PR are poorly understood. Thus, this study was designed to evaluate the effects of PR on calcium kinetics, basal and β-adrenergic contractility in murine ventricular cardiomyocytes. Methods: After breastfeeding male Fisher rats were distributed into a control group (CG, n = 20 and a protein-restricted group (PRG, n = 20, receiving isocaloric diets for 35 days containing 15% and 6% protein, respectively. Biometric and hemodynamic variables were measured. After euthanasia left ventricles (LV were collected for histopathological evaluation, SERCA2a expression, cardiomyocytes contractility and Ca2+sparks analysis. Results: PRG animals showed reduced general growth, increased heart rate and arterial pressure. These animals presented extracellular matrix expansion and disorganization, cardiomyocytes hypotrophy, reduced amplitudes of shortening and maximum velocity of contraction and relaxation at baseline and after β-adrenergic stimulation. Reduced SERCA2a expression as well as higher frequency and lower amplitude of Ca2+sparks were observed in PRG cardiomyocytes. Conclusion: The observations reveal that protein restriction induces marked myocardial morphofunctional damage. The pathological changes of cardiomyocyte mechanics suggest the potential involvement of the β-adrenergic system, which is possibly associated with changes in SERCA2a expression and disturbances in Ca2+ intracellular kinetics.

  11. BAG3 role in cardiomyocytes physiopathology

    De Marco, Margot

    2013-01-01

    2010 - 2011 The anti-apoptotic protein BAG3 is expressed at high levels in skeletal and cardiac muscle in vivo. Our group recently focused its interest on BAG3 role in myocardiocyte proliferation, survival and response to stressful stimuli. We found that BAG3 is upregulated during the differentiation of cardiomyoblasts. Our results prompted us to verify whether bag3 silencing could affect the differentiation state of cardiocytes and we found that bag3 silencing resulted in highly reducing ...

  12. The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles.

    Bing Xu

    2017-08-01

    Full Text Available Microparticles (MPs are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17 creates transverse-tubule (t-tubule membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA, and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT-III subunit charged multivesicular body protein 4B (CHMP4B colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily

  13. The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles.

    Xu, Bing; Fu, Ying; Liu, Yan; Agvanian, Sosse; Wirka, Robert C; Baum, Rachel; Zhou, Kang; Shaw, Robin M; Hong, TingTing

    2017-08-01

    Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT

  14. Immaturity of human stem-cell-derived cardiomyocytes in culture: fatal flaw or soluble problem?

    Veerman, Christiaan C.; Kosmidis, Georgios; Mummery, Christine L.; Casini, Simona; Verkerk, Arie O.; Bellin, Milena

    2015-01-01

    Cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are increasingly used to model cardiac disease, test drug efficacy and for safety pharmacology. Nevertheless, a major hurdle to more extensive use is their immaturity and similarity to fetal rather than adult cardiomyocytes. Here, we

  15. Microscale Generation of Cardiospheres Promotes Robust Enrichment of Cardiomyocytes Derived from Human Pluripotent Stem Cells

    Doan C. Nguyen

    2014-08-01

    Full Text Available Cardiomyocytes derived from human pluripotent stem cells (hPSCs are a promising cell source for regenerative medicine, disease modeling, and drug discovery, all of which require enriched cardiomyocytes, ideally ones with mature phenotypes. However, current methods are typically performed in 2D environments that produce immature cardiomyocytes within heterogeneous populations. Here, we generated 3D aggregates of cardiomyocytes (cardiospheres from 2D differentiation cultures of hPSCs using microscale technology and rotary orbital suspension culture. Nearly 100% of the cardiospheres showed spontaneous contractility and synchronous intracellular calcium transients. Strikingly, from starting heterogeneous populations containing ∼10%–40% cardiomyocytes, the cell population within the generated cardiospheres featured ∼80%–100% cardiomyocytes, corresponding to an enrichment factor of up to 7-fold. Furthermore, cardiomyocytes from cardiospheres exhibited enhanced structural maturation in comparison with those from a parallel 2D culture. Thus, generation of cardiospheres represents a simple and robust method for enrichment of cardiomyocytes in microtissues that have the potential use in regenerative medicine as well as other applications.

  16. Dystrophin-deficient cardiomyocytes derived from human urine: New biologic reagents for drug discovery

    Xuan Guan

    2014-03-01

    Full Text Available The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD. Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs. USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.

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

    Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.; van Meer, Berend; Ward-van Oostwaard, Dorien; Passier, Robert; Tertoolen, Leon G. J.; Mummery, Christine L.; Casini, Simona

    2015-01-01

    One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes

  18. Imaging alterations of cardiomyocyte cAMP microdomains in disease

    Alexander eFroese

    2015-08-01

    Full Text Available 3’,5’-cyclic adenosine monophosphate (cAMP is an important second messenger which regulates heart function by acting in distinct subcellular microdomains. Recent years have provided deeper mechanistic insights into compartmentalized cAMP signaling and its link to cardiac disease. In this mini review, we summarize newest developments in this field achieved by cutting-edge biochemical and biophysical techniques. We further compile the data from different studies into a bigger picture of so far uncovered alterations in cardiomyocyte cAMP microdomains which occur in compensated cardiac hypertrophy and chronic heart failure. Finally, future research directions and translational perspectives are briefly discussed.

  19. The primary cilium coordinates early cardiogenesis and hedgehog signaling in cardiomyocyte differentiation

    Clement, Christian A; Kristensen, Stine G; Møllgård, Kjeld

    2009-01-01

    Defects in the assembly or function of primary cilia, which are sensory organelles, are tightly coupled to developmental defects and diseases in mammals. Here, we investigated the function of the primary cilium in regulating hedgehog signaling and early cardiogenesis. We report that the pluripotent...... P19.CL6 mouse stem cell line, which can differentiate into beating cardiomyocytes, forms primary cilia that contain essential components of the hedgehog pathway, including Smoothened, Patched-1 and Gli2. Knockdown of the primary cilium by Ift88 and Ift20 siRNA or treatment with cyclopamine...... development. These data support the conclusion that cardiac primary cilia are crucial in early heart development, where they partly coordinate hedgehog signaling....

  20. Numerical investigation of perforated polymer microcantilever sensor for contractile behavior of cardiomyocytes

    Khoa Nguyen, Trieu; Lee, Dong-Weon; Lee, Bong-Kee

    2017-06-01

    In this study, a numerical investigation of microcantilever sensors for detecting the contractile behavior of cardiomyocytes (CMs) was performed. Recently, a novel surface-patterned perforated SU-8 microcantilever sensor has been developed for the preliminary screening of cardiac toxicity. From the contractile motion of the CMs cultured on the microcantilever surface, a macroscopic bending of the microcantilever was obtained, which is considered to reflect a physiological change. As a continuation of the previous research, a novel numerical method based on a surface traction model was proposed and verified to further understand the bending behavior of the microcantilevers. Effects of various factors, including surface traction magnitude, focal area of CMs, and stiffness of microcantilever, on the bending displacement were investigated. From static and transient analyses, the focal area was found to be the most crucial factor. In addition, the current result can provide a design guideline for various micromechanical devices based on the same principle.

  1. Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome

    Xu, Aibin; Liu, Jingyi; Liu, Peilin; Jia, Min; Wang, Han; Tao, Ling

    2014-01-01

    Highlights: • Metabolic syndrome exacerbated MI/R induced injury accompanied by decreased Nur77. • ROS led to Nur77 translocation in metabolic syndrome. • Inhibiting relocation of Nur77 to mitochondria reduced ROS-induced cardiomyocyte injury in metabolic syndrome. - Abstract: Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H 2 O 2 led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H 2 O 2 and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the process

  2. Exercise training prior to myocardial infarction attenuates cardiac deterioration and cardiomyocyte dysfunction in rats

    Luiz Henrique Marchesi Bozi

    2013-04-01

    Full Text Available OBJECTIVES: The present study was performed to investigate 1 whether aerobic exercise training prior to myocardial infarction would prevent cardiac dysfunction and structural deterioration and 2 whether the potential cardiac benefits of aerobic exercise training would be associated with preserved morphological and contractile properties of cardiomyocytes in post-infarct remodeled myocardium. METHODS: Male Wistar rats underwent an aerobic exercise training protocol for eight weeks. The rats were then assigned to sham surgery (SHAM, sedentary lifestyle and myocardial infarction or exercise training and myocardial infarction groups and were evaluated 15 days after the surgery. Left ventricular tissue was analyzed histologically, and the contractile function of isolated myocytes was measured. Student's t-test was used to analyze infarct size and ventricular wall thickness, and the other parameters were analyzed by the Kruskal-Wallis test followed by Dunn's test or a one-way analysis of variance followed by Tukey's test (p<0.05. RESULTS: Myocardial infarctions in exercise-trained animals resulted in a smaller myocardial infarction extension, a thicker infarcted wall and less collagen accumulation as compared to myocardial infarctions in sedentary animals. Myocardial infarction-induced left ventricular dilation and cardiac dysfunction, as evaluated by +dP/dt and -dP/dt, were both prevented by previous aerobic exercise training. Moreover, aerobic exercise training preserved cardiac myocyte shortening, improved the maximum shortening and relengthening velocities in infarcted hearts and enhanced responsiveness to calcium. CONCLUSION: Previous aerobic exercise training attenuated the cardiac dysfunction and structural deterioration promoted by myocardial infarction, and such benefits were associated with preserved cardiomyocyte morphological and contractile properties.

  3. Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome

    Xu, Aibin; Liu, Jingyi [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China); Institute of Cardiovascular Disease, General Hospital of Beijing Command, PLA, Beijing (China); Liu, Peilin; Jia, Min; Wang, Han [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China); Tao, Ling, E-mail: lingtao2006@gmail.com [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China)

    2014-04-18

    Highlights: • Metabolic syndrome exacerbated MI/R induced injury accompanied by decreased Nur77. • ROS led to Nur77 translocation in metabolic syndrome. • Inhibiting relocation of Nur77 to mitochondria reduced ROS-induced cardiomyocyte injury in metabolic syndrome. - Abstract: Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H{sub 2}O{sub 2} led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H{sub 2}O{sub 2} and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the

  4. Altering CO2 during reperfusion of ischemic cardiomyocytes modifies mitochondrial oxidant injury.

    Lavani, Romeen; Chang, Wei-Tien; Anderson, Travis; Shao, Zuo-Hui; Wojcik, Kimberly R; Li, Chang-Qing; Pietrowski, Robert; Beiser, David G; Idris, Ahamed H; Hamann, Kimm J; Becker, Lance B; Vanden Hoek, Terry L

    2007-07-01

    Acute changes in tissue CO2 and pH during reperfusion of the ischemic heart may affect ischemia/reperfusion injury. We tested whether gradual vs. acute decreases in CO2 after cardiomyocyte ischemia affect reperfusion oxidants and injury. Comparative laboratory investigation. Institutional laboratory. Embryonic chick cardiomyocytes. Microscope fields of approximately 500 chick cardiomyocytes were monitored throughout 1 hr of simulated ischemia (PO2 of 3-5 torr, PCO2 of 144 torr, pH 6.8), followed by 3 hrs of reperfusion (PO2 of 149 torr, PCO2 of 36 torr, pH 7.4), and compared with cells reperfused with relative hypercarbia (PCO2 of 71 torr, pH 6.8) or hypocarbia (PCO2 of 7 torr, pH 7.9). The measured outcomes included cell viability (via propidium iodide) and oxidant generation (reactive oxygen species via 2',7'-dichlorofluorescin oxidation and nitric oxide [NO] via 4,5-diaminofluorescein diacetate oxidation). Compared with normocarbic reperfusion, hypercarbia significantly reduced cell death from 54.8% +/- 4.0% to 26.3% +/- 2.8% (p < .001), significantly decreased reperfusion reactive oxygen species (p < .05), and increased NO at a later phase of reperfusion (p < .01). The NO synthase inhibitor N-nitro-L-arginine methyl ester (200 microM) reversed this oxidant attenuation (p < .05), NO increase (p < .05), and the cardioprotection conferred by hypercarbic reperfusion (increasing death to 54.3% +/- 6.0% [p < .05]). Conversely, hypocarbic reperfusion increased cell death to 80.4% +/- 4.5% (p < .01). It also increased reactive oxygen species by almost two-fold (p = .052), without affecting the NO level thereafter. Increased reactive oxygen species was attenuated by the mitochondrial complex III inhibitor stigmatellin (20 nM) when given at reperfusion (p < .05). Cell death also decreased from 85.9% +/- 4.5% to 52.2% +/- 6.5% (p < .01). The nicotinamide adenine dinucleotide phosphate oxidase inhibitor apocynin (300 microM) had no effect on reperfusion reactive oxygen

  5. FOG-2 mediated recruitment of the NuRD complex regulates cardiomyocyte proliferation during heart development.

    Garnatz, Audrey S; Gao, Zhiguang; Broman, Michael; Martens, Spencer; Earley, Judy U; Svensson, Eric C

    2014-11-01

    FOG-2 is a multi-zinc finger protein that binds the transcriptional activator GATA4 and modulates GATA4-mediated regulation of target genes during heart development. Our previous work has demonstrated that the Nucleosome Remodeling and Deacetylase (NuRD) complex physically interacts with FOG-2 and is necessary for FOG-2 mediated repression of GATA4 activity in vitro. However, the relevance of this interaction for FOG-2 function in vivo has remained unclear. In this report, we demonstrate the importance of FOG-2/NuRD interaction through the generation and characterization of mice homozygous for a mutation in FOG-2 that disrupts NuRD binding (FOG-2(R3K5A)). These mice exhibit a perinatal lethality and have multiple cardiac malformations, including ventricular and atrial septal defects and a thin ventricular myocardium. To investigate the etiology of the thin myocardium, we measured the rate of cardiomyocyte proliferation in wild-type and FOG-2(R3K5A) developing hearts. We found cardiomyocyte proliferation was reduced by 31±8% in FOG-2(R3K5A) mice. Gene expression analysis indicated that the cell cycle inhibitor Cdkn1a (p21(cip1)) is up-regulated 2.0±0.2-fold in FOG-2(R3K5A) hearts. In addition, we demonstrate that FOG-2 can directly repress the activity of the Cdkn1a gene promoter, suggesting a model by which FOG-2/NuRD promotes ventricular wall thickening by repression of this cell cycle inhibitor. Consistent with this notion, the genetic ablation of Cdkn1a in FOG-2(R3K5A) mice leads to an improvement in left ventricular function and a partial rescue of left ventricular wall thickness. Taken together, our results define a novel mechanism in which FOG-2/NuRD interaction is required for cardiomyocyte proliferation by directly down-regulating the cell cycle inhibitor Cdkn1a during heart development. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Developmental changes in electrophysiological characteristics of human induced Pluripotent Stem Cell-derived cardiomyocytes

    Ben-Ari, Meital; Naor, Shulamit; Zeevi-Levin, Naama; Schick, Revital; Ben Jehuda, Ronen; Reiter, Irina; Raveh, Amit; Grijnevitch, Inna; Barak, Omri; Rosen, Michael R.; Weissman, Amir; Binah, Ofer

    2016-01-01

    Background Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) only 3 types of action potentials (AP) exist: nodal, atrial and ventricular-like. Objective To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) during culture development a cardiac precursor cell is present that - depending on age - can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of nodal phenotype, transient appearance of atrial phenotype, evolution to ventricular phenotype, and persistence of transitional phenotypes. Methods To test these hypotheses we: (1) performed FACS analysis of nodal, atrial and ventricular markers; (2) recorded AP from 280 7-to-95 day old iPSC-CMs; (3) analyzed AP characteristics. Results The major findings were: (1) FACS analysis of 30 and 60-day old cultures showed that an iPSC-CMs population shifts from nodal into atrial/ventricular phenotype, while including significant transitional populations.(2) The AP population did not consist of 3 distinct phenotypes; (3) Culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57–70 days) appearance of atrial phenotype; (4) Beat Rate Variability was more prominent in nodal than ventricular cardiomyocytes while If density increased in older cultures. Conclusions From the onset of development the iPSC-CMs population includes nodal, atrial and ventricular AP and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial which appears only transiently, yet dominates at 57–70 days of evolution. PMID:27639456

  7. Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes.

    Ben-Ari, Meital; Naor, Shulamit; Zeevi-Levin, Naama; Schick, Revital; Ben Jehuda, Ronen; Reiter, Irina; Raveh, Amit; Grijnevitch, Inna; Barak, Omri; Rosen, Michael R; Weissman, Amir; Binah, Ofer

    2016-12-01

    Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs), only 3 types of action potentials (APs) exist: nodal-, atrial-, and ventricular-like. To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) During culture development a cardiac precursor cell is present that-depending on age-can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of a nodal phenotype, transient appearance of an atrial phenotype, evolution to a ventricular phenotype, and persistence of transitional phenotypes. To test these hypotheses, we (1) performed fluorescence-activated cell sorting analysis of nodal, atrial, and ventricular markers; (2) recorded APs from 280 7- to 95-day-old iPSC-CMs; and (3) analyzed AP characteristics. The major findings were as follows: (1) fluorescence-activated cell sorting analysis of 30- and 60-day-old cultures showed that an iPSC-CMs population shifts from the nodal to the atrial/ventricular phenotype while including significant transitional populations; (2) the AP population did not consist of 3 phenotypes; (3) culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of the atrial phenotype; and (4) beat rate variability was more prominent in nodal than in ventricular cardiomyocytes, while pacemaker current density increased in older cultures. From the onset of development in culture, the iPSC-CMs population includes nodal, atrial, and ventricular APs and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial, which appears only transiently yet dominates at 57-70 days of evolution. Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  8. Systematic review including re-analyses of 1148 individual data sets of central venous pressure as a predictor of fluid responsiveness

    Eskesen, T G; Wetterslev, M; Perner, A

    2016-01-01

    PURPOSE: Central venous pressure (CVP) has been shown to have poor predictive value for fluid responsiveness in critically ill patients. We aimed to re-evaluate this in a larger sample subgrouped by baseline CVP values. METHODS: In April 2015, we systematically searched and included all clinical...

  9. Characterization of Genotoxic Response to 15 Multiwalled Carbon Nanotubes with Variable Physicochemical Properties Including Surface Functionalizations in the FE1-Muta(TM) Mouse Lung Epithelial Cell Line

    Jackson, Petra; Kling, Kirsten; Jensen, Keld Alstrup

    2015-01-01

    Carbon nanotubes vary greatly in physicochemical properties. We compared cytotoxic and genotoxic response to 15 multiwalled carbon nanotubes (MWCNT) with varying physicochemical properties to identify drivers of toxic responses. The studied MWCNT included OECD Working Party on Manufactured...... Nanomaterials (WPMN) (NM-401, NM-402, and NM-403), materials (NRCWE-026 and MWCNT-XNRI-7), and three sets of surface-modified MWCNT grouped by physical characteristics (thin, thick, and short I-III, respectively). Each Groups I-III included pristine, hydroxylated and carboxylated MWCNT. Group III also included...... an amino-functionalized MWCNT. The level of surface functionalization of the MWCNT was low. The level and type of elemental impurities of the MWCNT varied by...

  10. Induced pluripotent stem cell derived cardiomyocytes as models for cardiac arrhythmias

    Maaike eHoekstra

    2012-08-01

    Full Text Available Cardiac arrhythmias are a major cause of morbidity and mortality. In younger patients, the majority of sudden cardiac deaths have an underlying Mendelian genetic cause. Over the last 15 years, enormous progress has been made in identifying the distinct clinical phenotypes and in studying the basic cellular and genetic mechanisms associated with the primary Mendelian (monogenic arrhythmia syndromes. Investigation of the electrophysiological consequences of an ion channel mutation is ideally done in the native cardiomyocyte environment. However, the majority of such studies so far have relied on heterologous expression systems in which single ion channel genes are expressed in non-cardiac cells. In some cases, transgenic mouse models haven been generated, but these also have significant shortcomings, primarily related to species differences.The discovery that somatic cells can be reprogrammed to pluripotency as induced pluripotent stem cells (iPSC has generated much interest since it presents an opportunity to generate patient- and disease-specific cell lines from which normal and diseased human cardiomyocytes can be obtained These genetically diverse human model systems can be studied in vitro and used to decipher mechanisms of disease and identify strategies and reagents for new therapies. Here we review the present state of the art with respect to cardiac disease models already generated using IPSC technology and which have been (partially characterized.Human iPSC (hiPSC models have been described for the cardiac arrhythmia syndromes, including LQT1, LQT2, LQT3-Brugada Syndrome, LQT8/Timothy syndrome and catecholaminergic polymorphic ventricular tachycardia. In most cases, the hiPSC-derived cardiomyoctes recapitulate the disease phenotype and have already provided opportunities for novel insight into cardiac pathophysiology. It is expected that the lines will be useful in the development of pharmacological agents for the management of these

  11. Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs).

    Sala, Luca; Ward-van Oostwaard, Dorien; Tertoolen, Leon G J; Mummery, Christine L; Bellin, Milena

    2017-05-12

    Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

  12. c-kitpos GATA-4 high rat cardiac stem cells foster adult cardiomyocyte survival through IGF-1 paracrine signalling.

    Nanako Kawaguchi

    2010-12-01

    Full Text Available Resident c-kit positive (c-kitpos cardiac stem cells (CSCs could be considered the most appropriate cell type for myocardial regeneration therapies. However, much is still unknown regarding their biological properties and potential.We produced clones of high and low expressing GATA-4 CSCs from long-term bulk-cultured c-kitpos CSCs isolated from adult rat hearts. When c-kitpos GATA-4 high expressing clonal CSCs (cCSCs were co-cultured with adult rat ventricular cardiomyocytes, we observed increased survival and contractility of the cardiomyocytes, compared to cardiomyocytes cultured alone, co-cultured with fibroblasts or c-kitpos GATA-4 low expressing cCSCs. When analysed by ELISA, the concentration of IGF-1 was significantly increased in the c-kitpos GATA-4 high cCSC/cardiomyocyte co-cultures and there was a significant correlation between IGF-1 concentration and cardiomyocyte survival. We showed the activation of the IGF-1 receptor and its downstream molecular targets in cardiomyocytes co-cultured with c-kitpos GATA-4 high cCSCs but not in cardiomyocytes that were cultured alone, co-cultured with fibroblasts or c-kitpos GATA-4 low cCSCs. Addition of a blocking antibody specific to the IGF-1 receptor inhibited the survival of cardiomyocytes and prevented the activation of its signalling in cardiomyocytes in the c-kitpos GATA-4 high cCSC/cardiomyocyte co-culture system. IGF-1 supplementation or IGF-1 high conditioned medium taken from the co-culture of c-kitpos GATA-4 high cCSCs plus cardiomyocytes did extend the survival and contractility of cardiomyocytes cultured alone and cardiomyocytes co-cultured with c-kitpos GATA-4 low cCSCs.c-kitpos GATA-4 high cCSCs exert a paracrine survival effect on cardiomyocytes through induction of the IGF-1R and signalling pathway.

  13. Predictive lethal proarrhythmic risk evaluation using a closed-loop-circuit cell network with human induced pluripotent stem cells derived cardiomyocytes

    Nomura, Fumimasa; Hattori, Akihiro; Terazono, Hideyuki; Kim, Hyonchol; Odaka, Masao; Sugio, Yoshihiro; Yasuda, Kenji

    2016-06-01

    For the prediction of lethal arrhythmia occurrence caused by abnormality of cell-to-cell conduction, we have developed a next-generation in vitro cell-to-cell conduction assay, i.e., a quasi in vivo assay, in which the change in spatial cell-to-cell conduction is quantitatively evaluated from the change in waveforms of the convoluted electrophysiological signals from lined-up cardiomyocytes on a single closed loop of a microelectrode of 1 mm diameter and 20 µm width in a cultivation chip. To evaluate the importance of the closed-loop arrangement of cardiomyocytes for prediction, we compared the change in waveforms of convoluted signals of the responses in the closed-loop circuit arrangement with that of the response of cardiomyocyte clusters using a typical human ether a go-go related gene (hERG) ion channel blocker, E-4031. The results showed that (1) waveform prolongation and fluctuation both in the closed loops and clusters increased depending on the E-4031 concentration increase. However, (2) only the waveform signals in closed loops showed an apparent temporal change in waveforms from ventricular tachycardia (VT) to ventricular fibrillation (VF), which is similar to the most typical cell-to-cell conductance abnormality. The results indicated the usefulness of convoluted waveform signals of a closed-loop cell network for acquiring reproducible results acquisition and more detailed temporal information on cell-to-cell conduction.

  14. Exposure to rosiglitazone, a PPAR-γ agonist, in late gestation reduces the abundance of factors regulating cardiac metabolism and cardiomyocyte size in the sheep fetus.

    Lie, Shervi; Hui, Melisa; McMillen, I Caroline; Muhlhausler, Beverly S; Posterino, Giuseppe S; Dunn, Stacey L; Wang, Kimberley C; Botting, Kimberley J; Morrison, Janna L

    2014-03-15

    It is unknown whether cardiomyocyte hypertrophy and the transition to fatty acid oxidation as the main source of energy after birth is dependent on the maturation of the cardiomyocytes' metabolic system, or on the limitation of substrate availability before birth. This study aimed to investigate whether intrafetal administration of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, during late gestation can stimulate the expression of factors regulating cardiac growth and metabolism in preparation for birth, and the consequences of cardiac contractility in the fetal sheep at ∼140 days gestation. The mRNA expression and protein abundance of key factors regulating growth and metabolism were quantified using quantitative RT-PCR and Western blot analysis, respectively. Cardiac contractility was determined by measuring the Ca(2+) sensitivity and maximum Ca(2+)-activated force of skinned cardiomyocyte bundles. Rosiglitazone-treated fetuses had a lower cardiac abundance of insulin-signaling molecules, including insulin receptor-β, insulin receptor substrate-1 (IRS-1), phospho-IRS-1 (Tyr-895), phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, PI3K catalytic subunit p110α, phospho-3-phosphoinositide-dependent protein kinase 1 (Ser-241), protein kinase B (Akt-1), phospho-Akt (Ser-273), PKCζ, phospho-PKCζ(Thr-410), Akt substrate 160 kDa (AS160), phospho-AS160 (Thr-642), and glucose transporter type-4. Additionally, cardiac abundance of regulators of fatty acid β-oxidation, including adiponectin receptor 1, AMPKα, phospho-AMPKα (Thr-172), phospho-acetyl CoA carboxylase (Ser-79), carnitine palmitoyltransferase-1, and PGC-1α was lower in the rosiglitazone-treated group. Rosiglitazone administration also resulted in a decrease in cardiomyocyte size. Rosiglitazone administration in the late-gestation sheep fetus resulted in a decreased abundance of factors regulating cardiac glucose uptake, fatty acid β-oxidation, and

  15. A Review of Human Pluripotent Stem Cell-Derived Cardiomyocytes for High-Throughput Drug Discovery, Cardiotoxicity Screening and Publication Standards

    Mordwinkin, Nicholas M.; Burridge, Paul W.; Wu, Joseph C.

    2012-01-01

    Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results, and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and ...

  16. Targeting Cardiomyocyte Ca2+ Homeostasis in Heart Failure

    Røe, Åsmund T.; Frisk, Michael; Louch, William E.

    2015-01-01

    Improved treatments for heart failure patients will require the development of novel therapeutic strategies that target basal disease mechanisms. Disrupted cardiomyocyte Ca2+ homeostasis is recognized as a major contributor to the heart failure phenotype, as it plays a key role in systolic and diastolic dysfunction, arrhythmogenesis, and hypertrophy and apoptosis signaling. In this review, we outline existing knowledge of the involvement of Ca2+ homeostasis in these deficits, and identify four promising targets for therapeutic intervention: the sarcoplasmic reticulum Ca2+ ATPase, the Na+-Ca2+ exchanger, the ryanodine receptor, and t-tubule structure. We discuss experimental data indicating the applicability of these targets that has led to recent and ongoing clinical trials, and suggest future therapeutic approaches. PMID:25483944

  17. A review of human pluripotent stem cell-derived cardiomyocytes for high-throughput drug discovery, cardiotoxicity screening, and publication standards.

    Mordwinkin, Nicholas M; Burridge, Paul W; Wu, Joseph C

    2013-02-01

    Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human-induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach.

  18. Uninterrupted monitoring of drug effects in human-induced pluripotent stem cell-derived cardiomyocytes with bioluminescence Ca2+ microscopy.

    Suzuki, Kazushi; Onishi, Takahito; Nakada, Chieko; Takei, Shunsuke; Daniels, Matthew J; Nakano, Masahiro; Matsuda, Tomoki; Nagai, Takeharu

    2018-05-18

    Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca 2+ indicator GmNL(Ca 2+ ), and its application in a customized microscope for high-throughput drug screening. GmNL(Ca 2+ ) gives a 140% signal change with Ca 2+ , and can image drug-induced changes of Ca 2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca 2+ ) with this adaptation, we could image spontaneous Ca 2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.

  19. A novel type of self-beating cardiomyocytes in adult mouse ventricles

    Omatsu-Kanbe, Mariko; Matsuura, Hiroshi

    2009-01-01

    This study was designed to investigate the presence of resident heart cells that are distinct from terminally-differentiated cardiomyocytes. Adult mouse heart was coronary perfused with collagenase, and ventricles were excised and further digested. After spinning cardiomyocyte-containing fractions down, the supernatant fraction was collected and cultured without adding any chemicals. Two to five days after plating, some of rounded cells adhered to the culture dish, gradually changed their shape and then started self-beating. These self-beating cells did not appreciably proliferate but underwent a further morphological maturation process to form highly branched shapes with many projections. These cells were mostly multinucleated, well sarcomeric-organized and expressed cardiac marker proteins, defined as atypically-shaped cardiomyocytes (ACMs). Patch-clamp experiments revealed that ACMs exhibited spontaneous action potentials arising from the preceding slow diastolic depolarization. We thus found a novel type of resident heart cells in adult cardiac ventricles that spontaneously develop into self-beating cardiomyocytes.

  20. CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart

    M. Gomez-Velazquez (Melisa); C. Badia-Careaga (Claudio); Lechuga-Vieco, A.V. (Ana Victoria); Nieto-Arellano, R. (Rocio); Tena, J.J. (Juan J.); Rollan, I. (Isabel); Alvarez, A. (Alba); Torroja, C. (Carlos); Caceres, E.F. (Eva F.); Roy, A. (Anna); N.J. Galjart (Niels); Delgado-Olguin, P. (Paul); F. Sánchez-Cabo (Fátima); Enriquez, J.A. (Jose Antonio); Gomez-Skarmeta, J.L. (Jose Luis); M. Manzanares (Miguel)

    2017-01-01

    textabstractCardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such

  1. Role of microRNA-195 in cardiomyocyte apoptosis induced by ...

    drinking water and sterilized standard diet. The mice were ... was performed with the in situ cell death detection kit ... facturer's protocol to detect apoptotic cardiomyocytes. The ..... ulate the leakage of Cyt-c and initiate apoptosis through the.

  2. Activation of β-Adrenoceptors by Dobutamine May Induce a Higher Expression of Peroxisome Proliferator-Activated Receptors δ (PPARδ in Neonatal Rat Cardiomyocytes

    Ming-Ting Chou

    2012-01-01

    Full Text Available Recent evidence showed the role of peroxisome proliferator-activated receptors (PPARs in cardiac function. Cardiac contraction induced by various agents is critical in restoring the activity of peroxisome proliferator-activated receptors δ (PPARδ in cardiac myopathy. Because dobutamine is an agent widely used to treat heart failure in emergency setting, this study is aimed to investigate the change of PPARδ in response to dobutamine. Neonatal rat cardiomyocytes were used to examine the effects of dobutamine on PPARδ expression levels and cardiac troponin I (cTnI phosphorylation via Western blotting analysis. We show that treatment with dobutamine increased PPARδ expression and cTnI phosphorylation in a time- and dose-dependent manner in neonatal rat cardiomyocytes. These increases were blocked by the antagonist of β1-adrenoceptors. Also, the action of dobutamine was related to the increase of calcium ions and diminished by chelating intracellular calcium. Additionally, dobutamine-induced action was reduced by the inhibition of downstream messengers involved in this calcium-related pathway. Moreover, deletion of PPARδ using siRNA generated the reduction of cTnI phosphorylation in cardiomyocytes treated with dobutamine. Thus, we concluded that PPARδ is increased by dobutamine in cardiac cells.

  3. Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury.

    Wang, Wei Eric; Li, Liangpeng; Xia, Xuewei; Fu, Wenbin; Liao, Qiao; Lan, Cong; Yang, Dezhong; Chen, Hongmei; Yue, Rongchuan; Zeng, Cindy; Zhou, Lin; Zhou, Bin; Duan, Dayue Darrel; Chen, Xiongwen; Houser, Steven R; Zeng, Chunyu

    2017-08-29

    Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca 2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca 2+ -dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca 2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca 2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia

  4. Deficiency of insulin-like growth factor 1 reduces vulnerability to chronic alcohol intake-induced cardiomyocyte mechanical dysfunction: role of AMPK.

    Ge, Wei; Li, Qun; Turdi, Subat; Wang, Xiao-Ming; Ren, Jun

    2011-08-01

    Circulating insulin-like growth factor I (IGF-1) levels are closely associated with cardiac performance although the role of IGF-1 in alcoholic cardiac dysfunction is unknown. This study was designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on chronic alcohol-induced cardiomyocyte contractile and intracellular Ca(2+) dysfunction. Adult male C57 and LID mice were placed on a 4% alcohol diet for 15 weeks. Cardiomyocyte contractile and intracellular Ca(2+) properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-relengthening (TR(90) ), change in fura-fluorescence intensity (ΔFFI) and intracellular Ca(2+) decay. Levels of apoptotic regulators caspase-3, Bcl-2 and c-Jun NH2-terminal kinase (JNK), the ethanol metabolizing enzyme mitochondrial aldehyde dehydrogenase (ALDH2), as well as the cellular fuel gauge AMP-activated protein kinase (AMPK) were evaluated. Chronic alcohol intake enlarged myocyte cross-sectional area, reduced PS, ± dL/dt and ΔFFI as well as prolonged TR(90) and intracellular Ca(2+) decay, the effect of which was greatly attenuated by IGF-1 deficiency. The beneficial effect of LID against alcoholic cardiac mechanical defect was ablated by IGF-1 replenishment. Alcohol intake increased caspase-3 activity/expression although it down-regulated Bcl-2, ALDH2 and pAMPK without affecting JNK and AMPK. IGF-1 deficiency attenuated alcoholism-induced responses in all these proteins with the exception of Bcl-2. In addition, the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside abrogated short-term ethanol incubation-elicited cardiac mechanical dysfunction. Taken together, these data suggested that IGF-1 deficiency may reduce the sensitivity to ethanol-induced myocardial mechanical dysfunction. Our data further depicted a likely role of Caspase-3, ALDH2 and AMPK activation in IGF-1 deficiency induced 'desensitization' of alcoholic cardiomyopathy. © 2011 The

  5. Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility.

    Sheeja Rajasingh

    Full Text Available Human induced pluripotent stem cells (iPSCs derived cardiomyocytes (iCMCs would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments, adult human skin fibroblasts (HSF and human umbilical vein endothelial cells (HUVECs were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages, including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0, a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal, ectodermal and mesodermal cells, including CMCs with >88% of cells being positive for troponin T (CTT and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs.

  6. Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility.

    Rajasingh, Sheeja; Thangavel, Jayakumar; Czirok, Andras; Samanta, Saheli; Roby, Katherine F; Dawn, Buddhadeb; Rajasingh, Johnson

    2015-01-01

    Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments, adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages, including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0, a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal, ectodermal and mesodermal cells, including CMCs with >88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs.

  7. Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

    Sundaravadivel Balasubramanian

    2010-07-01

    Full Text Available The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring

  8. Group B streptococcal beta-hemolysin/cytolysin directly impairs cardiomyocyte viability and function.

    Mary E Hensler

    Full Text Available BACKGROUND: Group B Streptococcus (GBS is a leading cause of neonatal sepsis where myocardial dysfunction is an important contributor to poor outcome. Here we study the effects of the GBS pore-forming beta-hemolysin/cytolysin (Bh/c exotoxin on cardiomyocyte viability, contractility, and calcium transients. METHODOLOGY/PRINCIPAL FINDINGS: HL-1 cardiomyocytes exposed to intact wild-type (WT or isogenic Deltabeta h/c mutant GBS, or to cell-free extracts from either strain, were assessed for viability by trypan blue exclusion and for apoptosis by TUNEL staining. Functionality of exposed cardiomyocytes was analyzed by visual quantitation of the rate and extent of contractility. Mitochondrial membrane polarization was measured in TMRE-loaded cells exposed to GBS beta h/c. Effects of GBS beta h/c on calcium transients were studied in fura-2AM-loaded primary rat ventricular cardiomyocytes. Exposure of HL-1 cardiomyocytes to either WT GBS or beta h/c extracts significantly reduced both rate and extent of contractility and later induced necrotic and apoptotic cell death. No effects on cardiomyocyte viability or function were observed after treatment with Deltabeta h/c mutant bacteria or extracts. The beta h/c toxin was associated with complete and rapid loss of detectable calcium transients in primary neonatal rat ventricular cardiomyocytes and induced a loss of mitochondrial membrane polarization. These effects on viability and function were abrogated by the beta h/c inhibitor, dipalmitoyl phosphatidylcholine (DPPC. CONCLUSIONS/SIGNIFICANCE: Our data show a rapid loss of cardiomyocyte viability and function induced by GBS beta h/c, and these deleterious effects are inhibited by DPPC, a normal constituent of human pulmonary surfactant.. These findings have clinical implications for the cardiac dysfunction observed in neonatal GBS infections.

  9. A water-based training program that include perturbation exercises to improve stepping responses in older adults: study protocol for a randomized controlled cross-over trial

    Tsedek Irit

    2008-08-01

    Full Text Available Abstract Background Gait and balance impairments may increase the risk of falls, the leading cause of accidental death in the elderly population. Fall-related injuries constitute a serious public health problem associated with high costs for society as well as human suffering. A rapid step is the most important protective postural strategy, acting to recover equilibrium and prevent a fall from initiating. It can arise from large perturbations, but also frequently as a consequence of volitional movements. We propose to use a novel water-based training program which includes specific perturbation exercises that will target the stepping responses that could potentially have a profound effect in reducing risk of falling. We describe the water-based balance training program and a study protocol to evaluate its efficacy (Trial registration number #NCT00708136. Methods/Design The proposed water-based training program involves use of unpredictable, multi-directional perturbations in a group setting to evoke compensatory and volitional stepping responses. Perturbations are made by pushing slightly the subjects and by water turbulence, in 24 training sessions conducted over 12 weeks. Concurrent cognitive tasks during movement tasks are included. Principles of physical training and exercise including awareness, continuity, motivation, overload, periodicity, progression and specificity were used in the development of this novel program. Specific goals are to increase the speed of stepping responses and improve the postural control mechanism and physical functioning. A prospective, randomized, cross-over trial with concealed allocation, assessor blinding and intention-to-treat analysis will be performed to evaluate the efficacy of the water-based training program. A total of 36 community-dwelling adults (age 65–88 with no recent history of instability or falling will be assigned to either the perturbation-based training or a control group (no training

  10. Spatially defined InsP3-mediated signaling in embryonic stem cell-derived cardiomyocytes.

    Nidhi Kapoor

    Full Text Available The functional role of inositol 1,4,5-trisphosphate (InsP3 signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP3 receptors can translate Ca(2+ release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs as a model system since their spontaneous electrical activity depends on InsP3-mediated Ca(2+ release. [InsP3]i was monitored with the FRET-based InsP3-biosensor FIRE-1 (Fluorescent InsP3 Responsive Element and heterogeneity in sub-cellular [InsP3]i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc or expression of InsP3 5-phosphatase (m43 localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca(2+ transients (Fluo-4/AM and action potentials (current clamp. During diastole, the diastolic depolarization was paralleled by an increase of [Ca(2+]i and spontaneous activity was modulated by [InsP3]i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L or phenylephrine (PE, 10 µmol/L, respectively. Buffering of InsP3 by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP3 signaling throughout the cell (FIRE-1, or at the plasma membrane (m43 resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca(2+ released from InsP3Rs is more effective than Ca(2+ released from RyRs to enhance INCX. The results support the hypothesis that in ESdCs InsP3Rs form a functional signaling domain with NCX that translates Ca(2+ release efficiently into a depolarization of the membrane potential.

  11. Blueberry polyphenols prevent cardiomyocyte death by preventing calpain activation and oxidative stress.

    Louis, Xavier Lieben; Thandapilly, Sijo Joseph; Kalt, Wilhelmina; Vinqvist-Tymchuk, Melinda; Aloud, Basma Milad; Raj, Pema; Yu, Liping; Le, Hoa; Netticadan, Thomas

    2014-08-01

    The purpose of this study was to examine the efficacy of an aqueous wild blueberry extract and five wild blueberry polyphenol fractions on an in vitro model of heart disease. Adult rat cardiomyocytes were pretreated with extract and fractions, and then exposed to norepinephrine (NE). Cardiomyocyte hypertrophy, cell death, oxidative stress, apoptosis and cardiomyocyte contractile function as well as the activities of calpain, superoxide dismutase (SOD) and catalase (CAT) were measured in cardiomyocytes treated with and without NE and blueberry fraction (BF). Four of five blueberry fractions prevented cell death and cardiomyocyte hypertrophy induced by NE. Total phenolic fraction was used for all further analysis. The NE-induced increase in oxidative stress, nuclear condensation, calpain activity and lowering of SOD and CAT activities were prevented upon pretreatment with BF. Reduced contractile function was also significantly improved with BF pretreatment. Blueberry polyphenols prevent NE-induced adult cardiomyocyte hypertrophy and cell death. The protective effects of BF may be in part attributed to a reduction in calpain activity and oxidative stress.

  12. Quantitative analysis of biological responses to low dose-rate γ-radiation, including dose, irradiation time, and dose-rate

    Magae, J.; Furukawa, C.; Kawakami, Y.; Hoshi, Y.; Ogata, H.

    2003-01-01

    Full text: Because biological responses to radiation are complex processes dependent on irradiation time as well as total dose, it is necessary to include dose, dose-rate and irradiation time simultaneously to predict the risk of low dose-rate irradiation. In this study, we analyzed quantitative relationship among dose, irradiation time and dose-rate, using chromosomal breakage and proliferation inhibition of human cells. For evaluation of chromosome breakage we assessed micronuclei induced by radiation. U2OS cells, a human osteosarcoma cell line, were exposed to gamma-ray in irradiation room bearing 50,000 Ci 60 Co. After the irradiation, they were cultured for 24 h in the presence of cytochalasin B to block cytokinesis, cytoplasm and nucleus were stained with DAPI and propidium iodide, and the number of binuclear cells bearing micronuclei was determined by fluorescent microscopy. For proliferation inhibition, cells were cultured for 48 h after the irradiation and [3H] thymidine was pulsed for 4 h before harvesting. Dose-rate in the irradiation room was measured with photoluminescence dosimeter. While irradiation time less than 24 h did not affect dose-response curves for both biological responses, they were remarkably attenuated as exposure time increased to more than 7 days. These biological responses were dependent on dose-rate rather than dose when cells were irradiated for 30 days. Moreover, percentage of micronucleus-forming cells cultured continuously for more than 60 days at the constant dose-rate, was gradually decreased in spite of the total dose accumulation. These results suggest that biological responses at low dose-rate, are remarkably affected by exposure time, that they are dependent on dose-rate rather than total dose in the case of long-term irradiation, and that cells are getting resistant to radiation after the continuous irradiation for 2 months. It is necessary to include effect of irradiation time and dose-rate sufficiently to evaluate risk

  13. IFNL4 ss469415590 Variant Is Associated with Treatment Response in Japanese HCV Genotype 1 Infected Individuals Treated with IFN-Including Regimens

    Tatsuo Miyamura

    2014-01-01

    Full Text Available Aim. Eradication of hepatitis C virus (HCV is still challenging even if interferon- (IFN- free regimens with direct-acting antiviral agents (DAAs for HCV-infected individuals are available in clinical practice. IFNL4 is a newly described protein, associated with human antiviral defenses. We investigated whether IFNL4 ss469415590 variant has an effect on the prediction of treatment response in HCV-infected patients treated with IFN-including regimens. Patients and Methods. In all, 185 patients infected with HCV genotype 1 treated with peg-IFN plus ribavirin, with or without telaprevir, were genotyped for IFNL4 ss469415590. We retrospectively investigated whether the role of IFNL4 ss469415590 variant and other factors could predict sustained virological response (SVR in Japanese patients infected with HCV genotype 1. Results. There were 65.7%, 31.5%, and 2.8% patients in the IFNL4 ss469415590 TT/TT, TT/-G, and -G/-G groups, respectively. SVR rates were 82.1% or 49.3% in patients treated with peg-IFN plus ribavirin with or without telaprevir, respectively. IFNL4 ss469415590 variant and HCV viral loads or IFNL4 ss469415590 variant and early virological response were better predictors of SVR in patients treated with peg-IFN plus ribavirin with or without telaprevir, respectively. Conclusion. In the era of DAAs, measurement of IFNL4 ss469415590 variant could help the prediction of SVR in Japanese HCV genotype 1 infected individuals treated with IFN-including regimens.

  14. Induction of HO-1 by carbon monoxide releasing molecule-2 attenuates thrombin-induced COX-2 expression and hypertrophy in primary human cardiomyocytes

    Chien, Peter Tzu-Yu; Lin, Chih-Chung; Hsiao, Li-Der; Yang, Chuen-Mao

    2015-01-01

    Carbon monoxide (CO) is one of the cytoprotective byproducts of heme oxygenase (HO)-1 and exerts anti-inflammatory action in various models. However, the detailed mechanisms underlying CO-induced HO-1 expression in primary human cardiomyocytes remain largely unidentified. We used primary left ventricle myocytes as a model and applied CO releasing molecule (CORM)-2 to investigate the relationship of CO and HO-1 expression. We herein used Western blot, real-time PCR, promoter activity and EIA to investigate the role of HO-1 expression protecting against thrombin-mediated responses. We found that thrombin-induced COX-2 expression, PGE 2 release and cardiomyocyte hypertrophy markers (increase in ANF/BNP, α-actin expression and cell surface area) was attenuated by pretreatment with CORM-2 which was partially reversed by hemoglobin (Hb) or ZnPP (an inhibitor of HO-1 activity), suggesting that HO-1/CO system may be of clinical importance to ameliorate heart failure through inhibition of inflammatory responses. CORM-2-induced HO-1 protein expression, mRNA and promoter was attenuated by pretreatment with the inhibitors of Pyk2 (PF431396), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), p38 (SB202530), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (mithramycin A). The involvement of these signaling components was further confirmed by transfection with respective siRNAs, consistent with those of pharmacological inhibitors. These results suggested that CORM-2-induced HO-1 expression is mediated through a Pyk2/PDGFR/PI3K/Akt/FoxO1/Sp1-dependent manner and exerts a cytoprotective effect in human cardiomyocytes. - Graphical abstract: In summary, CORM-2 treatment induces Pyk2 transactivated PDGFR, which induces PI3K/Akt/MAPK activation, and then recruits Sp1/Foxo1 transcriptional factors to regulate HO-1 gene expression in primary human cardiomyocytes. - Highlights: • CORM-2 induces HO-1 expression. • Pyk2-dependent PDGFR activates PI3K/Akt/MAPK pathway in CORM-2-induced HO-1

  15. Induction of HO-1 by carbon monoxide releasing molecule-2 attenuates thrombin-induced COX-2 expression and hypertrophy in primary human cardiomyocytes

    Chien, Peter Tzu-Yu [Department of Physiology and Pharmacology and Health Ageing Research Center, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan (China); Lin, Chih-Chung; Hsiao, Li-Der [Department of Anesthetics, Chang Gung Memorial Hospital at Lin-Kou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan (China); Yang, Chuen-Mao, E-mail: chuenmao@mail.cgu.edu.tw [Department of Physiology and Pharmacology and Health Ageing Research Center, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan (China); Research Center for Industry of Human Ecology and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Tao-Yuan, Taiwan (China)

    2015-12-01

    Carbon monoxide (CO) is one of the cytoprotective byproducts of heme oxygenase (HO)-1 and exerts anti-inflammatory action in various models. However, the detailed mechanisms underlying CO-induced HO-1 expression in primary human cardiomyocytes remain largely unidentified. We used primary left ventricle myocytes as a model and applied CO releasing molecule (CORM)-2 to investigate the relationship of CO and HO-1 expression. We herein used Western blot, real-time PCR, promoter activity and EIA to investigate the role of HO-1 expression protecting against thrombin-mediated responses. We found that thrombin-induced COX-2 expression, PGE{sub 2} release and cardiomyocyte hypertrophy markers (increase in ANF/BNP, α-actin expression and cell surface area) was attenuated by pretreatment with CORM-2 which was partially reversed by hemoglobin (Hb) or ZnPP (an inhibitor of HO-1 activity), suggesting that HO-1/CO system may be of clinical importance to ameliorate heart failure through inhibition of inflammatory responses. CORM-2-induced HO-1 protein expression, mRNA and promoter was attenuated by pretreatment with the inhibitors of Pyk2 (PF431396), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), p38 (SB202530), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (mithramycin A). The involvement of these signaling components was further confirmed by transfection with respective siRNAs, consistent with those of pharmacological inhibitors. These results suggested that CORM-2-induced HO-1 expression is mediated through a Pyk2/PDGFR/PI3K/Akt/FoxO1/Sp1-dependent manner and exerts a cytoprotective effect in human cardiomyocytes. - Graphical abstract: In summary, CORM-2 treatment induces Pyk2 transactivated PDGFR, which induces PI3K/Akt/MAPK activation, and then recruits Sp1/Foxo1 transcriptional factors to regulate HO-1 gene expression in primary human cardiomyocytes. - Highlights: • CORM-2 induces HO-1 expression. • Pyk2-dependent PDGFR activates PI3K/Akt/MAPK pathway in CORM-2-induced HO

  16. Pi3kcb links Hippo-YAP and PI3K-AKT signaling pathways to promote cardiomyocyte proliferation and survival.

    Lin, Zhiqiang; Zhou, Pingzhu; von Gise, Alexander; Gu, Fei; Ma, Qing; Chen, Jinghai; Guo, Haidong; van Gorp, Pim R R; Wang, Da-Zhi; Pu, William T

    2015-01-02

    Yes-associated protein (YAP), the nuclear effector of Hippo signaling, regulates cellular growth and survival in multiple organs, including the heart, by interacting with TEA (transcriptional enhancer activator)-domain sequence-specific DNA-binding proteins. Recent studies showed that YAP stimulates cardiomyocyte proliferation and survival. However, the direct transcriptional targets through which YAP exerts its effects are poorly defined. To identify direct YAP targets that mediate its mitogenic and antiapoptotic effects in the heart. We identified direct YAP targets by combining differential gene expression analysis in YAP gain- and loss-of-function with genome-wide identification of YAP-bound loci using chromatin immunoprecipitation and high throughput sequencing. This screen identified Pik3cb, encoding p110β, a catalytic subunit of phosphoinositol-3-kinase, as a candidate YAP effector that promotes cardiomyocyte proliferation and survival. YAP and TEA-domain occupied a conserved enhancer within the first intron of Pik3cb, and this enhancer drove YAP-dependent reporter gene expression. Yap gain- and loss-of-function studies indicated that YAP is necessary and sufficient to activate the phosphoinositol-3-kinase-Akt pathway. Like Yap, Pik3cb gain-of-function stimulated cardiomyocyte proliferation, and Pik3cb knockdown dampened YAP mitogenic activity. Reciprocally, impaired heart function in Yap loss-of-function was significantly rescued by adeno-associated virus-mediated Pik3cb expression. Pik3cb is a crucial direct target of YAP, through which the YAP activates phosphoinositol-3-kinase-AKT pathway and regulates cardiomyocyte proliferation and survival. © 2014 American Heart Association, Inc.

  17. The Extracts and Major Compounds Derived from Astragali Radix Alter Mitochondrial Bioenergetics in Cultured Cardiomyocytes: Comparison of Various Polar Solvents and Compounds

    Yun Huang

    2018-05-01

    Full Text Available Astragali Radix (AR is a widely used “Qi-invigorating” herb in China for its tonic effects in strengthening biological tissues. The extract of AR contains abundant antioxidants, including astragalosides and isoflavonoids. However, very few reports have systematically measured the effects of the major components of AR on cell mitochondrial bioenergetics. Here, a systemic approach employing an extracellular flux analyzer was developed to evaluate mitochondrial respiration in cultured cardiomyocyte cells H9C2. The effects of different polar extractives, as well as of the major compounds of AR, were compared. The contents of astragaloside IV, calycosin, formononetin, and genistein in the AR extracts obtained by using water, 50% ethanol, and 90% ethanol were measured by liquid chromatograph-mass spectrometer (LC–MS. The antioxidant activities of the AR extracts, as well as of their major compounds, were determined by measuring the free radical scavenging activity and protective effects in tert-butyl hydroperoxide (tBHP-treated H9C2 cells. By monitoring the real-time oxygen consumption rate (OCR in tBHP-treated cardiomyocytes with a Seahorse extracellular flux analyzer, the tonic effects of the AR extracts and of their main compounds on mitochondrial bioenergetics were evaluated. AR water extracts possessed the strongest antioxidant activity and protective effects in cardiomyocytes exposed to oxidative stress. The protection was proposed to be mediated via increasing the spare respiratory capacity and mitochondrial ATP production in the stressed cells. The major compounds of AR, astragaloside IV and genistein, showed opposite effects in regulating mitochondrial bioenergetics. These results demonstrate that highly polar extracts of AR, especially astragaloside-enriched extracts, possess better tonic effects on mitochondrial bioenergetics of cultured cardiomyocytes than extracts with a lower polarity.

  18. Protective effect of pioglitazone on cardiomyocyte apoptosis in low-dose streptozotocin & high-fat diet-induced type-2 diabetes in rats

    Uma Bhandari

    2015-01-01

    Full Text Available Background & objectives: Cardiomyocyte apoptosis is one of the pathologic phenomena associated with diabetes and related conditions including obesity, insulin resistance and hyperlipidaemia. In the present study, the protective effects of pioglitazone on cardiomyocyte apoptosis was evaluated in experimental diabetes induced by low dose of streptozoticin (STZ combined with high fat diet (HFD in rats. Methods: Male Wistar rats (150-200 g were injected with low-dose STZ (45 mg/kg, i.v., single dose and orally fed with a HFD (20 g/day/rat for a period of 28 days and simultaneously treated with pioglitazone (20 mg/kg/p.o. for a period of 21 days (from 8 th day to 28 th day. On 29 th day blood was collected, serum separated and used for biochemical parameters. Heart tissue was used for cardiomyocyte apoptosis measurement and also for histopathological examination. Results: Pioglitazone treatment resulted in a decrease in cardiomyocyte apoptosis as revealed by a decrease in cardiac caspase-3, lactate dehydrogenase (LDH levels and DNA fragmentation, and an increase in Na+K+ATPase levels in diabetic rats. Cardiac histology of diabetic control rats showed dense focal fatty infiltration in the myocardial cells whereas normal architecture with regular morphology and well preserved cytoplasm was observed with pioglitazone treatment. Pioglitazone treatment significantly reduced the heart rate, mean arterial blood pressure, body mass index (BMI and levels of serum glucose, leptin, insulin, HOMA-IR, total cholesterol (TC and triglycerides (TGs, apoliproprotein-B glycosylated haemoglobin (HbA1c levels and atherogenic index, and increased the levels of serum high density lipoprotein cholesterol (HDL-C and cardiac antioxidant enzymes. Interpretation & conclusions: The present study results suggest that pioglitazone possesses cardiac anti-apoptotic potential in diabetic rat model and can be further explored for its use for treatment of diabetic cardiomyopathy.

  19. Cardiomyocyte mitochondrial respiration is reduced by receptor for advanced glycation end-product signaling in a ceramide-dependent manner.

    Nelson, Michael B; Swensen, Adam C; Winden, Duane R; Bodine, Jared S; Bikman, Benjamin T; Reynolds, Paul R

    2015-07-01

    Cigarette smoke exposure is associated with an increased risk of cardiovascular complications. The role of advanced glycation end products (AGEs) is already well established in numerous comorbidities, including cardiomyopathy. Given the role of AGEs and their receptor, RAGE, in activating inflammatory pathways, we sought to determine whether ceramides could be a mediator of RAGE-induced altered heart mitochondrial function. Using an in vitro model, we treated H9C2 cardiomyocytes with the AGE carboxy-methyllysine before mitochondrial respiration assessment. We discovered that mitochondrial respiration was significantly impaired in AGE-treated cells, but not when cotreated with myriocin, an inhibitor of de novo ceramide biosynthesis. Moreover, we exposed wild-type and RAGE knockout mice to secondhand cigarette smoke and found reduced mitochondrial respiration in the left ventricular myocardium from wild-type mice, but RAGE knockout mice were protected from this effect. Finally, conditional overexpression of RAGE in the lungs of transgenic mice elicited a robust increase in left ventricular ceramides in the absence of smoke exposure. Taken together, these findings suggest a RAGE-ceramide axis as an important contributor to AGE-mediated disrupted cardiomyocyte mitochondrial function. Copyright © 2015 the American Physiological Society.

  20. MicroRNA-145 protects cardiomyocytes against hydrogen peroxide (H₂O₂-induced apoptosis through targeting the mitochondria apoptotic pathway.

    Ruotian Li

    Full Text Available MicroRNAs, a class of small and non-encoding RNAs that transcriptionally or post-transcriptionally modulate the expression of their target genes, has been implicated as critical regulatory molecules in many cardiovascular diseases, including ischemia/reperfusion induced cardiac injury. Here, we report microRNA-145, a tumor suppressor miRNA, can protect cardiomyocytes from hydrogen peroxide H₂O₂-induced apoptosis through targeting the mitochondrial pathway. Quantitative real-time PCR (qPCR demonstrated that the expression of miR-145 in either ischemia/reperfused mice myocardial tissues or H₂O₂-treated neonatal rat ventricle myocytes (NRVMs was markedly down-regulated. Over-expression of miR-145 significantly inhibited the H₂O₂-induced cellular apoptosis, ROS production, mitochondrial structure disruption as well as the activation of key signaling proteins in mitochondrial apoptotic pathway. These protective effects of miR-145 were abrogated by over-expression of Bnip3, an initiation factor of the mitochondrial apoptotic pathway in cardiomyocytes. Finally, we utilized both luciferase reporter assay and western blot analysis to identify Bnip3 as a direct target of miR-145. Our results suggest miR-145 plays an important role in regulating mitochondrial apoptotic pathway in heart challenged with oxidative stress. MiR-145 may represent a potential therapeutic target for treatment of oxidative stress-associated cardiovascular diseases, such as myocardial ischemia/reperfusion injury.

  1. Testosterone-mediated upregulation of delayed rectifier potassium channel in cardiomyocytes causes abbreviation of QT intervals in rats.

    Masuda, Kimiko; Takanari, Hiroki; Morishima, Masaki; Ma, FangFang; Wang, Yan; Takahashi, Naohiko; Ono, Katsushige

    2018-01-13

    Men have shorter rate-corrected QT intervals (QTc) than women, especially at the period of adolescence or later. The aim of this study was to elucidate the long-term effects of testosterone on cardiac excitability parameters including electrocardiogram (ECG) and potassium channel current. Testosterone shortened QT intervals in ECG in castrated male rats, not immediately after, but on day 2 or later. Expression of Kv7.1 (KCNQ1) mRNA was significantly upregulated by testosterone in cardiomyocytes of male and female rats. Short-term application of testosterone was without effect on delayed rectifier potassium channel current (I Ks ), whereas I Ks was significantly increased in cardiomyocytes treated with dihydrotestosterone for 24 h, which was mimicked by isoproterenol (24 h). Gene-selective inhibitors of a transcription factor SP1, mithramycin, abolished the effects of testosterone on Kv7.1. Testosterone increases Kv7.1-I Ks possibly through a pathway related to a transcription factor SP1, suggesting a genomic effect of testosterone as an active factor for cardiac excitability.

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

    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.

  3. Rate of Clinical Complete Response for 1 Year or More in Bone-Metastatic Breast Cancer after Comprehensive Treatments including Autologous Formalin-Fixed Tumor Vaccine.

    Kuranishi, Fumito; Imaoka, Yuki; Sumi, Yuusuke; Uemae, Yoji; Yasuda-Kurihara, Hiroko; Ishihara, Takeshi; Miyazaki, Tsubasa; Ohno, Tadao

    2018-01-01

    No effective treatment has been developed for bone-metastatic breast cancer. We found 3 cases with clinical complete response (cCR) of the bone metastasis and longer overall survival of the retrospectively examined cohort treated comprehensively including autologous formalin-fixed tumor vaccine (AFTV). AFTV was prepared individually for each patient from their own formalin-fixed and paraffin-embedded breast cancer tissues. Three patients maintained cCR status of the bone metastasis for 17 months or more. Rate of cCR for 1 year or more appeared to be 15% (3/20) after comprehensive treatments including AFTV. The median overall survival time (60.0 months) and the 3- to 8-year survival rates after diagnosis of bone metastasis were greater than those of historical control cohorts in Japan (1988-2002) and in the nationwide population-based cohort study of Denmark (1999-2007). Bone-metastatic breast cancer may be curable after comprehensive treatments including AFTV, although larger scale clinical trial is required.

  4. Rate of Clinical Complete Response for 1 Year or More in Bone-Metastatic Breast Cancer after Comprehensive Treatments including Autologous Formalin-Fixed Tumor Vaccine

    Fumito Kuranishi

    2018-01-01

    Full Text Available Introduction. No effective treatment has been developed for bone-metastatic breast cancer. We found 3 cases with clinical complete response (cCR of the bone metastasis and longer overall survival of the retrospectively examined cohort treated comprehensively including autologous formalin-fixed tumor vaccine (AFTV. Patients and Methods. AFTV was prepared individually for each patient from their own formalin-fixed and paraffin-embedded breast cancer tissues. Results. Three patients maintained cCR status of the bone metastasis for 17 months or more. Rate of cCR for 1 year or more appeared to be 15% (3/20 after comprehensive treatments including AFTV. The median overall survival time (60.0 months and the 3- to 8-year survival rates after diagnosis of bone metastasis were greater than those of historical control cohorts in Japan (1988–2002 and in the nationwide population-based cohort study of Denmark (1999–2007. Conclusion. Bone-metastatic breast cancer may be curable after comprehensive treatments including AFTV, although larger scale clinical trial is required.

  5. Overexpressed connective tissue growth factor in cardiomyocytes attenuates left ventricular remodeling induced by angiotensin II perfusion.

    Zhang, Ying; Yan, Hua; Guang, Gong-Chang; Deng, Zheng-Rong

    2017-01-01

    To evaluate the improving effects of specifically overexpressed connective tissue growth factor (CTGF) in cardiomyocytes on mice with hypertension induced by angiotensin II (AngII) perfusion, 24 transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) were divided into two equal groups that were perfused with acetic acid and AngII, respectively, for 7 days. Another 24 cage-control wild-type C57BL/6 mice (NLC) were divided and treated identically. Blood pressure was detected by caudal artery cannulation. Cardiac structural and functional changes were observed by echocardiography. Cardiac fibrosis was detected by Masson staining. After AngII perfusion, blood pressures of NLC and Tg-CTGF mice, especially those of the formers, significantly increased. Compared with NLC + AngII group, Tg-CTGF + AngII group had significantly lower left ventricular posterior wall thickness at end-diastole and left ventricular posterior wall thickness at end-systole as well as significantly higher left ventricular end-systolic diameter and left ventricular end-diastolic diameter (P tissues (P < 0.05). Tg-CTGF can protect AngII-induced cardiac remodeling of mice with hypertension by mitigating inflammatory response. CTGF may be a therapy target for hypertension-induced myocardial fibrosis, but the detailed mechanism still needs in-depth studies.

  6. TonEBP modulates the protective effect of taurine in ischemia-induced cytotoxicity in cardiomyocytes

    Yang, Y J; Han, Y Y; Chen, K; Zhang, Y; Liu, X; Li, S; Wang, K Q; Ge, J B; Liu, W; Zuo, J

    2015-01-01

    Taurine, which is found at high concentration in the heart, exerts several protective actions on myocardium. Physically, the high level of taurine in heart is maintained by a taurine transporter (TauT), the expression of which is suppressed under ischemic insult. Although taurine supplementation upregulates TauT expression, elevates the intracellular taurine content and ameliorates the ischemic injury of cardiomyocytes (CMs), little is known about the regulatory mechanisms of taurine governing TauT expression under ischemia. In this study, we describe the TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway involved in the taurine-regulated TauT expression in ischemic CMs. Taurine inhibited the ubiquitin-dependent proteasomal degradation of TonEBP, promoted the translocation of TonEBP into the nucleus, enhanced TauT promoter activity and finally upregulated TauT expression in CMs. In addition, we observed that TonEBP had an anti-apoptotic and anti-oxidative role in CMs under ischemia. Moreover, the protective effects of taurine on myocardial ischemia were TonEBP dependent. Collectively, our findings suggest that TonEBP is a core molecule in the protective mechanism of taurine in CMs under ischemic insult. PMID:26673669

  7. Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

    Nicolas Christoforou

    Full Text Available The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS cells, which once differentiated allow for the enrichment of Nkx2-5(+ cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+ cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological

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

    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.

  9. Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in 3D collagen matrix: Effects of niche cell supplementation and mechanical stimulation.

    Zhang, W; Kong, C W; Tong, M H; Chooi, W H; Huang, N; Li, R A; Chan, B P

    2017-02-01

    Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as a promising source for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. Here, we fabricate cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials. Supplementation of niche cells at 3% to the number of hESC-CMs enhance the maturation of the hESC-CMs in 3D tissue matrix. The benefits of adding mesenchymal stem cells (MSCs) are comparable to that of adding fibroblasts. These two cell types demonstrate similar effects in promoting the compaction and cell spreading, as well as expression of maturation markers at both gene and protein levels. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of twitch force, elastic modulus, sarcomere length and molecular signature, when comparing to static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture. Our results therefore suggest that this 3D model can be used for in vitro cardiac maturation study. Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as being a promising source of cells for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. In the current study, we have fabricated cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials and demonstrated that supplementation of mesenchymal niche cells as well as provision of mechanical loading particularly stretching have significantly promoted the maturation of the cardiomyocytes and hence improved the mechanical functional characteristics of the tissue strips

  10. Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations

    Cohen, J.D., E-mail: jennifer.cohen@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Babiarz, J.E., E-mail: joshua.babiarz@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Abrams, R.M., E-mail: rory.abrams@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Guo, L., E-mail: liang.guo@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Kameoka, S., E-mail: sei.kameoka@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Chiao, E., E-mail: eric.chiao@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Taunton, J., E-mail: taunton@cmp.ucsf.edu [Howard Hughes Medical Institute, Cellular and Molecular Pharmacology, University California San Francisco, San Francisco, CA 94158 (United States); Kolaja, K.L., E-mail: kyle.kolaja@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States)

    2011-11-15

    Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC{sub 50} in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 {mu}M concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 {mu}M. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity. -- Highlights: Black-Right-Pointing-Pointer Cytoxic effect of sunitinib on human stem cell derived cardiomyocytes Black

  11. Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations

    Cohen, J.D.; Babiarz, J.E.; Abrams, R.M.; Guo, L.; Kameoka, S.; Chiao, E.; Taunton, J.; Kolaja, K.L.

    2011-01-01

    Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC 50 in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 μM concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 μM. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity. -- Highlights: ► Cytoxic effect of sunitinib on human stem cell derived cardiomyocytes ► Sunitinib causes ATP depletion, LDH release, GSH

  12. The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype.

    Munier, C Mee Ling; van Bockel, David; Bailey, Michelle; Ip, Susanna; Xu, Yin; Alcantara, Sheilajen; Liu, Sue Min; Denyer, Gareth; Kaplan, Warren; Suzuki, Kazuo; Croft, Nathan; Purcell, Anthony; Tscharke, David; Cooper, David A; Kent, Stephen J; Zaunders, John J; Kelleher, Anthony D

    2016-10-17

    Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  13. Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells

    Zhang Fabao; Li Li; Fang Bo; Zhu Dingliang; Yang Huangtian; Gao Pingjin

    2005-01-01

    Mesenchymal stem cells (MSCs) have limited ability to differentiate into cardiomyocytes and the factors affect this process are not fully understood. In this study, we investigated the passage (P)-related transdifferentiation potential of MSCs into cardiomyocyte-like cells and its relationship to the proliferation ability. After 5-azacytidine treatment, only P4 but not P1 and P8 rat bone marrow MSCs (rMSCs) showed formation of myotube and expressed cardiomyocyte-associated markers. The growth property analysis showed P4 rMSCs had a growth-arrest appearance, while P1 and P8 rMSCs displayed an exponential growth pattern. When the rapid proliferation of P1 and P8 rMSCs was inhibited by 5-bromo-2-deoxyuridine, a mitosis inhibitor, only P1, not P8 rMSCs, differentiated into cardiomyocyte-like cells after 5-azacytidine treatment. These results demonstrate that the differentiation ability of rMSCs into cardiomyocytes is in proliferation ability-dependent and passage-restricted patterns. These findings reveal a novel regulation on the transdifferentiation of MSCs and provide useful information for exploiting the clinical therapeutic potential of MSCs

  14. Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways.

    Twu, Cheryl; Liu, Nancy Q; Popik, Waldemar; Bukrinsky, Michael; Sayre, James; Roberts, Jaclyn; Rania, Shammas; Bramhandam, Vishnu; Roos, Kenneth P; MacLellan, W Robb; Fiala, Milan

    2002-10-29

    We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-alpha. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-alpha and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways.

  15. Graphene Sheet-Induced Global Maturation of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

    Wang, Jiaxian; Cui, Chang; Nan, Haiyan; Yu, Yuanfang; Xiao, Yini; Poon, Ellen; Yang, Gang; Wang, Xijie; Wang, Chenchen; Li, Lingsong; Boheler, Kenneth Richard; Ma, Xu; Cheng, Xin; Ni, Zhenhua; Chen, Minglong

    2017-08-09

    Human induced pluripotent stem cells (hiPSCs) can proliferate infinitely. Their ability to differentiate into cardiomyocytes provides abundant sources for disease modeling, drug screening and regenerative medicine. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) display a low degree of maturation and fetal-like properties. Current in vitro differentiation methods do not mimic the structural, mechanical, or physiological properties of the cardiogenesis niche. Recently, we present an efficient cardiac maturation platform that combines hiPSCs monolayer cardiac differentiation with graphene substrate, which is a biocompatible and superconductive material. The hiPSCs lines were successfully maintained on the graphene sheets and were able to differentiate into functional cardiomyocytes. This strategy markedly increased the myofibril ultrastructural organization, elevated the conduction velocity, and enhanced both the Ca 2+ handling and electrophysiological properties in the absence of electrical stimulation. On the graphene substrate, the expression of connexin 43 increased along with the conduction velocity. Interestingly, the bone morphogenetic proteins signaling was also significantly activated during early cardiogenesis, confirmed by RNA sequencing analysis. Here, we reasoned that graphene substrate as a conductive biomimetic surface could facilitate the intrinsic electrical propagation, mimicking the microenvironment of the native heart, to further promote the global maturation of hiPSC-CMs. Our findings highlight the capability of electrically active substrates to influence cardiomyocyte development. We believe that application of graphene sheets will be useful for simple, fast, and scalable maturation of regenerated cardiomyocytes.

  16. Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy

    Gun-Sik Cho

    2017-01-01

    Full Text Available Summary: Pluripotent stem cells (PSCs offer unprecedented opportunities for disease modeling and personalized medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs. Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonatal hearts. PSC-CMs became similar to adult CMs in morphology, structure, and function within a month of transplantation into rats. The similarity was further supported by single-cell RNA-sequencing analysis. Moreover, this in vivo maturation allowed patient-derived PSC-CMs to reveal the disease phenotype of arrhythmogenic right ventricular cardiomyopathy, which manifests predominantly in adults. This study lays a foundation for understanding human CM maturation and pathogenesis and can be instrumental in PSC-based modeling of adult heart diseases. : Pluripotent stem cell (PSC-derived cells remain fetal like, and this has become a major impediment to modeling adult diseases. Cho et al. find that PSC-derived cardiomyocytes mature into adult cardiomyocytes when transplanted into neonatal rat hearts. This method can serve as a tool to understand maturation and pathogenesis in human cardiomyocytes. Keywords: cardiomyocyte, maturation, iPS, cardiac progenitor, neonatal, disease modeling, cardiomyopathy, ARVC, T-tubule, calcium transient, sarcomere shortening

  17. Exosomes from Cardiomyocyte Progenitor Cells and Mesenchymal Stem Cells Stimulate Angiogenesis Via EMMPRIN.

    Vrijsen, Krijn R; Maring, Janita A; Chamuleau, Steven A J; Verhage, Vera; Mol, Emma A; Deddens, Janine C; Metz, Corina H G; Lodder, Kirsten; van Eeuwijk, Esther C M; van Dommelen, Susan M; Doevendans, Pieter A; Smits, Anke M; Goumans, Marie-José; Sluijter, Joost P G

    2016-10-01

    To date, cellular transplantation therapy has not yet fulfilled its high expectations for cardiac repair. A major limiting factor is lack of long-term engraftment of the transplanted cells. Interestingly, transplanted cells can positively affect their environment via secreted paracrine factors, among which are extracellular vesicles, including exosomes: small bi-lipid-layered vesicles containing proteins, mRNAs, and miRNAs. An exosome-based therapy will therefore relay a plethora of effects, without some of the limiting factors of cell therapy. Since cardiomyocyte progenitor cells (CMPC) and mesenchymal stem cells (MSC) induce vessel formation and are frequently investigated for cardiac-related therapies, the pro-angiogenic properties of CMPC and MSC-derived exosome-like vesicles are investigated. Both cell types secrete exosome-like vesicles, which are efficiently taken up by endothelial cells. Endothelial cell migration and vessel formation are stimulated by these exosomes in in vitro models, mediated via ERK/Akt-signaling. Additionally, these exosomes stimulated blood vessel formation into matrigel plugs. Analysis of pro-angiogenic factors revealed high levels of extracellular matrix metalloproteinase inducer (EMMPRIN). Knockdown of EMMPRIN on CMPCs leads to a diminished pro-angiogenic effect, both in vitro and in vivo. Therefore, CMPC and MSC exosomes have powerful pro-angiogenic effects, and this effect is largely mediated via the presence of EMMPRIN on exosomes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Myeloid-derived suppressor cells in murine AIDS inhibit B-cell responses in part via soluble mediators including reactive oxygen and nitrogen species, and TGF-β

    Rastad, Jessica L. [Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756 (United States); Green, William R., E-mail: William.R.Green@dartmouth.edu [Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756 (United States); Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756 (United States)

    2016-12-15

    Monocytic myeloid-derived suppressor cells (M-MDSCs) were increased during LP-BM5 retroviral infection, and were capable of suppressing not only T-cell, but also B-cell responses. In addition to previously demonstrating iNOS- and VISTA-dependent M-MDSC mechanisms, in this paper, we detail how M-MDSCs utilized soluble mediators, including the reactive oxygen and nitrogen species superoxide, peroxynitrite, and nitric oxide, and TGF-β, to suppress B cells in a predominantly contact-independent manner. Suppression was independent of cysteine-depletion and hydrogen peroxide production. When two major mechanisms of suppression (iNOS and VISTA) were eliminated in double knockout mice, M-MDSCs from LP-BM5-infected mice were able to compensate using other, soluble mechanisms in order to maintain suppression of B cells. The IL-10 producing regulatory B-cell compartment was among the targets of M-MDSC-mediated suppression. -- Highlights: •LP-BM5-expanded M-MDSCs utilized soluble mediators nitric oxide, superoxide, peroxynitrite, and TGF-β to suppress B cells. •When two major mechanisms of suppression were eliminated through knockouts, M-MDSCs maintained suppression. •M-MDSCs from LP-BM5-infected mice decreased proliferation of IL-10 producing regulatory B cells.

  19. Developmental responses of bread wheat to changes in ambient temperature following deletion of a locus that includes FLOWERING LOCUS T1.

    Dixon, Laura E; Farré, Alba; Finnegan, E Jean; Orford, Simon; Griffiths, Simon; Boden, Scott A

    2018-01-04

    FLOWERING LOCUS T (FT) is a central integrator of environmental signals that regulates the timing of vegetative to reproductive transition in flowering plants. In model plants, these environmental signals have been shown to include photoperiod, vernalization, and ambient temperature pathways, and in crop species, the integration of the ambient temperature pathway remains less well understood. In hexaploid wheat, at least 5 FT-like genes have been identified, each with a copy on the A, B, and D genomes. Here, we report the characterization of FT-B1 through analysis of FT-B1 null and overexpression genotypes under different ambient temperature conditions. This analysis has identified that the FT-B1 alleles perform differently under diverse environmental conditions; most notably, the FT-B1 null produces an increase in spikelet and tiller number when grown at lower temperature conditions. Additionally, absence of FT-B1 facilitates more rapid germination under both light and dark conditions. These results provide an opportunity to understand the FT-dependent pathways that underpin key responses of wheat development to changes in ambient temperature. This is particularly important for wheat, for which development and grain productivity are sensitive to changes in temperature. © 2018 The Authors Plant, Cell & Environment Published by John Wiley & Sons Ltd.

  20. Myeloid-derived suppressor cells in murine AIDS inhibit B-cell responses in part via soluble mediators including reactive oxygen and nitrogen species, and TGF-β

    Rastad, Jessica L.; Green, William R.

    2016-01-01

    Monocytic myeloid-derived suppressor cells (M-MDSCs) were increased during LP-BM5 retroviral infection, and were capable of suppressing not only T-cell, but also B-cell responses. In addition to previously demonstrating iNOS- and VISTA-dependent M-MDSC mechanisms, in this paper, we detail how M-MDSCs utilized soluble mediators, including the reactive oxygen and nitrogen species superoxide, peroxynitrite, and nitric oxide, and TGF-β, to suppress B cells in a predominantly contact-independent manner. Suppression was independent of cysteine-depletion and hydrogen peroxide production. When two major mechanisms of suppression (iNOS and VISTA) were eliminated in double knockout mice, M-MDSCs from LP-BM5-infected mice were able to compensate using other, soluble mechanisms in order to maintain suppression of B cells. The IL-10 producing regulatory B-cell compartment was among the targets of M-MDSC-mediated suppression. -- Highlights: •LP-BM5-expanded M-MDSCs utilized soluble mediators nitric oxide, superoxide, peroxynitrite, and TGF-β to suppress B cells. •When two major mechanisms of suppression were eliminated through knockouts, M-MDSCs maintained suppression. •M-MDSCs from LP-BM5-infected mice decreased proliferation of IL-10 producing regulatory B cells.

  1. Examining the protective role of ErbB2 modulation in human-induced pluripotent stem cell-derived cardiomyocytes.

    Eldridge, Sandy; Guo, Liang; Mussio, Jodie; Furniss, Mike; Hamre, John; Davis, Myrtle

    2014-10-01

    Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as an in vitro model system in cardiac biology and in drug discovery (e.g., cardiotoxicity testing). Qualification of these cells for use in mechanistic investigations will require detailed evaluations of cardiomyocyte signaling pathways and cellular responses. ErbB signaling and the ligand neuregulin play critical roles in survival and functional integrity of cardiac myocytes. As such, we sought to characterize the expression and activity of the ErbB family of receptors. Antibody microarray analysis performed on cell lysates derived from maturing hiPSC-CMs detected expression of ∼570 signaling proteins. EGFR/ErbB1, HER2/ErbB2, and ErbB4, but not ErbB3 receptors, of the epidermal growth factor receptor family were confirmed by Western blot. Activation of ErbB signaling by neuregulin-1β (NRG, a natural ligand for ErbB4) and its modulation by trastuzumab (a monoclonal anti-ErbB2 antibody) and lapatinib (a small molecule ErbB2 tyrosine kinase inhibitor) were evaluated through assessing phosphorylation of AKT and Erk1/2, two major downstream kinases of ErbB signaling, using nanofluidic proteomic immunoassay. Downregulation of ErbB2 expression by siRNA silencing attenuated NRG-induced AKT and Erk1/2 phosphorylation. Activation of ErbB signaling with NRG, or inhibition with trastuzumab, alleviated or aggravated doxorubicin-induced cardiomyocyte damage, respectively, as assessed by a real-time cellular impedance analysis and ATP measurement. Collectively, these results support the expanded use of hiPSC-CMs to examine mechanisms of cardiotoxicity and support the value of using these cells in early assessments of cardiotoxicity or efficacy. Published by Oxford University Press on behalf of Toxicological Sciences 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  2. Electrophysiological analysis of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) using multi-electrode arrays (MEAs)

    Sala, Luca; Ward-van Oostwaard, Dorien; Tertoolen, Leon G.J.; Mummery, Christine L.; Bellin, Milena

    2017-01-01

    Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes.

  3. Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells

    Giacomelli, Elisa; Bellin, Milena; Sala, Luca; Van Meer, Berend J.; Tertoolen, Leon G.J.; Orlova, Valeria V.; Mummery, Christine L.

    2017-01-01

    Cardiomyocytes and endothelial cells in the heart are in close proximity and in constant dialogue. Endothelium regulates the size of the heart, supplies oxygen to the myocardium and secretes factors that support cardiomyocyte function. Robust and predictive cardiac disease models that faithfully

  4. Inhibition of Rho - ROCK signaling induces apoptotic and non-apoptotic PS exposure in cardiomyocytes via inhibition of flippase

    Krijnen, Paul A. J.; Sipkens, Jessica A.; Molling, Johan W.; Rauwerda, Jan A.; Stehouwer, Coen D. A.; Muller, Alice; Paulus, Walter J.; van Nieuw Amerongen, Geerten P.; Hack, C. Erik; Verhoeven, Arthur J.; van Hinsbergh, Victor W. M.; Niessen, Hans W. M.

    2010-01-01

    Subsequent to myocardial infarction, cardiomyocytes within the infarcted areas and border zones expose phosphatidylserine (PS) in the outer plasma membrane leaflet (flip-flop). We showed earlier that in addition to apoptosis, this flip-flop can be reversible in cardiomyocytes. We now investigated a

  5. Formation of Cell-To-Cell Connection between Bone Marrow Cells and Isolated Rat Cardiomyocytes in a Cocultivation Model

    Skopalík, J.; Pásek, Michal; Rychtárik, M.; Koristek, Z.; Gabrielová, E.; Sheer, P.; Matejovič, P.; Modrianský, M.; Klabusay, M.

    2014-01-01

    Roč. 5, č. 5 (2014), s. 1000185 ISSN 2157-7013 Institutional support: RVO:61388998 Keywords : bone marrow * mononuclear cells * isolated cardiomyocytes * cocultivation Subject RIV: BO - Biophysics http://omicsonline.org/ open - access /formation-of-celltocell-connection-between-bone-marrow-cells- and -isolated-rat-cardiomyocytes-2157-7013.1000185.php?aid=33364

  6. The practicality of including the systemic inflammatory response syndrome in the definition of polytrauma: experience of a level one trauma centre.

    Butcher, Nerida E; Balogh, Zsolt J

    2013-01-01

    The systemic inflammatory response syndrome (SIRS) has been advocated as a significant predictor of outcome in trauma. Recent trauma literature has proposed SIRS as a surrogate for physiological derangements characteristic of polytrauma with some authors recommending its inclusion into the definition of polytrauma. The practicality of daily SIRS collection outside of specifically designed prospective trials is unknown. The purpose of this study was to assess the availability of SIRS variables and its appropriateness for inclusion into a definition of polytrauma. We hypothesised SIRS variables would be readily available and easy to collect, thus represent an appropriate inclusion into the definition of polytrauma. A prospective observational study of all trauma team activation patients over 7-months (August 2009 to February 2010) at a University affiliated level-1 urban trauma centre. SIRS data (temperature>38°C or 90 bpm; RR>20/min or a PaCO(2)12.0×10(9)L(-1), or 10 immature bands) collected from presentation, at 24 h intervals until 72 h post injury. Inclusion criteria were all patients generating a trauma team activation response age >16. 336 patients met inclusion criteria. In 46% (155/336) serial SIRS scores could not be calculated due to missing data. Lowest rates of missing data observed on admission [3% (11/336)]. Stratified by ISS>15 (132/336), in 7% (9/132) serial SIRS scores could not be calculated due to missing data. In 123 patients ISS>15 with complete data, 81% (100/123) developed SIRS. For Abbreviated Injury Scale (AIS)>2 in at least 2 body regions (64/336) in 5% (3/64) serial SIRS scores could not be calculated, with 92% (56/61) of patients with complete data developing SIRS. For Direct ICU admissions [25% (85/336)] 5% (4/85) of patients could not have serial SIRS calculated [mean ISS 15(±11)] and 90% (73/81) developed SIRS at least once over 72 h. Based on the experience of our level-1 trauma centre, the practicability of including SIRS into the

  7. Cardiotoxicity evaluation using human embryonic stem cells and induced pluripotent stem cell-derived cardiomyocytes.

    Zhao, Qi; Wang, Xijie; Wang, Shuyan; Song, Zheng; Wang, Jiaxian; Ma, Jing

    2017-03-09

    Cardiotoxicity remains an important concern in drug discovery. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become an attractive platform to evaluate cardiotoxicity. However, the consistency between human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in prediction of cardiotoxicity has yet to be elucidated. Here we screened the toxicities of four representative drugs (E-4031, isoprenaline, quinidine, and haloperidol) using both hESC-CMs and hiPSC-CMs, combined with an impedance-based bioanalytical method. It showed that both hESC-CMs and hiPSC-CMs can recapitulate cardiotoxicity and identify the effects of well-characterized compounds. The combined platform of hPSC-CMs and an impedance-based bioanalytical method could improve preclinical cardiotoxicity screening, holding great potential for increasing drug development accuracy.

  8. Hsp60 and p70S6K form a complex in human cardiomyocytes

    Kroupskaya I. V.

    2011-02-01

    Full Text Available Molecular chaperon Hsp60 and protein kinase p70S6K play an important functional role in the regulation of cardiomyocytes vital function or apoptosis. Aim. To study a possibility of in vivo complex formation between Hsp60 and p70S6K in cardiomyocytes. Methods. Co-immunoprecipitation, Western-blot analysis. Results. We have identified in vivo interaction between molecular chaperone Hsp60 and two isoforms of proteinkinase p70S6K in human myocardium, normal and affected by cardiomyopathy. Conclusions. The results obtained suggest a possible participation of molecular chaperon Hsp60 in regulation of p70S6K activity in stressinduced apoptotic signaling pathway in cardiomyocytes.

  9. [Octanol preconditioning alleviates mouse cardiomyocyte swelling induced by simulated ischemia/reperfusion challenge in vitro].

    Luo, Yukun; Fang, Jun; Fan, Lin; Lin, Chaogui; Chen, Zhaoyang; Chen, Lianglong

    2012-10-01

    To investigate the role of connexin 43-formed hemichannels in cell volume regulation induced by simulated ischemia/reperfusion (SI/R). Mouse cardiomyocytes isolated on a Langendorff apparatus with enzyme solution were aliquoted into control, SI/R and SI/R +octanol groups. Calcein-AM was used to stain the cells and the cell volume was measured with confocal microscope by stack scanning. Trypan blue was used to measure the cell viability after the treatments. Calcein-AM staining and cofocal microscopy yielded stable and reproducible results for cell volume measurement. Mouse cardiomyocytes subjected to simulated SI/R showed obvious cell swelling as compared with the control cells [(126∓6)% vs 100%, Poctanol preconditioning significantly attenuated the cell swelling [(113∓6)%, Poctanol preconditioning obviously reduced the viability of the cells with SI/R challenge [(31∓2)%, Poctanol can alleviate the cell swelling to enhance the viability of the cardiomyocytes following SI/R.

  10. Decreased inward rectifier potassium current IK1 in dystrophin-deficient ventricular cardiomyocytes.

    Rubi, Lena; Koenig, Xaver; Kubista, Helmut; Todt, Hannes; Hilber, Karlheinz

    2017-03-04

    Kir2.x channels in ventricular cardiomyocytes (most prominently Kir2.1) account for the inward rectifier potassium current I K1 , which controls the resting membrane potential and the final phase of action potential repolarization. Recently it was hypothesized that the dystrophin-associated protein complex (DAPC) is important in the regulation of Kir2.x channels. To test this hypothesis, we investigated potential I K1 abnormalities in dystrophin-deficient ventricular cardiomyocytes derived from the hearts of Duchenne muscular dystrophy mouse models. We found that I K1 was substantially diminished in dystrophin-deficient cardiomyocytes when compared to wild type myocytes. This finding represents the first functional evidence for a significant role of the DAPC in the regulation of Kir2.x channels.

  11. Herpesvirus-Mediated Delivery of a Genetically Encoded Fluorescent Ca2+ Sensor to Canine Cardiomyocytes

    János Prorok

    2009-01-01

    Full Text Available We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescent Ca2+ sensor to adult canine cardiomyocytes. The efficacy of transduction was assessed by calculating the ratio of fluorescently labelled and nonlabelled cells in cell culture. Interaction of the virus vector with electrophysiological properties of cardiomyocytes was evaluated by the analysis of transient outward current (Ito, kinetics of the intracellular Ca2+ transients, and cell shortening. Functionality of transferred troponeon was verified by FRET analysis. We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%. We showed that even after four days neither the amplitude nor the kinetics of the Ito current was significantly changed and no major shifts occurred in parameters of [Ca2+]i transients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells.

  12. Effects of high-altitude exercise training on contractile function of rat skinned cardiomyocyte.

    Cazorla, O; Aït Mou, Y; Goret, L; Vassort, G; Dauzat, M; Lacampagne, A; Tanguy, S; Obert, P

    2006-09-01

    Previous studies have questioned whether there is an improved cardiac function after high-altitude training. Accordingly, the present study was designed specifically to test whether this apparent blunted response of the whole heart to training can be accounted for by altered mechanical properties at the cellular level. Adult rats were trained for 5 weeks under normoxic (N, NT for sedentary and trained animals, respectively) or hypobaric hypoxic (H, HT) conditions. Cardiac morphology and function were evaluated by echocardiography. Calcium Ca2+ sensitivity of the contractile machinery was estimated in skinned cardiomyocytes isolated from the left ventricular (LV) sub-epicardium (Epi) and sub-endocardium (Endo) at short and long sarcomere lengths (SL). Cardiac remodelling was harmonious (increase in wall thickness with chamber dilatation) in NT rats and disharmonious (hypertrophy without chamber dilatation) in HT rats. Contrary to NT rats, HT rats did not exhibit enhancement in global cardiac performance evaluated by echocardiography. Stretch- dependent Ca2+ sensitization of the myofilaments (cellular index of the Frank-Starling mechanism) increased from Epi to Endo in N rats. Training in normoxic conditions further increased this stretch-dependent Ca2+ sensitization. Chronic hypoxia did not significantly affect myofibrilar Ca2+ sensitivity. In contrast, high-altitude training decreased Ca2+ sensitivity of the myofilaments at both SL, mostly in Endo cells, resulting in a loss of the transmural gradient of the stretch-dependent Ca2+ sensitization. Expression of myosin heavy chain isoforms was affected both by training and chronic hypoxia but did not correlate with mechanical data. Training at sea level increased the transmural gradient of stretch-dependent Ca2+ sensitization of the myofilaments, accounting for an improved Frank-Starling mechanism. High-altitude training depressed myofilament response to Ca2+, especially in the Endo layer. This led to a reduction in

  13. Tuning the conductivity and inner structure of electrospun fibers to promote cardiomyocyte elongation and synchronous beating.

    Liu, Yaowen; Lu, Jinfu; Xu, Guisen; Wei, Jiaojun; Zhang, Zhibin; Li, Xiaohong

    2016-12-01

    The key to addressing the challenges facing cardiac tissue engineering is the integration of physical, chemical, and electrical cues into scaffolds. Aligned and conductive scaffolds have been fabricated as synthetic microenvironments to improve the function of cardiomyocytes. However, up to now, the influence of conductive capability and inner structure of fibrous scaffolds have not been determined on the cardiomyocyte morphologies and beating patterns. In the current study, highly aligned fibers were fabricated with loaded up to 6% of carbon nanotubes (CNTs) to modulate the electrical conductivity, while blend and coaxial electrospinning were utilized to create a bulk distribution of CNTs in fiber matrices and a spatial embedment in fiber cores, respectively. Conductive networks were formed in the fibrous scaffolds after the inoculation of over 3% CNTs, and the increase in the conductivity could maintain the cell viabilities, induce the cell elongation, enhance the production of sarcomeric α-actinin and troponin I, and promote the synchronous beating of cardiomyocytes. Although the conductivity of blend fibers is slightly higher than that of coaxial fibers with the same CNT loadings, the lower exposures to CNTs resulted in higher cell viability, elongation, extracellular matrix secretion and beating rates for cardiomyocytes on coaxial fibers. Taken altogether, core-sheath fibers with loaded 5% of CNTs in the fiber cores facilitated the cardiomyocyte growth with a production of organized contractile proteins and a pulsation frequency close to that of the atrium. It is suggested that electrospun scaffolds that couple conductivity and fibrous structure considerations may provide optimal stimuli to foster cell morphology and functions for myocardial regeneration or establishment of in vitro cardiomyocyte culture platform for drug screening. Copyright © 2016. Published by Elsevier B.V.

  14. Adrenergic Stress Protection of Human iPS Cell-Derived Cardiomyocytes by Fast Kv7.1 Recycling

    Ilaria Piccini

    2017-09-01

    Full Text Available The fight-or-flight response (FFR, a physiological acute stress reaction, involves positive chronotropic and inotropic effects on heart muscle cells mediated through β-adrenoceptor activation. Increased systolic calcium is required to enable stronger heart contractions whereas elevated potassium currents are to limit the duration of the action potentials and prevent arrhythmia. The latter effect is accomplished by an increased functional activity of the Kv7.1 channel encoded by KCNQ1. Current knowledge, however, does not sufficiently explain the full extent of rapid Kv7.1 activation and may hence be incomplete. Using inducible genetic KCNQ1 complementation in KCNQ1-deficient human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs, we here reinvestigate the functional role of Kv7.1 in adapting human CMs to adrenergic stress. Under baseline conditions, Kv7.1 was barely detectable at the plasma membrane of hiPSC-CMs, yet it fully protected these from adrenergic stress-induced beat-to-beat variability of repolarization and torsade des pointes-like arrhythmia. Furthermore, isoprenaline treatment increased field potential durations specifically in KCNQ1-deficient CMs to cause these adverse macroscopic effects. Mechanistically, we find that the protective action by Kv7.1 resides in a rapid translocation of channel proteins from intracellular stores to the plasma membrane, induced by adrenergic signaling. Gene silencing experiments targeting RAB GTPases, mediators of intracellular vesicle trafficking, showed that fast Kv7.1 recycling under acute stress conditions is RAB4A-dependent.Our data reveal a key mechanism underlying the rapid adaptation of human cardiomyocytes to adrenergic stress. These findings moreover aid to the understanding of disease pathology in long QT syndrome and bear important implications for safety pharmacological screening.

  15. Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome.

    Xu, Aibin; Liu, Jingyi; Liu, Peilin; Jia, Min; Wang, Han; Tao, Ling

    2014-04-18

    Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H2O2 led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H2O2 and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the process. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

    Inadomi, Chiaki, E-mail: inadomic@nagasaki-u.ac.jp [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan); Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Murata, Hiroaki [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan); Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Ihara, Yoshito [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Department of Biochemistry, Wakayama Medical University, Wakayama 641-8509 (Japan); Goto, Shinji; Urata, Yoshishige [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Yodoi, Junji [Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto 606-8507 (Japan); Kondo, Takahito [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Sumikawa, Koji [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan)

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. Black-Right-Pointing-Pointer NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. Black-Right-Pointing-Pointer Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

  17. Chymase inhibition prevents fibronectin and myofibrillar loss and improves cardiomyocyte function and LV torsion angle in dogs with isolated mitral regurgitation.

    Pat, Betty; Chen, Yuanwen; Killingsworth, Cheryl; Gladden, James D; Shi, Ke; Zheng, Junying; Powell, Pamela C; Walcott, Greg; Ahmed, Mustafa I; Gupta, Himanshu; Desai, Ravi; Wei, Chih-Chang; Hase, Naoki; Kobayashi, Tsunefumi; Sabri, Abdelkarim; Granzier, Henk; Denney, Thomas; Tillson, Michael; Dillon, A Ray; Husain, Ahsan; Dell'italia, Louis J

    2010-10-12

    The left ventricular (LV) dilatation of isolated mitral regurgitation (MR) is associated with an increase in chymase and a decrease in interstitial collagen and extracellular matrix. In addition to profibrotic effects, chymase has significant antifibrotic actions because it activates matrix metalloproteinases and kallikrein and degrades fibronectin. Thus, we hypothesize that chymase inhibitor (CI) will attenuate extracellular matrix loss and LV remodeling in MR. We studied dogs with 4 months of untreated MR (MR; n=9) or MR treated with CI (MR+CI; n=8). Cine MRI demonstrated a >40% increase in LV end-diastolic volume in both groups, consistent with a failure of CI to improve a 25% decrease in interstitial collagen in MR. However, LV cardiomyocyte fractional shortening was decreased in MR versus normal dogs (3.71±0.24% versus 4.81±0.31%; P<0.05) and normalized in MR+CI dogs (4.85±0.44%). MRI with tissue tagging demonstrated an increase in LV torsion angle in MR+CI versus MR dogs. CI normalized the significant decrease in fibronectin and FAK phosphorylation and prevented cardiomyocyte myofibrillar degeneration in MR dogs. In addition, total titin and its stiffer isoform were increased in the LV epicardium and paralleled the changes in fibronectin and FAK phosphorylation in MR+CI dogs. These results suggest that chymase disrupts cell surface-fibronectin connections and FAK phosphorylation that can adversely affect cardiomyocyte myofibrillar structure and function. The greater effect of CI on epicardial versus endocardial titin and noncollagen cell surface proteins may be responsible for the increase in torsion angle in chronic MR.

  18. Generation of functional cardiomyocytes from rat embryonic and induced pluripotent stem cells using feeder-free expansion and differentiation in suspension culture.

    Dahlmann, Julia; Awad, George; Dolny, Carsten; Weinert, Sönke; Richter, Karin; Fischer, Klaus-Dieter; Munsch, Thomas; Leßmann, Volkmar; Volleth, Marianne; Zenker, Martin; Chen, Yaoyao; Merkl, Claudia; Schnieke, Angelika; Baraki, Hassina; Kutschka, Ingo; Kensah, George

    2018-01-01

    The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.

  19. Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

    Inadomi, Chiaki; Murata, Hiroaki; Ihara, Yoshito; Goto, Shinji; Urata, Yoshishige; Yodoi, Junji; Kondo, Takahito; Sumikawa, Koji

    2012-01-01

    Highlights: ► GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. ► NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. ► Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

  20. Expression Profiling of Human Pluripotent Stem Cell-Derived Cardiomyocytes Exposed to Doxorubicin-Integration and Visualization of Multi-Omics Data.

    Holmgren, Gustav; Sartipy, Peter; Andersson, Christian X; Lindahl, Anders; Synnergren, Jane

    2018-05-01

    Anthracyclines, such as doxorubicin, are highly efficient chemotherapeutic agents against a variety of cancers. However, anthracyclines are also among the most cardiotoxic therapeutic drugs presently on the market. Chemotherapeutic-induced cardiomyopathy is one of the leading causes of disease and mortality in cancer survivors. The exact mechanisms responsible for doxorubicin-induced cardiomyopathy are not completely known, but the fact that the cardiotoxicity is dose-dependent and that there is a variation in time-to-onset of toxicity, and gender- and age differences suggests that several mechanisms may be involved. In this study, we investigated doxorubicin-induced cardiotoxicity in human pluripotent stem cell-derived cardiomyocytes using proteomics. In addition, different sources of omics data (protein, mRNA, and microRNA) from the same experimental setup were further combined and analyzed using newly developed methods to identify differential expression in data of various origin and types. Subsequently, the results were integrated in order to generate a combined visualization of the findings. In our experimental model system, we exposed cardiomyocytes derived from human pluripotent stem cells to doxorubicin for up to 2 days, followed by a wash-out period of additionally 12 days. Besides an effect on the cell morphology and cardiomyocyte functionality, the data show a strong effect of doxorubicin on all molecular levels investigated. Differential expression patterns that show a linkage between the proteome, transcriptome, and the regulatory microRNA network, were identified. These findings help to increase the understanding of the mechanisms behind anthracycline-induced cardiotoxicity and suggest putative biomarkers for this condition.

  1. The T Cell Response to Major Grass Allergens Is Regulated and Includes IL-10 Production in Atopic but Not in Non-Atopic Subjects

    Domdey, A.; Liu, A.; Millner, A.

    2010-01-01

    in allergen-specific responses. The aim was to determine whether major grass allergens induce production of suppressive cytokines in allergic and healthy subjects and to examine the inhibitory effect of these cytokines on allergic responses. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated...... from healthy and grass-allergic donors and stimulated with the major grass allergens Phl p 1 or Phl p 5. The effects of endogenous IL-10 and/or TGF-beta on proliferation and cytokine production were determined by use of blocking antibodies. In addition, the number of CD4(+)CD25(+) T cells...... in PBMCs in the two groups, but fewer cells from atopic donors were CD4(+)CD25(+)CCR4(+) and more cells were CD4(+)CD25(+)CLA(+) compared to healthy donors. Conclusion: Allergen-specific responses of grass allergic patients but not in non-atopic subjects are influenced by regulatory cytokines produced...

  2. Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis.

    Nakashima, Kazuo; Fujita, Yasunari; Katsura, Koji; Maruyama, Kyonoshin; Narusaka, Yoshihiro; Seki, Motoaki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2006-01-01

    ABA-responsive elements (ABREs) are cis-acting elements and basic leucine zipper (bZIP)-type ABRE-binding proteins (AREBs) are transcriptional activators that function in the expression of RD29B in vegetative tissue of Arabidopsis in response to abscisic acid (ABA) treatment. Dehydration-responsive elements (DREs) function as coupling elements of ABRE in the expression of RD29A in response to ABA. Expression analysis using abi3 and abi5 mutants showed that ABI3 and ABI5 play important roles in the expression of RD29B in seeds. Base-substitution analysis showed that two ABREs function strongly and one ABRE coupled with DRE functions weakly in the expression of RD29A in embryos. In a transient transactivation experiment, ABI3, ABI5 and AREB1 activated transcription of a GUS reporter gene driven by the RD29B promoter strongly but these proteins activated the transcription driven by the RD29A promoter weakly. In 35S::ABI3 Arabidopsis plants, the expression of RD29B was up-regulated strongly, but that of RD29A was up-regulated weakly. These results indicate that the expression of RD29B having ABREs in the promoter is up-regulated strongly by ABI3, whereas that of RD29A having one ABRE coupled with DREs in the promoter is up-regulated weakly by ABI3. We compared the expression of 7000 Arabidopsis genes in response to ABA treatment during germination and in the vegetative growth stage, and that in 35S::ABI3 plants using a full-length cDNA microarray. The expression of ABI3- and/or ABA-responsive genes and cis-elements in the promoters are discussed.

  3. Assessment of drug-induced arrhythmic risk using limit cycle and autocorrelation analysis of human iPSC-cardiomyocyte contractility

    Kirby, R. Jason [Sanford Burnham Prebys Medical Discovery Institute, Conrad Prebys Center for Chemical Genomics, 6400 Sanger Rd, Orlando, FL 32827 (United States); Qi, Feng [Sanford Burnham Prebys Medical Discovery Institute, Applied Bioinformatics Facility, 6400 Sanger Rd, Orlando, FL 32827 (United States); Phatak, Sharangdhar; Smith, Layton H. [Sanford Burnham Prebys Medical Discovery Institute, Conrad Prebys Center for Chemical Genomics, 6400 Sanger Rd, Orlando, FL 32827 (United States); Malany, Siobhan, E-mail: smalany@sbpdiscovery.org [Sanford Burnham Prebys Medical Discovery Institute, Conrad Prebys Center for Chemical Genomics, 6400 Sanger Rd, Orlando, FL 32827 (United States)

    2016-08-15

    Cardiac safety assays incorporating label-free detection of human stem-cell derived cardiomyocyte contractility provide human relevance and medium throughput screening to assess compound-induced cardiotoxicity. In an effort to provide quantitative analysis of the large kinetic datasets resulting from these real-time studies, we applied bioinformatic approaches based on nonlinear dynamical system analysis, including limit cycle analysis and autocorrelation function, to systematically assess beat irregularity. The algorithms were integrated into a software program to seamlessly generate results for 96-well impedance-based data. Our approach was validated by analyzing dose- and time-dependent changes in beat patterns induced by known proarrhythmic compounds and screening a cardiotoxicity library to rank order compounds based on their proarrhythmic potential. We demonstrate a strong correlation for dose-dependent beat irregularity monitored by electrical impedance and quantified by autocorrelation analysis to traditional manual patch clamp potency values for hERG blockers. In addition, our platform identifies non-hERG blockers known to cause clinical arrhythmia. Our method provides a novel suite of medium-throughput quantitative tools for assessing compound effects on cardiac contractility and predicting compounds with potential proarrhythmia and may be applied to in vitro paradigms for pre-clinical cardiac safety evaluation. - Highlights: • Impedance-based monitoring of human iPSC-derived cardiomyocyte contractility • Limit cycle analysis of impedance data identifies aberrant oscillation patterns. • Nonlinear autocorrelation function quantifies beat irregularity. • Identification of hERG and non-hERG inhibitors with known risk of arrhythmia • Automated software processes limit cycle and autocorrelation analyses of 96w data.

  4. Assessment of drug-induced arrhythmic risk using limit cycle and autocorrelation analysis of human iPSC-cardiomyocyte contractility

    Kirby, R. Jason; Qi, Feng; Phatak, Sharangdhar; Smith, Layton H.; Malany, Siobhan

    2016-01-01

    Cardiac safety assays incorporating label-free detection of human stem-cell derived cardiomyocyte contractility provide human relevance and medium throughput screening to assess compound-induced cardiotoxicity. In an effort to provide quantitative analysis of the large kinetic datasets resulting from these real-time studies, we applied bioinformatic approaches based on nonlinear dynamical system analysis, including limit cycle analysis and autocorrelation function, to systematically assess beat irregularity. The algorithms were integrated into a software program to seamlessly generate results for 96-well impedance-based data. Our approach was validated by analyzing dose- and time-dependent changes in beat patterns induced by known proarrhythmic compounds and screening a cardiotoxicity library to rank order compounds based on their proarrhythmic potential. We demonstrate a strong correlation for dose-dependent beat irregularity monitored by electrical impedance and quantified by autocorrelation analysis to traditional manual patch clamp potency values for hERG blockers. In addition, our platform identifies non-hERG blockers known to cause clinical arrhythmia. Our method provides a novel suite of medium-throughput quantitative tools for assessing compound effects on cardiac contractility and predicting compounds with potential proarrhythmia and may be applied to in vitro paradigms for pre-clinical cardiac safety evaluation. - Highlights: • Impedance-based monitoring of human iPSC-derived cardiomyocyte contractility • Limit cycle analysis of impedance data identifies aberrant oscillation patterns. • Nonlinear autocorrelation function quantifies beat irregularity. • Identification of hERG and non-hERG inhibitors with known risk of arrhythmia • Automated software processes limit cycle and autocorrelation analyses of 96w data

  5. Mapping of redox state of mitochondrial cytochromes in live cardiomyocytes using Raman microspectroscopy

    Brazhe, Nadezda A; Treiman, Marek; Brazhe, Alexey R

    2012-01-01

    This paper presents a nonivasive approach to study redox state of reduced cytochromes [Formula: see text], [Formula: see text] and [Formula: see text] of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach ...

  6. Cardiomyocyte behavior on biodegradable polyurethane/gold nanocomposite scaffolds under electrical stimulation

    Ganji, Yasaman [Faculty of Biomedical Engineering, Amirkabir University of Technology, 424 Hafez Ave, Tehran (Iran, Islamic Republic of); Institute for Materials Science, Dept. Biocompatible Nanomaterials, University of Kiel, Kaiserstr. 2, D-24143 Kiel (Germany); Li, Qian [Institute for Materials Science, Dept. Biocompatible Nanomaterials, University of Kiel, Kaiserstr. 2, D-24143 Kiel (Germany); Quabius, Elgar Susanne [Dept. of Otorhinolaryngology, Head and Neck Surgery, University of Kiel, Arnold-Heller-Str. 3, Building 27, D-24105 Kiel (Germany); Institute of Immunology, University of Kiel, Arnold-Heller-Str. 3, Building 17, D-24105 Kiel (Germany); Böttner, Martina [Department of Anatomy, University of Kiel, Otto-Hahn-Platz 8, 24118 Kiel (Germany); Selhuber-Unkel, Christine, E-mail: cse@tf.uni-kiel.de [Institute for Materials Science, Dept. Biocompatible Nanomaterials, University of Kiel, Kaiserstr. 2, D-24143 Kiel (Germany); Kasra, Mehran [Faculty of Biomedical Engineering, Amirkabir University of Technology, 424 Hafez Ave, Tehran (Iran, Islamic Republic of)

    2016-02-01

    Following a myocardial infarction (MI), cardiomyocytes are replaced by scar tissue, which decreases ventricular contractile function. Tissue engineering is a promising approach to regenerate such damaged cardiomyocyte tissue. Engineered cardiac patches can be fabricated by seeding a high density of cardiac cells onto a synthetic or natural porous polymer. In this study, nanocomposite scaffolds made of gold nanotubes/nanowires incorporated into biodegradable castor oil-based polyurethane were employed to make micro-porous scaffolds. H9C2 cardiomyocyte cells were cultured on the scaffolds for one day, and electrical stimulation was applied to improve cell communication and interaction in neighboring pores. Cells on scaffolds were examined by fluorescence microscopy and scanning electron microscopy, revealing that the combination of scaffold design and electrical stimulation significantly increased cell confluency of H9C2 cells on the scaffolds. Furthermore, we showed that the gene expression levels of Nkx2.5, atrial natriuretic peptide (ANF) and natriuretic peptide precursor B (NPPB), which are functional genes of the myocardium, were up-regulated by the incorporation of gold nanotubes/nanowires into the polyurethane scaffolds, in particular after electrical stimulation. - Highlights: • Biodegradable polyurethane/gold nanocomposites for cardiomyocyte adhesion are proposed. • The nanocomposite scaffolds are porous and electrical stimulation enhances cell adhesion. • Expression levels of functional myocardium genes were upregulated after electrical stimulation.

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

    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.

  8. PGC-1α and Reactive Oxygen Species Regulate Human Embryonic Stem Cell-Derived Cardiomyocyte Function

    Birket, Matthew J.; Casini, Simona; Kosmidis, Georgios; Elliott, David A.; Gerencser, Akos A.; Baartscheer, Antonius; Schumacher, Cees; Mastroberardino, Pier G.; Elefanty, Andrew G.; Stanley, Ed G.; Mummery, Christine L.

    2013-01-01

    Diminished mitochondrial function is causally related to some heart diseases. Here, we developed a human disease model based on cardiomyocytes from human embryonic stem cells (hESCs), in which an important pathway of mitochondrial gene expression was inactivated. Repression of PGC-1α, which is

  9. Human heart disease : lessons from human pluripotent stem cell-derived cardiomyocytes

    Giacomelli, E.; Mummery, C.L.; Bellin, M.

    2017-01-01

    Technical advances in generating and phenotyping cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are now driving their wider acceptance as in vitro models to understand human heart disease and discover therapeutic targets that may lead to new compounds for clinical use. Current

  10. Identification and functionality of proteomes secreted by rat cardiac stem cells and neonatal cardiomyocytes

    Šťastná, Miroslava; Chimenti, I.; Marban, E.; Van Eyk, J.E.

    2010-01-01

    Roč. 10, č. 2 (2010), s. 245-253 ISSN 1615-9853 Institutional research plan: CEZ:AV0Z40310501 Keywords : animal proteomics * cardiac stem cells * neonatal cardiomyocytes Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.815, year: 2010

  11. Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates

    Kijlstra, Jan David; Hu, Dongjian; van der Meer, Peter; Domian, Ibrahim J

    2017-01-01

    Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and

  12. Dehydrosilybin attenuates the production of ROS in rat cardiomyocyte mitochondria with an uncoupler-like mechanism

    Gabrielová, E.; Jabůrek, Martin; Gažák, Radek; Vostálová, J.; Ježek, Jan; Křen, Vladimír; Modrianský, M.

    2010-01-01

    Roč. 42, č. 6 (2010), s. 499-509 ISSN 0145-479X R&D Projects: GA ČR(CZ) GA303/08/0658 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z50200510 Keywords : Reactive oxygen species * Cardiomyocytes * Dehydrosilybin Subject RIV: CE - Biochemistry Impact factor: 3.637, year: 2010

  13. Functional interaction between bicarbonate transporters and carbonic anhydrase modulates lactate uptake into mouse cardiomyocytes.

    Peetz, Jan; Barros, L Felipe; San Martín, Alejandro; Becker, Holger M

    2015-07-01

    Blood-derived lactate is a precious energy substrate for the heart muscle. Lactate is transported into cardiomyocytes via monocarboxylate transporters (MCTs) together with H(+), which couples lactate uptake to cellular pH regulation. In this study, we have investigated how the interplay between different acid/base transporters and carbonic anhydrases (CA), which catalyze the reversible hydration of CO2, modulates the uptake of lactate into isolated mouse cardiomyocytes. Lactate transport was estimated both as lactate-induced acidification and as changes in intracellular lactate levels measured with a newly developed Förster resonance energy transfer (FRET) nanosensor. Recordings of intracellular pH showed an increase in the rate of lactate-induced acidification when CA was inhibited by 6-ethoxy-2-benzothiazolesulfonamide (EZA), while direct measurements of lactate flux demonstrated a decrease in MCT transport activity, when CA was inhibited. The data indicate that catalytic activity of extracellular CA increases lactate uptake and counteracts intracellular lactate-induced acidification. We propose a hypothetical model, in which HCO3 (-), formed from cell-derived CO2 at the outer surface of the cardiomyocyte plasma membrane by membrane-anchored, extracellular CA, is transported into the cell via Na(+)/HCO3 (-) cotransport to counteract intracellular acidification, while the remaining H(+) stabilizes extracellular pH at the surface of the plasma membrane during MCT activity to enhance lactate influx into cardiomyocytes.

  14. Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development

    Silvia Mazzotta

    2016-10-01

    Full Text Available Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis, and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development.

  15. Evaluation of electrical propagation delay with cardiomyocytes by photosensitization reaction in vitro

    Doi, Marika; Ogawa, Emiyu; Arai, Tsunenori

    2017-02-01

    In order to study cardiomyocyte electrical conduction damage by a photosensitization reaction (PR) mostly comes from outside of the cells in a few minutes after the PR, we studied propagation delay of contact action potential with cardiomyocyte by the PR. To determine appropriate PR condition for tachyarrhythmia ablation, a precise electrophysiological experiment in vitro has been preferable. We measured the contact action potential using a microelectrode array system of which information may be correct than conventional Ca2+ measurement. We investigated the propagation delays of an evoked potential to evaluate the electrical conduction damage by the PR. Rat cardiomyocytes were cultivated for 5-7 days on a dish with which 64 electrodes were patterned, in an incubator controlled to 37°C, 5% CO2. The following conditions were used for the PR: 40 μg/ml talapordfin sodium and 290 mW/cm2, 40-78 J/cm2 for an irradiation. A 2D map was obtained to visualize the propagation delays of the evoked potential. The propagation speed, which was calculated based on the measured propagation delays, was decreased by about 30-50% on average of all electrodes after the PR. Therefore, we think 2D propagation delays measurement of the evoked potential with contact action potential measuring system might be available to evaluate the acute electrical conduction damage of cardiomyocyte by the PR.

  16. Bioreactor cultivation enhances NTEB formation and differentiation of NTES cells into cardiomyocytes.

    Lü, Shuanghong; Liu, Sheng; He, Wenjun; Duan, Cuimi; Li, Yanmin; Liu, Zhiqiang; Zhang, Ye; Hao, Tong; Wang, Yanmeng; Li, Dexue; Wang, Changyong; Gao, Shaorong

    2008-09-01

    Autogenic embryonic stem cells established from somatic cell nuclear transfer (SCNT) embryos have been proposed as unlimited cell sources for cell transplantation-based treatment of many genetic and degenerative diseases, which can eliminate the immune rejection that occurs after transplantation. In the present study, pluripotent nuclear transfer ES (NTES) cell lines were successfully established from different strains of mice. One NTES cell line, NT1, with capacity of germline transmission, was used to investigate in vitro differentiation into cardiomyocytes. To optimize differentiation conditions for mass production of embryoid bodies (NTEBs) from NTES cells, a slow-turning lateral vessel (STLV) rotating bioreactor was used for culturing the NTES cells to produce NTEBs compared with a conventional static cultivation method. Our results demonstrated that the NTEBs formed in STLV bioreactor were more uniform in size, and no large necrotic centers with most of the cells in NTEBs were viable. Differentiation of the NTEBs formed in both the STLV bioreactor and static culture into cardiomyocytes was induced by ascorbic acid, and the results demonstrated that STLV-produced NTEBs differentiated into cardiomyocytes more efficiently. Taken together, our results suggested that STLV bioreactor provided a more ideal culture condition, which can facilitate the formation of better quality NTEBs and differentiation into cardiomyocytes more efficiently in vitro.

  17. Cardiomyocyte Hypocontractility and Reduced Myofibril Density in End-Stage Pediatric Cardiomyopathy

    Ilse A. E. Bollen

    2017-12-01

    Full Text Available Dilated cardiomyopathy amongst children (pediatric cardiomyopathy, pediatric CM is associated with a high morbidity and mortality. Because little is known about the pathophysiology of pediatric CM, treatment is largely based on adult heart failure therapy. The reason for high morbidity and mortality is largely unknown as well as data on cellular pathomechanisms is limited. Here, we assessed cardiomyocyte contractility and protein expression to define cellular pathomechanisms in pediatric CM. Explanted heart tissue of 11 pediatric CM patients and 18 controls was studied. Contractility was measured in single membrane-permeabilized cardiomyocytes and protein expression was assessed with gel electrophoresis and western blot analysis. We observed increased Ca2+-sensitivity of myofilaments which was due to hypophosphorylation of cardiac troponin I, a feature commonly observed in adult DCM. We also found a significantly reduced maximal force generating capacity of pediatric CM cardiomyocytes, as well as a reduced passive force development over a range of sarcomere lengths. Myofibril density was reduced in pediatric CM compared to controls. Correction of maximal force and passive force for myofibril density normalized forces in pediatric CM cardiomyocytes to control values. This implies that the hypocontractility was caused by the reduction in myofibril density. Unlike in adult DCM we did not find an increase in compliant titin isoform expression in end-stage pediatric CM. The limited ability of pediatric CM patients to maintain myofibril density might have contributed to their early disease onset and severity.

  18. Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS mediated cardiomyocyte hypertrophy

    Tigchelaar, Wardit; Yu, Hongjuan; De Jong, Anne Margreet; van Gilst, Wiek H; van der Harst, Pim; Westenbrink, B Daan; de Boer, Rudolf A; Sillje, Herman H W

    2015-01-01

    Recently, a genetic variant in the mitochondrial exo/endo nuclease EXOG, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and

  19. HALT & REVERSE: Hsf1 activators lower cardiomyocyt damage; towards a novel approach to REVERSE atrial fibrillation

    E. Lanters (Eva); D.M.S. Marion (Denise M. S.); C. Kik (Charles); H. Steen (Herman); A.J.J.C. Bogers (Ad); M.A. Allessie (Maurits); B.J.J.M. Brundel (Bianca); N.M.S. de Groot (Natasja)

    2015-01-01

    textabstractBackground: Atrial fibrillation is a progressive arrhythmia, the exact mechanism underlying the progressive nature of recurrent AF episodes is still unknown. Recently, it was found that key players of the protein quality control system of the cardiomyocyte, i.e. Heat Shock Proteins,

  20. NanoSIMS Analysis of Intravascular Lipolysis and Lipid Movement across Capillaries and into Cardiomyocytes

    He, Cuiwen; Weston, Thomas A; Jung, Rachel S

    2018-01-01

    , mice were given an injection of [2H]triglyceride-enriched TRLs, and the movement of 2H-labeled lipids across capillaries and into cardiomyocytes was examined by NanoSIMS. TRL processing and lipid movement in tissues were extremely rapid. Within 30 s, TRL-derived lipids appeared in the subendothelial...

  1. Cardiomyocyte Hypocontractility and Reduced Myofibril Density in End-Stage Pediatric Cardiomyopathy.

    Bollen, Ilse A E; van der Meulen, Marijke; de Goede, Kyra; Kuster, Diederik W D; Dalinghaus, Michiel; van der Velden, Jolanda

    2017-01-01

    Dilated cardiomyopathy amongst children (pediatric cardiomyopathy, pediatric CM) is associated with a high morbidity and mortality. Because little is known about the pathophysiology of pediatric CM, treatment is largely based on adult heart failure therapy. The reason for high morbidity and mortality is largely unknown as well as data on cellular pathomechanisms is limited. Here, we assessed cardiomyocyte contractility and protein expression to define cellular pathomechanisms in pediatric CM. Explanted heart tissue of 11 pediatric CM patients and 18 controls was studied. Contractility was measured in single membrane-permeabilized cardiomyocytes and protein expression was assessed with gel electrophoresis and western blot analysis. We observed increased Ca 2+ -sensitivity of myofilaments which was due to hypophosphorylation of cardiac troponin I, a feature commonly observed in adult DCM. We also found a significantly reduced maximal force generating capacity of pediatric CM cardiomyocytes, as well as a reduced passive force development over a range of sarcomere lengths. Myofibril density was reduced in pediatric CM compared to controls. Correction of maximal force and passive force for myofibril density normalized forces in pediatric CM cardiomyocytes to control values. This implies that the hypocontractility was caused by the reduction in myofibril density. Unlike in adult DCM we did not find an increase in compliant titin isoform expression in end-stage pediatric CM. The limited ability of pediatric CM patients to maintain myofibril density might have contributed to their early disease onset and severity.

  2. Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient–derived cardiomyocytes

    Susi Zatti

    2014-01-01

    Full Text Available Duchenne muscular dystrophy (DMD–associated cardiac diseases are emerging as a major cause of morbidity and mortality in DMD patients, and many therapies for treatment of skeletal muscle failed to improve cardiac function. The reprogramming of patients' somatic cells into pluripotent stem cells, combined with technologies for correcting the genetic defect, possesses great potential for the development of new treatments for genetic diseases. In this study, we obtained human cardiomyocytes from DMD patient–derived, induced pluripotent stem cells genetically corrected with a human artificial chromosome carrying the whole dystrophin genomic sequence. Stimulation by cytokines was combined with cell culturing on hydrogel with physiological stiffness, allowing an adhesion-dependent maturation and a proper dystrophin expression. The obtained cardiomyocytes showed remarkable sarcomeric organization of cardiac troponin T and α-actinin, expressed cardiac-specific markers, and displayed electrically induced calcium transients lasting less than 1 second. We demonstrated that the human artificial chromosome carrying the whole dystrophin genomic sequence is stably maintained throughout the cardiac differentiation process and that multiple promoters of the dystrophin gene are properly activated, driving expression of different isoforms. These dystrophic cardiomyocytes can be a valuable source for in vitro modeling of DMD-associated cardiac disease. Furthermore, the derivation of genetically corrected, patient-specific cardiomyocytes represents a step toward the development of innovative cell and gene therapy approaches for DMD.

  3. Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

    Salick, Max R.

    The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

  4. Protective effects of novel single compound, Hirsutine on hypoxic neonatal rat cardiomyocytes.

    Wu, Li Xin; Gu, Xian Feng; Zhu, Yi Chun; Zhu, Yi Zhun

    2011-01-10

    Uncaria rhynchophylla is a traditional Chinese herb that has been applied in China for treatment of ailments of the cardiovascular system, but little is known about its active constituents and effect in cardiomyocytes. In present study, we investigated the cardioprotective effect of 0.1μΜ, 1μΜ and 10μΜ Hirsutine isolated from the methanolic extracts of Uncaria rhynchophylla by high performance liquid chromatography (HPLC) on neonatal rat cardiomyocytes treated with hypoxia to determine the mechanism underlying the protective effect with regard to cardiac anti-oxidant enzymes and apoptosis genes. Hirsutine significantly increased the viability of cardiomyocytes injured by hypoxia. Gene expression levels of proapoptotic genes (Bax, Fas and caspase-3) were significantly downregulated compared with the hypoxic control group (P<0.05), whereas the expression level of Bcl-2 was upregulated following Hirsutine treatment (P<0.05). Correspondingly, Hirsutine treatment increased Bcl-2 protein level and decreased Bax protein level. Assay investigating cardiac anti-oxidant enzymes provided further evidence for the protective effect of Hirsutine, as indicated by the induction of the anti-oxidant enzymes superoxide dismutase. The results of present study suggest that the mechanism of action of Hirsutine in hypoxic neonatal rat cardiomyocytes may be related to its anti-oxidant and anti-apoptotic properties. This may open an avenue for developing novel candidate compounds with cardioprotectiveeffect from unique Chinese plant. Copyright © 2010 Elsevier B.V. All rights reserved.

  5. The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease

    Yalin Liao

    2016-08-01

    Full Text Available RNA functions through the dynamic formation of complexes with RNA-binding proteins (RBPs in all clades of life. We determined the RBP repertoire of beating cardiomyocytic HL-1 cells by jointly employing two in vivo proteomic methods, mRNA interactome capture and RBDmap. Together, these yielded 1,148 RBPs, 391 of which are shared with all other available mammalian RBP repertoires, while 393 are thus far unique to cardiomyocytes. RBDmap further identified 568 regions of RNA contact within 368 RBPs. The cardiomyocyte mRNA interactome composition reflects their unique biology. Proteins with roles in cardiovascular physiology or disease, mitochondrial function, and intermediary metabolism are all highly represented. Notably, we identified 73 metabolic enzymes as RBPs. RNA-enzyme contacts frequently involve Rossmann fold domains with examples in evidence of both, mutual exclusivity of, or compatibility between RNA binding and enzymatic function. Our findings raise the prospect of previously hidden RNA-mediated regulatory interactions among cardiomyocyte gene expression, physiology, and metabolism.

  6. Mena associates with Rac1 and modulates connexin 43 remodeling in cardiomyocytes.

    Ram, Rashmi; Wescott, Andrew P; Varandas, Katherine; Dirksen, Robert T; Blaxall, Burns C

    2014-01-01

    Mena, a member of the Ena/VASP family of actin regulatory proteins, modulates microfilaments and interacts with cytoskeletal proteins associated with heart failure. Mena is localized at the intercalated disc (ICD) of adult cardiac myocytes, colocalizing with numerous cytoskeletal proteins. Mena's role in the maintainence of mechanical myocardial stability at the cardiomyocyte ICD remains unknown. We hypothesized that Mena may modulate signals from the sarcolemma to the actin cytoskeleton at the ICD to regulate the expression and localization of connexin 43 (Cx43). The small GTPase Rac1 plays a pivotal role in the regulation of actin cytoskeletal reorganization and mediating morphological and transcriptional changes in cardiomyocytes. We found that Mena is associated with active Rac1 in cardiomyocytes and that RNAi knockdown of Mena increased Rac1 activity significantly. Furthermore, Mena knockdown increased Cx43 expression and altered Cx43 localization and trafficking at the ICD, concomitant with faster intercellular communication, as assessed by dye transfer between cardiomyocyte pairs. In mice overexpressing constitutively active Rac1, left ventricular Mena expression was increased significantly, concomitant with lateral redistribution of Cx43. These results suggest that Mena is a critical regulator of the ICD and is required for normal localization of Cx43 in part via regulation of Rac1.

  7. MYR1-Dependent Effectors Are the Major Drivers of a Host Cell’s Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects

    Adit Naor

    2018-04-01

    Full Text Available The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5 and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma, we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT, RHΔmyr1, and RHΔasp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, “hidden” responses arising in RHΔmyr1- but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite’s ability to co-opt host cell functions.

  8. Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured over Different Spatial Scales.

    Huebsch, Nathaniel; Loskill, Peter; Mandegar, Mohammad A; Marks, Natalie C; Sheehan, Alice S; Ma, Zhen; Mathur, Anurag; Nguyen, Trieu N; Yoo, Jennie C; Judge, Luke M; Spencer, C Ian; Chukka, Anand C; Russell, Caitlin R; So, Po-Lin; Conklin, Bruce R; Healy, Kevin E

    2015-05-01

    Contractile motion is the simplest metric of cardiomyocyte health in vitro, but unbiased quantification is challenging. We describe a rapid automated method, requiring only standard video microscopy, to analyze the contractility of human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CM). New algorithms for generating and filtering motion vectors combined with a newly developed isogenic iPSC line harboring genetically encoded calcium indicator, GCaMP6f, allow simultaneous user-independent measurement and analysis of the coupling between calcium flux and contractility. The relative performance of these algorithms, in terms of improving signal to noise, was tested. Applying these algorithms allowed analysis of contractility in iPS-CM cultured over multiple spatial scales from single cells to three-dimensional constructs. This open source software was validated with analysis of isoproterenol response in these cells, and can be applied in future studies comparing the drug responsiveness of iPS-CM cultured in different microenvironments in the context of tissue engineering.

  9. Preparation of a recombinant adenoviral encoding human NIS gene and its specific expression in cardiomyocytes

    Wang Lihua; Zhang Miao; Guo Rui; Shi Shuo; Li Biao

    2012-01-01

    Objective: To construct a recombinant adenovirus vector containing the human NIS gene with the myosin light chain-2(MLC-2v) gene as the promoter and evaluate its specific expression and feasibility as a reporter gene in cardiomyocytes. Methods: MLC-2v promoter and NIS were subcloned into an adenovirus shuttle vector, and forwarded by homologous recombination in the bacteria BJ5183 containing AdEasy-1 plasmid. Positive recombinant adenovirus vector was selected, packaged and amplified in the HEK293 cells to obtain recombinant adenovirus Ad-MLC-NIS. Ad-cytomegalovirus (CMV)-NIS with cytomegalovirus as the promoter, Ad-MLC without NIS and Ad-NIS without promoter were constructed as the controls. Cardiomyocytes and non-cardiomyocytes were then infected by the adenovirus. The protein expression was tested by Western blot analysis. The function and features of NIS protein were evaluated by dynamic iodide uptake and NaClO 4 iodine uptake inhibition test in vitro. The viability and proliferation of cardiomyocytes after adenovirus transfection and radioiodine incubation were checked by trypan blue staining. Results: Recombinant NIS adenovirus was successfully constructed. Western blot analysis showed that the NIS protein was highly expressed in cardiomyocytes transfected with Ad-MLC-NIS, and all cells transfected with Ad-CMV-NIS. However, in non-cardiomyocytes transfected with Ad-MLC-NIS, little NIS protein was detected. Dynamic iodine uptake tests showed that the peaks of iodide uptake of the three different cell lines (H9C2, A549, U87 cell) transfected with Ad-MLC-NIS were 5844.0, 833.6 and 846.0 counts · min -1 , respectively. The iodide uptake function of H9C2 was inhibited by NaClO 4 . There was almost no change in cell viability and proliferation when the MOI was 100. Conclusions: Ad-MLC-NIS allows myocardial specific expression of an external gene, and the cardiomyocytes with NIS expression are capable of iodine uptake. Further research of NIS as a reporter gene in

  10. Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes.

    Pasquier, Jennifer; Gupta, Renuka; Rioult, Damien; Hoarau-Véchot, Jessica; Courjaret, Raphael; Machaca, Khaled; Al Suwaidi, Jassim; Stanley, Edouard G; Rafii, Shahin; Elliott, David A; Abi Khalil, Charbel; Rafii, Arash

    2017-06-01

    Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes. We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4 + ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. After 8 days in culture, 94% ± 6% of the NKX2-5GFP + cells were beating when hESCs embryonic bodies were plated on E4 + ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP + cardiomyocytes were close to the E4 + ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges. The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony. Copyright © 2017. Published by Elsevier Inc.

  11. Response to Therapy and Outcomes in Oropharyngeal Cancer Are Associated With Biomarkers Including Human Papillomavirus, Epidermal Growth Factor Receptor, Gender, and Smoking

    Kumar, Bhavna; Cordell, Kitrina G.; Lee, Julia S.; Prince, Mark E.; Tran, Huong H.; Wolf, Gregory T.; Urba, Susan G.; Worden, Francis P.; Chepeha, Douglas B.; Teknos, Theodoros N.; Eisbruch, Avraham; Tsien, Christina I.; Taylor, Jeremy; D'Silva, Nisha J.; Yang, Kun; Kurnit, David M.; Bradford, Carol R.

    2007-01-01

    Induction chemotherapy and concurrent chemoradiation for responders or immediate surgery for non-responders is an effective treatment strategy head and neck squamous cell carcinoma (HNSCC) of the larynx and oropharynx. Biomarkers that predict outcome would be valuable in selecting patients for therapy. In this study, the presence and titer of high risk human papilloma virus (HPV) and expression of epidermal growth factor receptor (EGFR) in pre-treatment biopsies, as well as smoking and gender were examined in oropharynx cancer patients enrolled in an organ sparing trial. HPV16 copy number was positively associated with response to therapy and with overall and disease specific survival, whereas EGFR expression, current or former smoking behavior, and female gender (in this cohort) were associated with poor response and poor survival in multivariate analysis. Smoking cessation and strategies to target EGFR may be useful adjuncts for therapy to improve outcome in the cases with the poorest biomarker profile

  12. [Rat cardiomyocyte remodeling after neonatal cryptosporidiosis. II. Elongation, excessive polyploidization and HIF-1alpha overexpression].

    Anatskaia, O V; Sidorenko, N V; Matveev, I V; Kropotov, A V; Vinogradov, A E

    2012-01-01

    Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization.

  13. Heme Oxygenase-1/Carbon Monoxide System and Embryonic Stem Cell Differentiation and Maturation into Cardiomyocytes

    Suliman, Hagir B.; Zobi, Fabio

    2016-01-01

    Abstract Aims: The differentiation of embryonic stem (ES) cells into energetically efficient cardiomyocytes contributes to functional cardiac repair and is envisioned to ameliorate progressive degenerative cardiac diseases. Advanced cell maturation strategies are therefore needed to create abundant mature cardiomyocytes. In this study, we tested whether the redox-sensitive heme oxygenase-1/carbon monoxide (HO-1/CO) system, operating through mitochondrial biogenesis, acts as a mechanism for ES cell differentiation and cardiomyocyte maturation. Results: Manipulation of HO-1/CO to enhance mitochondrial biogenesis demonstrates a direct pathway to ES cell differentiation and maturation into beating cardiomyocytes that express adult structural markers. Targeted HO-1/CO interventions up- and downregulate specific cardiogenic transcription factors, transcription factor Gata4, homeobox protein Nkx-2.5, heart- and neural crest derivatives-expressed protein 1, and MEF2C. HO-1/CO overexpression increases cardiac gene expression for myosin regulatory light chain 2, atrial isoform, MLC2v, ANP, MHC-β, and sarcomere α-actinin and the major mitochondrial fusion regulators, mitofusin 2 and MICOS complex subunit Mic60. This promotes structural mitochondrial network expansion and maturation, thereby supporting energy provision for beating embryoid bodies. These effects are prevented by silencing HO-1 and by mitochondrial reactive oxygen species scavenging, while disruption of mitochondrial biogenesis and mitochondrial DNA depletion by loss of mitochondrial transcription factor A compromise infrastructure. This leads to failure of cardiomyocyte differentiation and maturation and contractile dysfunction. Innovation: The capacity to augment cardiomyogenesis via a defined mitochondrial pathway has unique therapeutic potential for targeting ES cell maturation in cardiac disease. Conclusion: Our findings establish the HO-1/CO system and redox regulation of mitochondrial biogenesis as

  14. Overexpression of BAG3 Attenuates Hypoxia-Induced Cardiomyocyte Apoptosis by Inducing Autophagy.

    Zhang, Jiankai; He, Zhangyou; Xiao, Wenjian; Na, Qingqing; Wu, Tianxiu; Su, Kaixin; Cui, Xiaojun

    2016-01-01

    Hypoxia is a well-known factor in the promotion of apoptosis, which contributes to the development of numerous cardiac diseases, such as heart failure and myocardial infarction. Inhibiting apoptosis is an important therapeutic strategy for the treatment of related heart diseases caused by ischemia/hypoxic injury. Previous studies have demonstrated that BAG3 plays an important role in cardiomyocyte apoptosis and survival. However, the role of BAG3 in hypoxia-induced cardiomyocyte apoptosis remains to be clarified. Here, we demonstrate that BAG3 is induced by hypoxia stimuli in cultured cardiomyocytes. BAG3 expression level was measured in H9c2 cells treated with hypoxia for 48 h. Cell proliferation and apoptosis were tested using MTT assay and Annexin V FITC-PI staining assay, respectively. The mRNA or protein expression level of BAG3, LC3-I, LC3-II, Atg5, NF-x03BA;B p65 and phosphorylated NF-x03BA;B p65 were assessed by qRT-PCR and western blot assay, respectively. Resluts: Overexpression of BAG3 inhibited cell apoptosis and promoted proliferation in hypoxia-injured H9c2 cells. Furthermore, autophagy and NF-x03BA;B were activated by BAG3 overexpression, and the NF-x03BA;B inhibitor PDTC could inhibit the activation of autophagy induced by BAG3 overexpression. In addition, the autophagy inhibitor 3-MA partly impeded the inhibitory effect of BAG3 on hypoxia-induced cardiomyocyte apoptosis. these results suggested that overexpression of BAG3 promoted cell proliferation and inhibited apoptosis by activating autophagy though the NF-x03BA;B signaling pathway in hypoxia-injured cardiomyocytes. © 2016 The Author(s) Published by S. Karger AG, Basel.

  15. Overexpression of BAG3 Attenuates Hypoxia-Induced Cardiomyocyte Apoptosis by Inducing Autophagy

    Jiankai Zhang

    2016-07-01

    Full Text Available Background: Hypoxia is a well-known factor in the promotion of apoptosis, which contributes to the development of numerous cardiac diseases, such as heart failure and myocardial infarction. Inhibiting apoptosis is an important therapeutic strategy for the treatment of related heart diseases caused by ischemia/hypoxic injury. Previous studies have demonstrated that BAG3 plays an important role in cardiomyocyte apoptosis and survival. However, the role of BAG3 in hypoxia-induced cardiomyocyte apoptosis remains to be clarified. Here, we demonstrate that BAG3 is induced by hypoxia stimuli in cultured cardiomyocytes. Methods: BAG3 expression level was measured in H9c2 cells treated with hypoxia for 48 h. Cell proliferation and apoptosis were tested using MTT assay and Annexin V FITC-PI staining assay, respectively. The mRNA or protein expression level of BAG3, LC3-I, LC3-II, Atg5, NF-κB p65 and phosphorylated NF-κB p65 were assessed by qRT-PCR and western blot assay, respectively. Resluts: Overexpression of BAG3 inhibited cell apoptosis and promoted proliferation in hypoxia-injured H9c2 cells. Furthermore, autophagy and NF-κB were activated by BAG3 overexpression, and the NF-κB inhibitor PDTC could inhibit the activation of autophagy induced by BAG3 overexpression. In addition, the autophagy inhibitor 3-MA partly impeded the inhibitory effect of BAG3 on hypoxia-induced cardiomyocyte apoptosis. Conclusion: these results suggested that overexpression of BAG3 promoted cell proliferation and inhibited apoptosis by activating autophagy though the NF-κB signaling pathway in hypoxia-injured cardiomyocytes.

  16. Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade.

    Altomare, Claudia; Pianezzi, Enea; Cervio, Elisabetta; Bolis, Sara; Biemmi, Vanessa; Benzoni, Patrizia; Camici, Giovanni G; Moccetti, Tiziano; Barile, Lucio; Vassalli, Giuseppe

    2016-12-01

    Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na +  current (I Na ), nifedipine, a blocker of L-type Ca 2+  current (I CaL ), and E4031, a blocker of the rapid component of delayed rectifier K +  current (I Kr ). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K +  current (I Ks ). In hiPSC-derived cardiomyocytes of cardiac origin, I Na , I CaL , I Kr , and I Ks were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic

  17. Hypoxia changes the expression of the epidermal growth factor (EGF) system in human hearts and cultured cardiomyocytes

    Munk, Mathias; Memon, Ashfaque Ahmed; Goetze, Jens Peter

    2012-01-01

    by treatment with trastuzumab (20 nM). This resulted in inhibition of cardiomyocyte proliferation, but interestingly only in hypoxic cells. Co-treatment of HL-1 cells with HB-EGF (10 nM) but not with NRG-1 (5 ng/ml) rescued the cardiomyocytes from HER2 inhibition. HL-1 cardiomyocytes exposed to hypoxia...... revealed nuclear translocation of activated MAPK and the activity of this downstream signaling molecule was decreased by HER2 inhibition (20 nM trastuzumab), and re-established by HB-EGF (10 nM). CONCLUSIONS/SIGNIFICANCE: Hypoxia in the human heart alters the expression of the EGF system. Mimicking the HER...

  18. Cardiotoxic (Arrhythmogenic) Effects of 1,1-Difluoroethane Due to Electrolyte Imbalance and Cardiomyocyte Damage.

    Joshi, Kaushal; Barletta, Michael; Wurpel, John

    2017-06-01

    Inhalant abuse is the intentional inhalation of chemical vapors to attain euphoric effects. Many common household products are abused by inhalation and one is 1,1-difluoroethane (DFE), which is a halogenated hydrocarbon used in refrigeration, dust-off spray, and airbrush painting. Although many human DFE abuse cases have been studied, the etiology and mechanism of sudden death is still unknown. In this study, an animal model was used to simulate the human conditions of DFE inhalation abuse that results in sudden death.Current research targets mechanistic studies involving electrolyte changes and cardiomyocyte damage after DFE administration in vivo. To investigate these changes, Sprague Dawley rats (N = 6) were exposed to 30 seconds of 20 L/min of DFE in multiple doses. Isoflurane acted as a control. Two additional groups, epinephrine and epinephrine + DFE, were included to simulate the clinical condition of DFE abuse. Plasma sodium, potassium, calcium, and magnesium levels were measured, followed by lactate dehydrogenase, creatine kinase, and cardiac troponin I levels. In addition, oxidative stress markers were also evaluated in all animal groups. Electrolyte levels showed a significant rise in plasma potassium and magnesium levels for the treated groups. In addition, lactate dehydrogenase, creatine kinase, and cardiac troponin I levels in DFE and epinephrine + DFE administered rats were significantly elevated as compared with control. Some oxidative stress makers were also elevated significantly in treatment groups. Furthermore, histopathological analysis showed hyperemia/congestion in treated rats.These results support cardiotoxic effects indicating that DFE results in fatal arrhythmias, and the study can be important during clinical cases involving inhalant abuse.

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

    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.

  20. Usefulness of cardiotoxicity assessment using calcium transient in human induced pluripotent stem cell-derived cardiomyocytes.

    Watanabe, Hitoshi; Honda, Yayoi; Deguchi, Jiro; Yamada, Toru; Bando, Kiyoko

    2017-01-01

    Monitoring dramatic changes in intracellular calcium ion levels during cardiac contraction and relaxation, known as calcium transient, in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would be an attractive strategy for assessing compounds on cardiac contractility. In addition, as arrhythmogenic compounds are known to induce characteristic waveform changes in hiPSC-CMs, it is expected that calcium transient would allow evaluation of not only compound-induced effects on cardiac contractility, but also compound arrhythmogenic potential. Using a combination of calcium transient in hiPSC-CMs and a fast kinetic fluorescence imaging detection system, we examined in this study changes in calcium transient waveforms induced by a series of 17 compounds that include positive/negative inotropic agents as well as cardiac ion channel activators/inhibitors. We found that all positive inotropic compounds induced an increase in peak frequency and/or peak amplitude. The effects of a negative inotropic compound could clearly be detected in the presence of a β-adrenergic receptor agonist. Furthermore, most arrhythmogenic compounds raised the ratio of peak decay time to peak rise time (D/R ratio) in calcium transient waveforms. Compound concentrations at which these parameters exceeded cutoff values correlated well with systemic exposure levels at which arrhythmias were reported to be evoked. In conclusion, we believe that peak analysis of calcium transient and determination of D/R ratio are reliable methods for assessing compounds' cardiac contractility and arrhythmogenic potential, respectively. Using these approaches would allow selection of compounds with low cardiotoxic potential at the early stage of drug discovery.

  1. Propofol attenuates H2O2-induced oxidative stress and apoptosis via the mitochondria- and ER-medicated pathways in neonatal rat cardiomyocytes.

    Liu, Xue-Ru; Cao, Lu; Li, Tao; Chen, Lin-Lin; Yu, Yi-Yan; Huang, Wen-Jun; Liu, Li; Tan, Xiao-Qiu

    2017-05-01

    Previous studies have shown that propofol, an intravenous anesthetic commonly used in clinical practice, protects the myocardium from injury. Mitochondria- and endoplasmic reticulum (ER)-mediated oxidative stress and apoptosis are two important signaling pathways involved in myocardial injury and protection. The present study aimed to test the hypothesis that propofol could exert a cardio-protective effect via the above two pathways. Cultured neonatal rat cardiomyocytes were treated with culture medium (control group), H 2 O 2 at 500 μM (H 2 O 2 group), propofol at 50 μM (propofol group), and H 2 O 2 plus propofol (H 2 O 2  + propofol group), respectively. The oxidative stress, mitochondrial membrane potential (ΔΨm) and apoptosis of the cardiomyocytes were evaluated by a series of assays including ELISA, flow cytometry, immunofluorescence microscopy and Western blotting. Propofol significantly suppressed the H 2 O 2 -induced elevations in the activities of caspases 3, 8, 9 and 12, the ratio of Bax/Bcl-2, and cell apoptosis. Propofol also inhibited the H 2 O 2 -induced reactive oxygen species (ROS) generation, lactic dehydrogenase (LDH) release and mitochondrial transmembrane potential (ΔΨm) depolarization, and restored the H 2 O 2 -induced reductions of glutathione (GSH) and superoxide dismutase (SOD). In addition, propofol decreased the expressions of glucose-regulated protein 78 kDa (Grp78) and inositol-requiring enzyme 1α (IRE1α), two important signaling molecules in the ER-mediated apoptosis pathway. Propofol protects cardiomyocytes from H 2 O 2 -induced injury by inhibiting the mitochondria- and ER-mediated apoptosis signaling pathways.

  2. MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.

    Naor, Adit; Panas, Michael W; Marino, Nicole; Coffey, Michael J; Tonkin, Christopher J; Boothroyd, John C

    2018-04-03

    The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma , we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔ myr1 , and RHΔ asp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔ myr1 - but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions. IMPORTANCE Toxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1

  3. Functional and Pharmacological Analysis of Cardiomyocytes Differentiated from Human Peripheral Blood Mononuclear-Derived Pluripotent Stem Cells

    Michael Riedel

    2014-07-01

    Full Text Available Advances in induced pluripotent stem cell (iPSC technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of PBMC-derived iPSC CM are generally similar to those of iPSC CM derived from other somatic cells, using patch-clamp, calcium transient, and multielectrode array (MEA analyses. Distinct iPSC lines derived from a single patient display similar electrophysiological features and pharmacological responses. Finally, we demonstrate that human iPSC CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for an in-vitro-tachypacing model system for the study of human tachyarrhythmias.

  4. Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells.

    Wang, Xiaomei; Su, Jin; Sherman, Alexandra; Rogers, Geoffrey L; Liao, Gongxian; Hoffman, Brad E; Leong, Kam W; Terhorst, Cox; Daniell, Henry; Herzog, Roland W

    2015-04-09

    Coagulation factor replacement therapy for the X-linked bleeding disorder hemophilia is severely complicated by antibody ("inhibitor") formation. We previously found that oral delivery to hemophilic mice of cholera toxin B subunit-coagulation factor fusion proteins expressed in chloroplasts of transgenic plants suppressed inhibitor formation directed against factors VIII and IX and anaphylaxis against factor IX (FIX). This observation and the relatively high concentration of antigen in the chloroplasts prompted us to evaluate the underlying tolerance mechanisms. The combination of oral delivery of bioencapsulated FIX and intravenous replacement therapy induced a complex, interleukin-10 (IL-10)-dependent, antigen-specific systemic immune suppression of pathogenic antibody formation (immunoglobulin [Ig] 1/inhibitors, IgE) in hemophilia B mice. Tolerance induction was also successful in preimmune mice but required prolonged oral delivery once replacement therapy was resumed. Orally delivered antigen, initially targeted to epithelial cells, was taken up by dendritic cells throughout the small intestine and additionally by F4/80(+) cells in the duodenum. Consistent with the immunomodulatory responses, frequencies of tolerogenic CD103(+) and plasmacytoid dendritic cells were increased. Ultimately, latency-associated peptide expressing CD4(+) regulatory T cells (CD4(+)CD25(-)LAP(+) cells with upregulated IL-10 and transforming growth factor-β (TGF-β) expression) as well as conventional CD4(+)CD25(+) regulatory T cells systemically suppressed anti-FIX responses. © 2015 by The American Society of Hematology.

  5. Effect of bionic electrical stimulation on the differentiation of embryonic stem cells into cardiomyocytes in the presence myocardial cells in vitro

    Li-na ZHENG

    2011-08-01

    Full Text Available Objective To investigate the effects of electrical stimulation on the differentiation of embryonic stem cells(ESCs into cardiomyocytes in the presence of myocardial cells in vitro.Methods ESCs and neonate rat cardiomyocytes were isolated and cultured.These cells of primary culture were divided into 5 groups according to whether or not electric stimulation was given and the presence of cardiomyocytes: control group,stimulation group,cardiomyocytes group,stimulation+ cardiomyocyte conditioned medium group,and stimulation+cardiomyocytes group.Expression of troponin T(cTnT in the differentiated cells from ESCs was examined by immunofluoresence on the 5th,7th and 14th day.Results In the group co-cultured with myocardial cell and electrical stimulation,the differentiating ratio of cardiomyocytes derived from ESCs and expressing cTnT was 40.00%±2.39%,and it was higher than that in control group(2.00%±1.60%,stimulation group(3.00%±2.00%,cardiomyocytes group(28.70%±4.06%,stimulation+cardiomyocyte conditioned medium group(17.10%±2.23%,P < 0.05.Conclusion Bionic electric stimulation promotes the differentiation of ESCs into cardiomyocyte in a microenvironment consisting of myocardial cells.

  6. Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.

    Hiroko Kita-Matsuo

    Full Text Available Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

  7. Impact of miR-208 and its Target Gene Nemo-Like Kinase on the Protective Effect of Ginsenoside Rb1 in Hypoxia/Ischemia Injuried Cardiomyocytes

    Xu Yan

    2016-09-01

    Full Text Available Background/Aims: Ginsenoside Rb1 (GS-Rb1 is one of the most important active pharmacological extracts of the Traditional Chinese Medicine ginseng, with extensive evidence of its cardioprotective properties. Mir-208 has been shown to act as a biomarker of acute myocardial infarction in vivo studies including man. However the impact of miR-208 on the protective effect of GS-Rb1 in hypoxia/ischemia injured cardiomyocytes remains unclear. The current study aims to investigate the target gene of miR-208 and the impact on the protective effect of GS-Rb1 in hypoxia/ischemia (H/I injuried cardiomyocytes. Materials and Methods: Primary cultures of neonatal rat cardiomyocytes (NRCMs was subjected to the H/I conditions with or without GS-Rb1. Cell viability was calculated by MTT assay and confirmed by flow cytometry analysis. Mir-208 was then detected by qRT-PCR. Luciferase reporter assay was carried out to detect the target gene of Mir-208. Then the NRCMs were transfected with miR-208 mimics and inhibitors to evaluate the impact on cardioprotective properties of Rb1. Results: The miR-208 expression level was clearly upregulated in the H/I treated NRCMs accompanied by the percentage of the apoptotic cells which could be reversed by GS-Rb1 pretreatment. The nemo-like kinase (NLK mRNA and protein expression levels were decreased in H/I group measured by RT-PCR and western blotting. Luciferase activity assay was then carried out to identify that NLK may be a direct target of mir-208. MTT assay showed that miR-208 inhibitor slightly decreased the protective effect of Rb1 on the H/I impaired NRCMs. However, results showed no statistical difference. Conclusions: These findings proved that NLK was a direct target of mir-208 and miR-208 act indirectly during Rb1 protecting H/I impaired NRCMs and further researches were needed to explore the relationship that microRNAs and other signal pathways in the protective effect of GS-Rb1 on the hypoxia/ischemia injuries in

  8. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

    Jordan S. Leyton-Mange

    2014-02-01

    Full Text Available In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity.

  9. In EXOG-depleted cardiomyocytes cell death is marked by a decreased mitochondrial reserve capacity of the electron transport chain

    Tigchelaar, Wardit; De Jong, Anne Margreet; van Gilst, Wiek H.; De Boer, Rudolf A.; Sillje, Herman H. W.

    Depletion ofmitochondrial endo/exonuclease G-like (EXOG) in cultured neonatal cardiomyocytes stimulates mitochondrial oxygen consumption rate (OCR) and induces hypertrophy via reactive oxygen species (ROS). Here, we show that neurohormonal stress triggers cell death in endo/exonuclease

  10. A Hybrid Model for Safety Pharmacology on an Automated Patch Clamp Platform: Using Dynamic Clamp to Join iPSC-Derived Cardiomyocytes and Simulations of Ik1 Ion Channels in Real-Time

    Birgit Goversen

    2018-01-01

    Full Text Available An important aspect of the Comprehensive In Vitro Proarrhythmia Assay (CiPA proposal is the use of human stem cell-derived cardiomyocytes and the confirmation of their predictive power in drug safety assays. The benefits of this cell source are clear; drugs can be tested in vitro on human cardiomyocytes, with patient-specific genotypes if needed, and differentiation efficiencies are generally excellent, resulting in a virtually limitless supply of cardiomyocytes. There are, however, several challenges that will have to be surmounted before successful establishment of hSC-CMs as an all-round predictive model for drug safety assays. An important factor is the relative electrophysiological immaturity of hSC-CMs, which limits arrhythmic responses to unsafe drugs that are pro-arrhythmic in humans. Potentially, immaturity may be improved functionally by creation of hybrid models, in which the dynamic clamp technique joins simulations of lacking cardiac ion channels (e.g., IK1 with hSC-CMs in real-time during patch clamp experiments. This approach has been used successfully in manual patch clamp experiments, but throughput is low. In this study, we combined dynamic clamp with automated patch clamp of iPSC-CMs in current clamp mode, and demonstrate that IK1 conductance can be added to iPSC-CMs on an automated patch clamp platform, resulting in an improved electrophysiological maturity.

  11. TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation.

    Nadine Kirschmer

    Full Text Available The Transient Receptor Potential Channel Subunit 4 (TRPC4 has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II stimulation. NRCs infected with TRPC4β (Ad-C4β also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not

  12. Chymase mediates injury and mitochondrial damage in cardiomyocytes during acute ischemia/reperfusion in the dog.

    Zheng, Junying; Wei, Chih-Chang; Hase, Naoki; Shi, Ke; Killingsworth, Cheryl R; Litovsky, Silvio H; Powell, Pamela C; Kobayashi, Tsunefumi; Ferrario, Carlos M; Rab, Andras; Aban, Inmaculada; Collawn, James F; Dell'Italia, Louis J

    2014-01-01

    Cardiac ischemia and reperfusion (I/R) injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI). 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1) and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore-unrecognized chymase

  13. Chymase mediates injury and mitochondrial damage in cardiomyocytes during acute ischemia/reperfusion in the dog.

    Junying Zheng

    Full Text Available Cardiac ischemia and reperfusion (I/R injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI. 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1 and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore

  14. Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

    Feng, Shan-Li [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Sun, Ming-Rui [Department of Pharmacology, Qiqihaer Medical College, Qiqihaer 160001 (China); Li, Ting-Ting; Yin, Xin [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Xu, Chang-Qing [Department of Pathophysiology, Harbin Medical University, Harbin 150086 (China); Sun, Yi-Hua, E-mail: syh200415@126.com [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China)

    2011-03-11

    Research highlights: {yields} Calcium-sensing receptor (CaR) activation stimulates TRP channels. {yields} CaR promoted transient receptor potential C3 (TRPC3) expression. {yields} Adult rat ventricular myocytes display capacitative calcium entry (CCE), which was operated by TRPCs. {yields} TRPC channels activation induced by CaR activator sustained the increased [Ca{sup 2+}]{sub i} to evoke cardiomyocytes apoptosis. -- Abstract: Transient receptor potential (TRP) channels are expressed in cardiomyocytes, which gate a type of influx of extracellular calcium, the capacitative calcium entry. TRP channels play a role in mediating Ca{sup 2+} overload in the heart. Calcium-sensing receptors (CaR) are also expressed in rat cardiac tissue and promote the apoptosis of cardiomyocytes by Ca{sup 2+} overload. However, data about the link between CaR and TRP channels in rat heart are few. In this study, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of the TRP canonical proteins TRPC1 and TRPC3 in adult and neonatal rat cardiomyocytes. Laser scan confocal microscopy was used to detect intracellular [Ca{sup 2+}]{sub i} levels in isolated adult rat ventricular myocytes. The results showed that, in adult rat cardiomyocytes, the depletion of Ca{sup 2+} stores in the endoplasmic/sarcoplasmic reticulum (ER/SR) by thapsigargin induced a transient increase in [Ca{sup 2+}]{sub i} in the absence of [Ca{sup 2+}]{sub o} and the subsequent restoration of [Ca{sup 2+}]{sub o} sustained the increased [Ca{sup 2+}]{sub i} for a few minutes, whereas, the persisting elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of the TRPC inhibitor SKF96365. The stimulation of CaR by its activator gadolinium chloride (GdCl{sub 3}) or spermine also resulted in the same effect and the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of [Ca{sup 2+}]{sub o}. In adult and neonatal rat cardiomyocytes, GdCl{sub 3

  15. Deciphering the mechanisms involved in Portulaca oleracea (C4) response to drought: metabolic changes including crassulacean acid-like metabolism induction and reversal upon re-watering.

    D'Andrea, Rodrigo Matías; Andreo, Carlos Santiago; Lara, María Valeria

    2014-11-01

    Portulaca oleracea is a C(4) plant; however, under drought it can change its carbon fixation metabolism into a crassulacean acid metabolism (CAM)-like one. While the C(3) -CAM shift is well known, the C(4) -CAM transition has only been described in Portulaca. Here, a CAM-like metabolism was induced in P. oleracea by drought and then reversed by re-watering. Physiological and biochemical approaches were undertaken to evaluate the drought and recovery responses. In CAM-like plants, chlorophyll fluorescence parameters were transitory affected and non-radiative energy dissipation mechanisms were induced. Induction of flavonoids, betalains and antioxidant machinery may be involved in photosynthetic machinery protection. Metabolic analysis highlights a clear metabolic shift, when a CAM-like metabolism is induced and then reversed. Increases in nitrogenous compounds like free amino acids and urea, and of pinitol could contribute to withstand drought. Reciprocal variations in arginase and urease in drought-stressed and in re-watered plants suggest urea synthesis is strictly regulated. Recovery of C(4) metabolism was accounted by CO(2) assimilation pattern and malate levels. Increases in glycerol and in polyamines would be of importance of re-watered plants. Collectively, in P. oleracea multiple strategies, from induction of several metabolites to the transitory development of a CAM-like metabolism, participate to enhance its adaptation to drought. © 2014 Scandinavian Plant Physiology Society.

  16. Thymosin beta 4 protects cardiomyocytes from oxidative stress by targeting anti-oxidative enzymes and anti-apoptotic genes.

    Chuanyu Wei

    Full Text Available Thymosin beta-4 (Tβ4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H(2O(2 induced cardiac damage.Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H(2O(2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H(2O(2-induced cardiac damage was evaluated.Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl(2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4.This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the myocardium under oxidative stress.

  17. Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

    Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo

    2006-04-01

    Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

  18. Metabolic profiling using HPLC allows classification of drugs according to their mechanisms of action in HL-1 cardiomyocytes

    Strigun, Alexander; Wahrheit, Judith; Beckers, Simone; Heinzle, Elmar; Noor, Fozia

    2011-01-01

    Along with hepatotoxicity, cardiotoxic side effects remain one of the major reasons for drug withdrawals and boxed warnings. Prediction methods for cardiotoxicity are insufficient. High content screening comprising of not only electrophysiological characterization but also cellular molecular alterations are expected to improve the cardiotoxicity prediction potential. Metabolomic approaches recently have become an important focus of research in pharmacological testing and prediction. In this study, the culture medium supernatants from HL-1 cardiomyocytes after exposure to drugs from different classes (analgesics, antimetabolites, anthracyclines, antihistamines, channel blockers) were analyzed to determine specific metabolic footprints in response to the tested drugs. Since most drugs influence energy metabolism in cardiac cells, the metabolite 'sub-profile' consisting of glucose, lactate, pyruvate and amino acids was considered. These metabolites were quantified using HPLC in samples after exposure of cells to test compounds of the respective drug groups. The studied drug concentrations were selected from concentration response curves for each drug. The metabolite profiles were randomly split into training/validation and test set; and then analysed using multivariate statistics (principal component analysis and discriminant analysis). Discriminant analysis resulted in clustering of drugs according to their modes of action. After cross validation and cross model validation, the underlying training data were able to predict 50%-80% of conditions to the correct classification group. We show that HPLC based characterisation of known cell culture medium components is sufficient to predict a drug's potential classification according to its mode of action.

  19. Insulin protects apoptotic cardiomyocytes from hypoxia/reoxygenation injury through the sphingosine kinase/sphingosine 1-phosphate axis.

    Huan Yu

    Full Text Available OBJECTIVE: Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the mechanisms underlying this effect are still unknown. In this study, the ability of insulin to protect apoptotic cardiomyocytes from hypoxia/reoxygenation injury using the sphingosine kinase/sphingosine 1-phosphate axis was investigated. METHODS AND RESULTS: Rat cardiomyocytes were isolated and subjected to hypoxia and reoxygenation. [γ-32P] ATP was used to assess sphingosine kinase activity. Insulin was found to increase sphingosine kinase activity. Immunocytochemistry and Western blot analysis showed changes in the subcellular location of sphingosine kinase 1 from cytosol to the membrane in cardiomyocytes. Insulin caused cardiomyocytes to accumulate of S1P in a dose-dependent manner. FRET efficiency showed that insulin also transactivates the S1P1 receptor. TUNEL staining showed that administration of insulin during reoxygenation could to reduce the rate of reoxygenation-induced apoptosis, which is a requirement for SphK 1 activity. It also reduced the rate of activation of the S1P receptor and inhibited hypoxia/reoxygenation-induced cell death in cardiomyocytes. CONCLUSION: The sphingosine kinase 1/sphingosine 1-phosphate/S1P receptor axis is one pathway through which insulin protects rat cardiomyocytes from apoptosis induced by hypoxia/reoxygenation injury.

  20. Use of computer aids including expert systems to enhance diagnosis of NPP safety status and operator response. VDU displays in accidents - Interact

    Humble, P.; Welbourne, D.

    1998-01-01

    This report describes NNC development of a demonstration concept called Interact of Visual Display Unit (VDU) displays, integrating on-screen control of plant actions. Most plant vendors now propose on-screen control and it is being included on some plants. The integration of Station Operating Instructions (SOI) into VDU presentation of plants is being developed rapidly. With on-screen control, SOIs can be displayed with control targets able to initiate plant control, directly as called for in the SOIs. Interact displays information and control options, using a cursor to simulate on-screen display and plant control. The displays show a method which integrates soft control and SOI information into a single unified presentation. They simulate the SOI for an accident, on-screen, with simulated inserted plant values

  1. Audiovisual synchrony enhances BOLD responses in a brain network including multisensory STS while also enhancing target-detection performance for both modalities

    Marchant, Jennifer L; Ruff, Christian C; Driver, Jon

    2012-01-01

    The brain seeks to combine related inputs from different senses (e.g., hearing and vision), via multisensory integration. Temporal information can indicate whether stimuli in different senses are related or not. A recent human fMRI study (Noesselt et al. [2007]: J Neurosci 27:11431–11441) used auditory and visual trains of beeps and flashes with erratic timing, manipulating whether auditory and visual trains were synchronous or unrelated in temporal pattern. A region of superior temporal sulcus (STS) showed higher BOLD signal for the synchronous condition. But this could not be related to performance, and it remained unclear if the erratic, unpredictable nature of the stimulus trains was important. Here we compared synchronous audiovisual trains to asynchronous trains, while using a behavioral task requiring detection of higher-intensity target events in either modality. We further varied whether the stimulus trains had predictable temporal pattern or not. Synchrony (versus lag) between auditory and visual trains enhanced behavioral sensitivity (d') to intensity targets in either modality, regardless of predictable versus unpredictable patterning. The analogous contrast in fMRI revealed BOLD increases in several brain areas, including the left STS region reported by Noesselt et al. [2007: J Neurosci 27:11431–11441]. The synchrony effect on BOLD here correlated with the subject-by-subject impact on performance. Predictability of temporal pattern did not affect target detection performance or STS activity, but did lead to an interaction with audiovisual synchrony for BOLD in inferior parietal cortex. PMID:21953980

  2. Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

    Ivana Acimovic

    2014-01-01

    Full Text Available Human pluripotent stem cells (hPSCs, namely, embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs, with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytes in vitro as well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs.

  3. Atomic force microscopy observation of lipopolysaccharide-induced cardiomyocyte cytoskeleton reorganization.

    Wang, Liqun; Chen, Tangting; Zhou, Xiang; Huang, Qiaobing; Jin, Chunhua

    2013-08-01

    We applied atomic force microscopy (AFM) to observe lipopolysaccharide (LPS)-induced intracellular cytoskeleton reorganization in primary cardiomyocytes from neonatal mouse. The nonionic detergent Triton X-100 was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized by AFM. Using three-dimensional technique of AFM, we were able to quantify the changes of cytoskeleton by the "density" and total "volume" of the cytoskeleton fibers. Compared to the control group, the density of cytoskeleton was remarkably decreased and the volume of cytoskeleton was significantly increased after LPS treatment, which suggests that LPS may induce the cytoskeleton reorganization and change the cardiomyocyte morphology. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. ISL1 protein transduction promotes cardiomyocyte differentiation from human embryonic stem cells.

    Hananeh Fonoudi

    Full Text Available BACKGROUND: Human embryonic stem cells (hESCs have the potential to provide an unlimited source of cardiomyocytes, which are invaluable resources for drug or toxicology screening, medical research, and cell therapy. Currently a number of obstacles exist such as the insufficient efficiency of differentiation protocols, which should be overcome before hESC-derived cardiomyocytes can be used for clinical applications. Although the differentiation efficiency can be improved by the genetic manipulation of hESCs to over-express cardiac-specific transcription factors, these differentiated cells are not safe enough to be applied in cell therapy. Protein transduction has been demonstrated as an alternative approach for increasing the efficiency of hESCs differentiation toward cardiomyocytes. METHODS: We present an efficient protocol for the differentiation of hESCs in suspension by direct introduction of a LIM homeodomain transcription factor, Islet1 (ISL1 recombinant protein into the cells. RESULTS: We found that the highest beating clusters were derived by continuous treatment of hESCs with 40 µg/ml recombinant ISL1 protein during days 1-8 after the initiation of differentiation. The treatment resulted in up to a 3-fold increase in the number of beating areas. In addition, the number of cells that expressed cardiac specific markers (cTnT, CONNEXIN 43, ACTININ, and GATA4 doubled. This protocol was also reproducible for another hESC line. CONCLUSIONS: This study has presented a new, efficient, and reproducible procedure for cardiomyocytes differentiation. Our results will pave the way for scaled up and controlled differentiation of hESCs to be used for biomedical applications in a bioreactor culture system.

  5. Pharmacological activation of mitochondrial BKCa channels protects isolated cardiomyocytes against simulated reperfusion-induced injury

    Borchert, Gudrun H.; Hlaváčková, Markéta; Kolář, František

    2013-01-01

    Roč. 238, č. 2 (2013), s. 233-241 ISSN 1535-3702 R&D Projects: GA AV ČR(CZ) IAA500110804; GA ČR(CZ) GAP303/12/1162 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : potassium channels * cardiomyocytes * mitochondria * ischemia/reperfusion * cytoprotection * reactive oxygen species Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 2.226, year: 2013

  6. Ca2+-regulatory proteins in cardiomyocytes from the right ventricle in children with congenital heart disease

    Wu Yihe

    2012-04-01

    Full Text Available Abstract Background Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease (CHD which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Methods Right ventricle tissues were collected from children with tetralogy of Fallot [n = 25, hypoxia and hypertrophy group (HH group], pulmonary stenosis [n = 25, hypertrophy group (H group], or small isolated ventricular septal defect [n = 25, control group (C group] during open-heart surgery. Paraffin sections of tissues were stained with 3,3′-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a, ryanodine receptor (RyR2, sodiumcalcium exchanger (NCX, sarcolipin (SLN and phospholamban (PLN] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase (PP1 were evaluated using western blot. Results Mild cardiomyocyte hypertrophy of the right ventricle in H and HH groups was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P16-phosphorylated PLN was down-regulated (PP Conclusions The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser16 dephosphorylation. Increased PP1 could result in the decreased PLN-Ser16 and inhibition of PP1 is a potential therapeutic target for heart dysfunction in pediatrics.

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

    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...

  8. High Fibroblast Growth Factor 23 concentrations in experimental renal failure impair calcium handling in cardiomyocytes.

    Verkaik, Melissa; Oranje, Maarten; Abdurrachim, Desiree; Goebel, Max; Gam, Zeineb; Prompers, Jeanine J; Helmes, Michiel; Ter Wee, Pieter M; van der Velden, Jolanda; Kuster, Diederik W; Vervloet, Marc G; Eringa, Etto C

    2018-04-01

    The overwhelming majority of patients with chronic kidney disease (CKD) die prematurely before reaching end-stage renal disease, mainly due to cardiovascular causes, of which heart failure is the predominant clinical presentation. We hypothesized that CKD-induced increases of plasma FGF23 impair cardiac diastolic and systolic function. To test this, mice were subjected to 5/6 nephrectomy (5/6Nx) or were injected with FGF23 for seven consecutive days. Six weeks after surgery, plasma FGF23 was higher in 5/6Nx mice compared to sham mice (720 ± 31 vs. 256 ± 3 pg/mL, respectively, P = 0.034). In cardiomyocytes isolated from both 5/6Nx and FGF23 injected animals the rise of cytosolic calcium during systole was slowed (-13% and -19%, respectively) as was the decay of cytosolic calcium during diastole (-15% and -21%, respectively) compared to controls. Furthermore, both groups had similarly decreased peak cytosolic calcium content during systole. Despite lower cytosolic calcium contents in CKD or FGF23 pretreated animals, no changes were observed in contractile parameters of cardiomyocytes between the groups. Expression of calcium handling proteins and cardiac troponin I phosphorylation were similar between groups. Blood pressure, the heart weight:tibia length ratio, α-MHC/β-MHC ratio and ANF mRNA expression, and systolic and diastolic function as measured by MRI did not differ between groups. In conclusion, the rapid, CKD-induced rise in plasma FGF23 and the similar decrease in cardiomyocyte calcium transients in modeled kidney disease and following 1-week treatment with FGF23 indicate that FGF23 partly mediates cardiomyocyte dysfunction in CKD. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  9. Electrospun Gelatin–Chondroitin Sulfate Scaffolds Loaded with Platelet Lysate Promote Immature Cardiomyocyte Proliferation

    Francesca Saporito

    2018-02-01

    Full Text Available The aim of the present work was the development of heart patches based on gelatin (G and chondroitin sulfate (CS to be used as implants to improve heart recovery after corrective surgery for critical congenital heart defects (CHD. Patches were prepared by means of electrospinning to obtain nanofibrous scaffolds and they were loaded with platelet lysate (PL as a source of growth factors to further enhance the repair process. Scaffolds were characterized for morphology and mechanical properties and for the capability to support in vitro adhesion and proliferation of dermal fibroblasts in order to assess the system’s general biocompatibility. Adhesion and proliferation of endothelial cells and cardiac cells (cardiomyocytes and cardiac fibroblasts from rat fetuses onto PL-loaded patches was evaluated. Patches presented good elasticity and high stiffness suitable for in vivo adaptation to heart contraction. CS improved adhesion and proliferation of dermal fibroblasts, as proof of their biocompatibility. Moreover, they enhanced the adhesion and proliferation of endothelial cells, a crucial mediator of cardiac repair. Cell adhesion and proliferation could be related to elastic properties, which could favor cell motility. The presence of platelet lysate and CS was crucial for the adhesion and proliferation of cardiac cells and, in particular, of cardiomyocytes: G/CS scaffold embedded with PL appeared to selectively promote proliferation in cardiomyocytes but not cardiac fibroblasts. In conclusion, G/CS scaffold seems to be a promising system to assist myocardial-repair processes in young patient, preserving cardiomyocyte viability and preventing cardiac fibroblast proliferation, likely reducing subsequent uncontrolled collagen deposition by fibroblasts following repair.

  10. Induced pluripotent stem cell-derived cardiomyocytes for cardiovascular disease modeling and drug screening

    Sharma, Arun; Wu, Joseph C; Wu, Sean M

    2013-01-01

    Human induced pluripotent stem cells (hiPSCs) have emerged as a novel tool for drug discovery and therapy in cardiovascular medicine. hiPSCs are functionally similar to human embryonic stem cells (hESCs) and can be derived autologously without the ethical challenges associated with hESCs. Given the limited regenerative capacity of the human heart following myocardial injury, cardiomyocytes derived from hiPSCs (hiPSC-CMs) have garnered significant attention from basic and translational scienti...

  11. Inhibition of ErbB2 by receptor tyrosine kinase inhibitors causes myofibrillar structural damage without cell death in adult rat cardiomyocytes

    Pentassuglia, Laura; Graf, Michael; Lane, Heidi; Kuramochi, Yukio; Cote, Gregory; Timolati, Francesco; Sawyer, Douglas B.; Zuppinger, Christian; Suter, Thomas M.

    2009-01-01

    Inhibition of ErbB2 (HER2) with monoclonal antibodies, an effective therapy in some forms of breast cancer, is associated with cardiotoxicity, the pathophysiology of which is poorly understood. Recent data suggest, that dual inhibition of ErbB1 (EGFR) and ErbB2 signaling is more efficient in cancer therapy, however, cardiac safety of this therapeutic approach is unknown. We therefore tested an ErbB1-(CGP059326) and an ErbB1/ErbB2-(PKI166) tyrosine kinase inhibitor in an in-vitro system of adult rat ventricular cardiomyocytes and assessed their effects on 1. cell viability, 2. myofibrillar structure, 3. contractile function, and 4. MAPK- and Akt-signaling alone or in combination with Doxorubicin. Neither CGP nor PKI induced cardiomyocyte necrosis or apoptosis. PKI but not CGP caused myofibrillar structural damage that was additive to that induced by Doxorubicin at clinically relevant doses. These changes were associated with an inhibition of excitation-contraction coupling. PKI but not CGP decreased p-Erk1/2, suggesting a role for this MAP-kinase signaling pathway in the maintenance of myofibrils. These data indicate that the ErbB2 signaling pathway is critical for the maintenance of myofibrillar structure and function. Clinical studies using ErbB2-targeted inhibitors for the treatment of cancer should be designed to include careful monitoring for cardiac dysfunction.

  12. Sympathetic neurons modulate the beat rate of pluripotent cell-derived cardiomyocytes in vitro.

    Takeuchi, Akimasa; Shimba, Kenta; Mori, Masahide; Takayama, Yuzo; Moriguchi, Hiroyuki; Kotani, Kiyoshi; Lee, Jong-Kook; Noshiro, Makoto; Jimbo, Yasuhiko

    2012-12-01

    Although stem cell-derived cardiomyocytes have great potential for the therapy of heart failure, it is unclear whether their function after grafting can be controlled by the host sympathetic nervous system, a component of the autonomic nervous system (ANS). Here we demonstrate the formation of functional connections between rat sympathetic superior cervical ganglion (SCG) neurons and pluripotent (P19.CL6) cell-derived cardiomyocytes (P19CMs) in compartmentalized co-culture, achieved using photolithographic microfabrication techniques. Formation of synapses between sympathetic neurons and P19CMs was confirmed by immunostaining with antibodies against β-3 tubulin, synapsin I and cardiac troponin-I. Changes in the beat rate of P19CMs were triggered after electrical stimulation of the co-cultured SCG neurons, and were affected by the pulse frequency of the electrical stimulation. Such changes in the beat rate were prevented when propranolol, a β-adrenoreceptor antagonist, was added to the culture medium. These results suggest that the beat rate of differentiated cardiomyocytes can be modulated by electrical stimulation of connected sympathetic neurons.

  13. Succinate modulates Ca(2+) transient and cardiomyocyte viability through PKA-dependent pathway.

    Aguiar, Carla J; Andrade, Vanessa L; Gomes, Enéas R M; Alves, Márcia N M; Ladeira, Marina S; Pinheiro, Ana Cristina N; Gomes, Dawidson A; Almeida, Alvair P; Goes, Alfredo M; Resende, Rodrigo R; Guatimosim, Silvia; Leite, M Fatima

    2010-01-01

    GPR91 is an orphan G-protein-coupled receptor (GPCR) that has been characterized as a receptor for succinate, a citric acid cycle intermediate, in several tissues. In the heart, the role of succinate is unknown. We now report that rat ventricular cardiomyocytes express GPR91. We found that succinate, through GPR91, increases the amplitude and the rate of decline of global Ca(2+) transient, by increasing the phosphorylation levels of ryanodine receptor and phospholamban, two well known Ca(2+) handling proteins. The effects of succinate on Ca(2+) transient were abolished by pre-treatment with adenylyl cyclase and cAMP-dependent protein kinase (PKA) inhibitors. Direct PKA activation by succinate was further confirmed using a FRET-based A-kinase activity reporter. Additionally, succinate decreases cardiomyocyte viability through a caspase-3 activation pathway, effect also prevented by PKA inhibition. Taken together, these observations show that succinate acts as a signaling molecule in cardiomyocytes, modulating global Ca(2+) transient and cell viability through a PKA-dependent pathway. 2009 Elsevier Ltd. All rights reserved.

  14. Automated grouping of action potentials of human embryonic stem cell-derived cardiomyocytes.

    Gorospe, Giann; Zhu, Renjun; Millrod, Michal A; Zambidis, Elias T; Tung, Leslie; Vidal, Rene

    2014-09-01

    Methods for obtaining cardiomyocytes from human embryonic stem cells (hESCs) are improving at a significant rate. However, the characterization of these cardiomyocytes (CMs) is evolving at a relatively slower rate. In particular, there is still uncertainty in classifying the phenotype (ventricular-like, atrial-like, nodal-like, etc.) of an hESC-derived cardiomyocyte (hESC-CM). While previous studies identified the phenotype of a CM based on electrophysiological features of its action potential, the criteria for classification were typically subjective and differed across studies. In this paper, we use techniques from signal processing and machine learning to develop an automated approach to discriminate the electrophysiological differences between hESC-CMs. Specifically, we propose a spectral grouping-based algorithm to separate a population of CMs into distinct groups based on the similarity of their action potential shapes. We applied this method to a dataset of optical maps of cardiac cell clusters dissected from human embryoid bodies. While some of the nine cell clusters in the dataset are presented with just one phenotype, the majority of the cell clusters are presented with multiple phenotypes. The proposed algorithm is generally applicable to other action potential datasets and could prove useful in investigating the purification of specific types of CMs from an electrophysiological perspective.

  15. Mast cell stabilization decreases cardiomyocyte and LV function in dogs with isolated mitral regurgitation.

    Pat, Betty; Killingsworth, Cheryl; Chen, Yuanwen; Gladden, James D; Walcott, Greg; Powell, Pamela C; Denney, Thomas; Gupta, Himanshu; Desai, Ravi; Tillson, Michael; Dillon, A Ray; Dell'italia, Louis J

    2010-09-01

    Mast cells are increased in isolated mitral regurgitation (MR) in the dog and may mediate extracellular matrix loss and left ventricular (LV) dilatation. We tested the hypothesis that mast cell stabilization would attenuate LV remodeling and improve function in the MR dog. MR was induced in adult dogs randomized to no treatment (MR, n = 5) or to the mast cell stabilizer, ketotifen (MR + MCS, n = 4) for 4 months. LV hemodynamics were obtained at baseline and after 4 months of MR and magnetic resonance imaging (MRI) was performed at sacrifice. MRI-derived, serial, short-axis LV end-diastolic (ED) and end-systolic (ES) volumes, LVED volume/mass ratio, and LV 3-dimensional radius/wall thickness were increased in MR and MR + MCS dogs compared with normal dogs (n = 6) (P < .05). Interstitial collagen was decreased by 30% in both MR and MR + MCS versus normal dogs (P < .05). LV contractility by LV maximum time-varying elastance was significantly depressed in MR and MR + MCS dogs. Furthermore, cardiomyocyte fractional shortening was decreased in MR versus normal dogs and further depressed in MR + MCS dogs (P < .05). In vitro administration of ketotifen to normal cardiomyocytes also significantly decreased fractional shortening and calcium transients. Chronic mast cell stabilization did not attenuate eccentric LV remodeling or collagen loss in MR. However, MCS therapy had a detrimental effect on LV function because of a direct negative inotropic effect on cardiomyocyte function. Published by Elsevier Inc.

  16. 17β-Estradiol-induced interaction of ERα with NPPA regulates gene expression in cardiomyocytes.

    Mahmoodzadeh, Shokoufeh; Pham, Thi Hang; Kuehne, Arne; Fielitz, Britta; Dworatzek, Elke; Kararigas, Georgios; Petrov, George; Davidson, Mercy M; Regitz-Zagrosek, Vera

    2012-12-01

    17β-Oestradiol (E2) and its receptors (ERα and ERβ) are important regulators of physiological and pathological processes in the cardiovascular system. ER act in concert with other regulatory factors mediating oestrogenic effects. However, the underlying mechanisms modulating ER transcriptional activity are not fully elucidated. To gain better understanding of E2-induced ERα action in the human heart, we aimed to identify and functionally analyse interaction partners of ERα. Using yeast two-hybrid assays with a human heart cDNA library, we identified atrial natriuretic peptide precursor A (NPPA), a well-known cardiac hypertrophy marker, as a novel ERα interaction partner interacting in an E2-dependent manner. Mutation analyses and immunofluorescence data indicated that the LXXLL motif within NPPA is necessary for its E2-induced interaction with ERα, its action as a co-repressor of ERα, and its translocation into the nucleus of human and rat cardiomyocytes. Expression analysis and chromatin immunoprecipitation assays in a human left ventricular cardiomyocyte cell line, AC16, showed that NPPA interacts with E2/ERα, suppressing the transcriptional activity of ERα on E2-target genes, such as NPPA, connexin43, αactinin-2, nuclear factor of activated T-cells, and collagens I and III. We characterize for the first time an E2-regulated interaction of NPPA with ERα in cardiomyocytes, that may be crucial in physiological and/or pathological cardiac processes, thereby representing a potential therapeutic target.

  17. Adult Murine Skeletal Muscle Contains Cells That Can Differentiate into Beating Cardiomyocytes In Vitro

    Winitsky Steve O

    2005-01-01

    Full Text Available It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

  18. Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro.

    Steve O Winitsky

    2005-04-01

    Full Text Available It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

  19. Effects of Multivitamins and Known Teratogens on Chick Cardiomyocytes Micromass Culture Assay

    Samreen Memon

    2013-09-01

    Full Text Available   Objective(s: This study aimed to find out whether the chick cardiomyocyte micromass (MM system could be employed to predict the teratogenecity of common environmental factors. Different multivitamins and over the counter drugs were used in this study.   Materials and Methods: White Leghorn 5-day-old embryo hearts were dissected and trypsinized to produce a cardiomyocyte cell suspension in Dulbecco's Modified Eagle's Medium. The cultures were incubated at 370C in 5% CO2 in air, and observations were made at 24, 48 and 144 hr, for the detection of cell beating. Cellular viability was assessed using the resazurin assay and cell protein content was assessed by the kenacid blue assay. It was observed that while not affecting total cell number folic acid, vitamin C, sodium fluoride and ginseng did not significantly reduced cell activity and beating. However cadmium chloride significantly reduced the beating, cell viability and cell protein content in micromass cultures. Results: The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter morphology and function, which may result in either teratogenic outcome or cytotoxicity. Conclusion: This could form part of a screen for developmental toxicity related to cardiac function

  20. N-n-butyl haloperidol iodide protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy.

    Wang, Bin; Zhong, Shuping; Zheng, Fuchun; Zhang, Yanmei; Gao, Fenfei; Chen, Yicun; Lu, Binger; Xu, Han; Shi, Ganggang

    2015-09-22

    N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. In this study, we hypothesized the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by inhibiting autophagy in H9c2 cells. The degree of autophagy by treatment with F2 exposed to H/R in H9c2 cell was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Our results indicated that treatment with F2 inhibited autophagy in H9c2 cells exposed to H/R. 3-methyladenine, an inhibitor of autophagy, suppressed H/R-induced autophagy, and decreased apoptosis, whereas rapamycin, a classical autophagy sensitizer, increased autophagy and apoptosis. Mechanistically, macrophage migration inhibitory factor (MIF) was inhibited by F2 treatment after H/R. Accordingly, small interfering RNA (siRNA)-mediated MIF knockdown decreased H/R-induced autophagy. In summary, F2 protects cardiomyocytes during H/R injury through suppressing autophagy activation. Our results provide a new mechanistic insight into a functional role of F2 against H/R-induced cardiomyocyte injury and death.

  1. Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction.

    Stoelzle, Sonja; Haythornthwaite, Alison; Kettenhofen, Ralf; Kolossov, Eugen; Bohlen, Heribert; George, Michael; Brüggemann, Andrea; Fertig, Niels

    2011-09-01

    Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

  2. Protective effect of pomegranate seed oil against H2O2 -induced oxidative stress in cardiomyocytes

    Mehdi Bihamta

    2017-01-01

    Full Text Available Objective: It has been well documented that oxidative stress is involved in the pathogenesis of cardiac diseases. Previous studies have shown that pomegranate seed oil (PSO has antioxidant properties. This study was designed to investigate probable protective effects of PSO against hydrogen peroxide (H2O2-induced damage in H9c2 cardiomyocytes.Materials and Methods: The cells were pretreated 24 hr with PSO 1 hr before exposure to 200 µM H2O2. Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium (MTT assay. The level of reactive oxygen species (ROS and lipid peroxidation were measured by fluorimetric methods.Results: H2O2 significantly decreased cell viability which was accompanied by an increase in ROS production and lipid peroxidation and a decline in superoxide dismutase activity. Pretreatment with PSO increased viability of cardiomyocytes and decrease the elevated ROS production and lipid peroxidation. Also, PSO was able to restore superoxide dismutase activity.Conclusion: PSO has protective effect against oxidative stress-induced damage in cardiomyocytes and can be considered as a natural cardioprotective agent to prevent cardiovascular diseases.

  3. EFFECTS OF AEROBIC TRAINING ON THE CARDIOMYOCYTES OF THE RIGHT ATRIUM OF MICE

    Vanessa Gonçalves Coutinho de Oliveira

    Full Text Available ABSTRACT Introduction: Polypeptide hormones (natriuretic peptides, NPs are secreted by the cardiac atria and play an important role in the regulation of blood pressure. Objective: To evaluate the effects of aerobic training on the secretory apparatus of NPs in cardiomyocytes of the right atrium. Methods: Nine-month-old mice were divided in two groups (n=10: control group (CG and trained group (TG. The training protocol was performed on a motor treadmill for 8 weeks. Systolic blood pressure was measured at the beginning of the experiment (9 months of age and at moment of the sacrifice (11 months of age. Electron micrographs were used to quantify the following variables: the quantitative density and area of NP granules, the relative volumes of the mitochondria, endoplasmic reticulum, and Golgi complex and the relative volume of euchromatin in the nucleus and the number of pores per 10 µm of the nuclear membrane. The results were compared by Student's t test (p< 0.05. Results: The cardiomyocytes obtained from TG mice showed increased density and sectional area of secretory granules of NP, higher relative volume of endoplasmic reticulum, mitochondria, and Golgi complex compared with the CG mice. Furthermore, the quantitative density of nuclear pores and the relative volume of euchromatin in the nucleus were significantly higher compared with the CG mice. Conclusion: Aerobic training caused hypertrophy of the secretory apparatus in the cardiomyocytes of right atrium, which could explain the intense synthesis of natriuretic peptides in trained mice with respect to the untrained mice.

  4. SurR9C84A protects and recovers human cardiomyocytes from hypoxia induced apoptosis

    Ashok, Ajay [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia); Department of Pathology, Case Western Reserve University, 2103 Cornell Rd. WRB 5128, Cleveland, OH 44106-7288 (United States); Kanwar, Jagat Rakesh [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia); Krishnan, Uma Maheswari [Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), School of Chemical & Biotechnology (SCBT), SASTRA University, Thanjavur 613401 (India); Kanwar, Rupinder Kaur, E-mail: rupinder.kanwar@deakin.edu.au [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia)

    2017-01-01

    Survivin, as an anti-apoptotic protein and a cell cycle regulator, is recently gaining importance for its regenerative potential in salvaging injured hypoxic cells of vital organs such as heart. Different strategies are being employed to upregulate survivin expression in dying hypoxic cardiomyocytes. We investigated the cardioprotective potential of a cell permeable survivin mutant protein SurR9C84A, for the management of hypoxia mediated cardiomyocyte apoptosis, in a novel and clinically relevant model employing primary human cardiomyocytes (HCM). The aim of this research work was to study the efficacy and mechanism of SurR9C84A facilitated cardioprotection and regeneration in hypoxic HCM. To mimic hypoxic microenvironment in vitro, well characterized HCM were treated with 100 µm (48 h) cobalt chloride to induce hypoxia. Hypoxia induced (HI) HCM were further treated with SurR9C84A (1 µg/mL) in order to analyse its cardioprotective efficacy. Confocal microscopy showed rapid internalization of SurR9C84A and scanning electron microscopy revealed the reinstatement of cytoskeleton projections in HI HCM. SurR9C84A treatment increased cell viability, reduced cell death via, apoptosis (Annexin-V assay), and downregulated free cardiac troponin T and MMP-9 expression. SurR9C84A also upregulated the expression of proliferation markers (PCNA and Ki-67) and downregulated mitochondrial depolarization and ROS levels thereby, impeding cell death. Human Apoptosis Array further revealed that SurR9C84A downregulated expression of pro-apoptotic markers and augmented expression of HSPs and HTRA2/Omi. SurR9C84A treatment led to enhanced levels of survivin, VEGF, PI3K and pAkt. SurR9C84A proved non-toxic to normoxic HCM, as validated through unaltered cell proliferation and other marker levels. Its pre-treatment exhibited lesser susceptibility to hypoxia/damage. SurR9C84A holds a promising clinical potential for human cardiomyocyte survival and proliferation following hypoxic injury

  5. Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters

    Rami Shinnawi

    2015-10-01

    Full Text Available The advent of the human-induced pluripotent stem cell (hiPSC technology has transformed biomedical research, providing new tools for human disease modeling, drug development, and regenerative medicine. To fulfill its unique potential in the cardiovascular field, efficient methods should be developed for high-resolution, large-scale, long-term, and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs. To achieve this goal, we combined the hiPSC technology with genetically encoded voltage (ArcLight and calcium (GCaMP5G fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels, respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders, developmental biology, and drug development and testing.

  6. Low Resting Membrane Potential and Low Inward Rectifier Potassium Currents Are Not Inherent Features of hiPSC-Derived Cardiomyocytes.

    Horváth, András; Lemoine, Marc D; Löser, Alexandra; Mannhardt, Ingra; Flenner, Frederik; Uzun, Ahmet Umur; Neuber, Christiane; Breckwoldt, Kaja; Hansen, Arne; Girdauskas, Evaldas; Reichenspurner, Hermann; Willems, Stephan; Jost, Norbert; Wettwer, Erich; Eschenhagen, Thomas; Christ, Torsten

    2018-03-13

    Human induced pluripotent stem cell (hiPSC) cardiomyocytes (CMs) show less negative resting membrane potential (RMP), which is attributed to small inward rectifier currents (I K1 ). Here, I K1 was measured in hiPSC-CMs (proprietary and commercial cell line) cultured as monolayer (ML) or 3D engineered heart tissue (EHT) and, for direct comparison, in CMs from human right atrial (RA) and left ventricular (LV) tissue. RMP was measured in isolated cells and intact tissues. I K1 density in ML- and EHT-CMs from the proprietary line was similar to LV and RA, respectively. I K1 density in EHT-CMs from the commercial line was 2-fold smaller than in the proprietary line. RMP in EHT of both lines was similar to RA and LV. Repolarization fraction and I K,ACh response discriminated best between RA and LV and indicated predominantly ventricular phenotype in hiPSC-CMs/EHT. The data indicate that I K1 is not necessarily low in hiPSC-CMs, and technical issues may underlie low RMP in hiPSC-CMs. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Garlic extracts prevent oxidative stress, hypertrophy and apoptosis in cardiomyocytes: a role for nitric oxide and hydrogen sulfide

    2012-01-01

    Background In ancient times, plants were recognized for their medicinal properties. Later, the arrival of synthetic drugs pushed it to the backstage. However, from being merely used for food, plants are now been widely explored for their therapeutic value. The current study explores the potential of skin and flesh extracts from a hard-necked Rocambole variety of purple garlic in preventing cardiomyocyte hypertrophy and cell death. Methods Norepinephrine (NE) was used to induce hypertrophy in adult rat cardiomyocytes pretreated with garlic skin and flesh extracts. Cell death was measured as ratio of rod to round shaped cardiomyocytes. Fluorescent probes were used to measure apoptosis and oxidative stress in cardiomyocytes treated with and without extracts and NE. Pharmacological blockade of nitric oxide (NO) and hydrogen sulfide (H2S) were used to elucidate the mechanism of action of garlic extracts. Garlic extract samples were also tested for alliin and allicin concentrations. Results Exposure of cardiomyocytes to NE induced an increase in cell size and cell death; this increase was significantly prevented upon treatment with garlic skin and flesh extracts. Norepinephrine increased apoptosis and oxidative stress in cardiomyocytes which was prevented upon pretreatment with skin and flesh extracts; NO, and H2S blockers significantly inhibited this beneficial effect. Allicin and alliin concentration were significantly higher in garlic flesh extract when compared to the skin extract. Conclusion These results suggest that both skin and flesh garlic extracts are effective in preventing NE induced cardiomyocyte hypertrophy and cell death. Reduction in oxidative stress may also play an important role in the anti-hypertrophic and anti-apoptotic properties of garlic extracts. These beneficial effects may in part be mediated by NO and H2S. PMID:22931510

  8. Axin1 up-regulated 1 accelerates stress-induced cardiomyocytes apoptosis through activating Wnt/β-catenin signaling.

    Ye, Xing; Lin, Junyi; Lin, Zebin; Xue, Aimin; Li, Liliang; Zhao, Ziqin; Liu, Li; Shen, Yiwen; Cong, Bin

    2017-10-15

    Stress-induced cardiomyocyte apoptosis contributes to the pathogenesis of a variety of cardiovascular diseases, but how stress induces cardiomyocyte apoptosis remains largely unclear. The present study aims to investigate the effects of Axin1 up-regulated 1 (Axud1), a novel pro-apoptotic protein, on the cardiomyocyte survival and the underlying mechanisms. To this end, a rat model under restraint stress (RS) was established and in vitro stress-induced cardiomyocytes culture was achieved. Our data showed that Axud1 was upregulated in the rat myocardia after exposure to RS. Anti-apoptotic Bcl-2 was decreased, whereas pro-apoptotic Bax and Cleaved caspase-3 (Cc3) were increased in a time-dependent manner. The Wnt/β-catenin signaling was observed to be interestingly activated in heart undergoing RS. In addition, the treatment of norepinephrine (NE) to in vitro cardiomyocytes increased Axud1 level and induced cell apoptosis. Wnt/β-catenin signaling was consistently activated. Knockdown of Axud1 using specific siRNA blunted NE-induced cardiomyocytes apoptosis and also inactivated the Wnt/β-catenin signaling. XAV-939, an inhibitor of Wnt/β-catenin signaling, partially reversed the pro-apoptotic effect of NE. In conclusion, Axud1 accelerated stress-induced cardiomyocytes apoptosis through activation of Wnt/β-catenin signaling pathway. Our data provided novel evidence that therapeutic strategies against Axud1 or Wnt/β-catenin signaling might be promising in relation to RS-induced myocardial injury. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Evaluation of nefazodone-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes

    Lee, Sujeong, E-mail: crystalee@gmail.com [Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-343 (Korea, Republic of); Lee, Hyang-Ae, E-mail: hyangaelee@gmail.com [Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-343 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799 (Korea, Republic of); Human and Environmental Toxicology Program, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 305-350 (Korea, Republic of); Choi, Sung Woo, E-mail: djmaya@snu.ac.kr [Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799 (Korea, Republic of); Kim, Sung Joon, E-mail: sjoonkim@snu.ac.kr [Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799 (Korea, Republic of); Kim, Ki-Suk, E-mail: idkks00@gmail.com [Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-343 (Korea, Republic of); Human and Environmental Toxicology Program, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 305-350 (Korea, Republic of)

    2016-04-01

    The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which express the major cardiac ion channels and recapitulate spontaneous mechanical and electrical activities, may provide a possible solution for the lack of in vitro human-based cardiotoxicity testing models. Cardiotoxicity induced by the antidepressant nefazodone was previously revealed to cause an acquired QT prolongation by hERG channel blockade. To elucidate the cellular mechanisms underlying the cardiotoxicity of nefazodone beyond hERG, its effects on cardiac action potentials (APs) and ion channels were investigated using hiPSC-CMs with whole-cell patch clamp techniques. In a proof of principle study, we examined the effects of cardioactive channel blockers on the electrophysiological profile of hiPSC-CMs in advance of the evaluation of nefazodone. Nefazodone dose-dependently prolonged the AP duration at 90% (APD{sub 90}) and 50% (APD{sub 50}) repolarization, reduced the maximum upstroke velocity (dV/dt{sub max}) and induced early after depolarizations. Voltage-clamp studies of hiPSC-CMs revealed that nefazodone inhibited various voltage-gated ion channel currents including I{sub Kr}, I{sub Ks}, I{sub Na}, and I{sub Ca}. Among them, I{sub Kr} and I{sub Na} showed relatively higher sensitivity to nefazodone, consistent with the changes in the AP parameters. In summary, hiPSC-CMs enabled an integrated approach to evaluate the complex interactions of nefazodone with cardiac ion channels. These results suggest that hiPSC-CMs can be an effective model for detecting drug-induced arrhythmogenicity beyond the current standard assay of heterologously expressed hERG K{sup +} channels. - Highlights: • Nefazodone prolonged APD and decreased upstroke velocity of APs in hiPSC-CMs. • Nefazodone inhibited cardiac ion channels, especially I{sub Kr} and I{sub Na}, in hiPSC-CMs. • Nefazodone-induced AP changes are mainly the result of I{sub Kr} and I{sub Na} inhibition

  10. Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.

    Hideki Uosaki

    Full Text Available RATIONALE: Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs. METHOD AND RESULT: We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2-positive cardiomyocytes appeared robustly with 30-70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1 antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7-8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95-98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium, CD140b (pericytes and TRA-1-60 (undifferentiated hESCs/hiPSCs. 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5-10×10(5 VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines. CONCLUSION: We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from h

  11. Cardiomyocyte hypertrophy induced by Endonuclease G deficiency requires reactive oxygen radicals accumulation and is inhibitable by the micropeptide humanin.

    Blasco, Natividad; Cámara, Yolanda; Núñez, Estefanía; Beà, Aida; Barés, Gisel; Forné, Carles; Ruíz-Meana, Marisol; Girón, Cristina; Barba, Ignasi; García-Arumí, Elena; García-Dorado, David; Vázquez, Jesús; Martí, Ramon; Llovera, Marta; Sanchis, Daniel

    2018-06-01

    The endonuclease G gene (Endog), which codes for a mitochondrial nuclease, was identified as a determinant of cardiac hypertrophy. How ENDOG controls cardiomyocyte growth is still unknown. Thus, we aimed at finding the link between ENDOG activity and cardiomyocyte growth. Endog deficiency induced reactive oxygen species (ROS) accumulation and abnormal growth in neonatal rodent cardiomyocytes, altering the AKT-GSK3β and Class-II histone deacethylases (HDAC) signal transduction pathways. These effects were blocked by ROS scavengers. Lack of ENDOG reduced mitochondrial DNA (mtDNA) replication independently of ROS accumulation. Because mtDNA encodes several subunits of the mitochondrial electron transport chain, whose activity is an important source of cellular ROS, we investigated whether Endog deficiency compromised the expression and activity of the respiratory chain complexes but found no changes in these parameters nor in ATP content. MtDNA also codes for humanin, a micropeptide with possible metabolic functions. Nanomolar concentrations of synthetic humanin restored normal ROS levels and cell size in Endog-deficient cardiomyocytes. These results support the involvement of redox signaling in the control of cardiomyocyte growth by ENDOG and suggest a pathway relating mtDNA content to the regulation of cell growth probably involving humanin, which prevents reactive oxygen radicals accumulation and hypertrophy induced by Endog deficiency. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Melatonin attenuates angiotensin II-induced cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway.

    Su, Hongyan; Li, Jingyuan; Chen, Tongshuai; Li, Na; Xiao, Jie; Wang, Shujian; Guo, Xiaobin; Yang, Yi; Bu, Peili

    2016-11-01

    Melatonin is well known for its cardioprotective effects; however, whether melatonin exerts therapeutic effects on cardiomyocyte hypertrophy remains to be investigated, as do the mechanisms underlying these effects, if they exist. Cyclophilin A (CyPA) and its corresponding receptor, CD147, which exists in a variety of cells, play crucial roles in modulating reactive oxygen species (ROS) production. In this study, we explored the role of the CyPA/CD147 signaling pathway in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy and the protective effects exerted by melatonin against Ang II-induced injury in cultured H9C2 cells. Cyclosporine A, a specific CyPA/CD147 signaling pathway inhibitor, was used to manipulate CyPA/CD147 activity. H9C2 cells were then subjected to Ang II or CyPA treatment in either the absence or presence of melatonin. Our results indicate that Ang II induces cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway and promotes ROS production, which can be blocked by melatonin pretreatment in a concentration-dependent manner, in cultured H9C2 cells and that CyPA/CD147 signaling pathway inhibition protects against Ang II-induced cardiomyocyte hypertrophy. The protective effects of melatonin against Ang II-induced cardiomyocyte hypertrophy depend at least partially on CyPA/CD147 inhibition.

  13. Selenium deficiency aggravates T-2 toxin-induced injury of primary neonatal rat cardiomyocytes through ER stress.

    Xu, Jing; Pan, Shengchi; Gan, Fang; Hao, Shu; Liu, Dandan; Xu, Haibin; Huang, Kehe

    2018-04-01

    Keshan disease is a potentially fatal cardiomyopathy in humans. Selenium deficiency, T-2 toxin, and myocarditis virus are thought to be the major factors contributing to Keshan disease. But the relationship among these three factors is poorly described. This study aims to explore whether selenium deficiency aggravates T-2 toxin-induced cardiomyocyte injury and its underlying mechanism. Cardiomyocytes were isolated from neonatal rat and cultured at the physiological (2.0 μM) or lower concentrations of selenium with different concentrations of T-2 toxin. Our results showed that selenium deficiencies aggravated T-2 toxin-induced cardiomyocyte injury in a concentration-dependent manner as demonstrated by MTT bioassay, LDH activity, reactive oxygen species levels and caspase 3 protein expressions. T-2 toxin treatment significantly increased mRNA expressions for stress proteins GRP78 and CHOP in cardiomyocytes compared with the control. Selenium deficiencies further promoted GRP78, CHOP and p-eIF2α expressions. Knockdown of CHOP by the specific small interfering RNA eliminated the effect of selenium deficiencies on T-2 toxin-induced injury. It could be concluded that selenium deficiency aggravates T-2 toxin-induced cardiomyocyte injury through initiating more aggressive endoplasmic reticulum stress. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

    Stephanie Friedrichs

    2015-01-01

    Full Text Available Disease-specific induced pluripotent stem (iPS cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

  15. MicroRNA-1 Regulates the Differentiation of Adipose-Derived Stem Cells into Cardiomyocyte-Like Cells

    Can Chen

    2018-01-01

    Full Text Available Stem cell transplantation is one of most valuable methods in the treatment of myocardial infarction, and adipose-derived stem cells (ASCs are becoming a hot topic in medical research. Previous studies have shown that ASCs can be differentiated into cardiomyocyte-like cells, but the efficiency and survival rates are low. We investigated the role and mechanism of microRNA-1 (miR-1 in the differentiation of ASCs into cardiomyocyte-like cells. ASCs and cardiomyocytes were isolated from neonatal rats. We constructed lentivirus for overexpressing miR-1 and used DAPT, an antagonist of the Notch1 pathway, for in vitro analyses. We performed cocultures with ASCs and cardiomyocytes. The differentiation efficiency of ASCs was detected by cell-specific surface antigens. Our results showed that miR-1 can promote the expression of Notch1 and reduce the expression of Hes1, a Notch pathway factor, and overexpression of miR-1 can promote the differentiation of ASCs into cardiomyocyte-like cells, which may occur by regulating Notch1 and Hes1.

  16. SIRT1 Functions as an Important Regulator of Estrogen-Mediated Cardiomyocyte Protection in Angiotensin II-Induced Heart Hypertrophy

    Tao Shen

    2014-01-01

    Full Text Available Background. Sirtuin 1 (SIRT1 is a member of the sirtuin family, which could activate cell survival machinery and has been shown to be protective in regulation of heart function. Here, we determined the mechanism by which SIRT1 regulates Angiotensin II- (AngII- induced cardiac hypertrophy and injury in vivo and in vitro. Methods. We analyzed SIRT1 expression in the hearts of control and AngII-induced mouse hypertrophy. Female C57BL/6 mice were ovariectomized and pretreated with 17β-estradiol to measure SIRT1 expression. Protein synthesis, cardiomyocyte surface area analysis, qRT-PCR, TUNEL staining, and Western blot were performed on AngII-induced mouse heart hypertrophy samples and cultured neonatal rat ventricular myocytes (NRVMs to investigate the function of SIRT1. Results. SIRT1 expression was slightly upregulated in AngII-induced mouse heart hypertrophy in vivo and in vitro, accompanied by elevated cardiomyocyte apoptosis. SIRT1 overexpression relieves AngII-induced cardiomyocyte hypertrophy and apoptosis. 17β-Estradiol was able to protect cardiomyocytes from AngII-induced injury with a profound upregulation of SIRT1 and activation of AMPK. Moreover, estrogen receptor inhibitor ICI 182,780 and SIRT1 inhibitor niacinamide could block SIRT1’s protective effect. Conclusions. These results indicate that SIRT1 functions as an important regulator of estrogen-mediated cardiomyocyte protection during AngII-induced heart hypertrophy and injury.

  17. Dynamic link between histone H3 acetylation and an increase in the functional characteristics of human ESC/iPSC-derived cardiomyocytes.

    Tomomi G Otsuji

    Full Text Available Cardiomyocytes (CMs derived from human embryonic stem cells (hESCs or human induced pluripotent stem cells (hiPSCs are functionally heterogeneous, display insufficient biological efficacy and generally possess the electrophysiological properties seen in fetal CMs. However, a homogenous population of hESC/hiPSC-CMs, with properties similar to those of adult human ventricular cells, is required for use in drug cardiotoxicity screening. Unfortunately, despite the requirement for the functional characteristics of post-mitotic beating cell aggregates to mimic the behavior of mature cardiomyocytes in vitro, few technological improvements have been made in this field to date. Previously, we showed that culturing hESC-CMs under low-adhesion conditions with cyclic replating confers continuous contractility on the cells, leading to a functional increase in cardiac gene expression and electrophysiological properties over time. The current study reveals that culturing hESC/hiPSC-CMs under non-adhesive culture conditions enhances the electrophysiological properties of the CMs through an increase in the acetylation of histone H3 lysine residues, as confirmed by western blot analyses. Histone H3 acetylation was induced chemically by treating primitive hESC/hiPSC-CMs with Trichostatin A (TSA, a histone deacetylase (HDAC inhibitor, resulting in an immediate increase in global cardiac gene expression. In functional analyses using multi-electrode array (MEA recordings, TSA-treated hESC/hiPSC-CM colonies showed appropriate responses to particular concentrations of known potassium ion channel inhibitors. Thus, the combination of a cell-autonomous functional increase in response to non-adhesive culture and short-term TSA treatment of hESC/hiPSC-CM colonies cultured on MEA electrodes will help to make cardiac toxicity tests more accurate and reproducible via genome-wide chromatin activation.

  18. Longstanding Hyperthyroidism Is Associated with Normal or Enhanced Intrinsic Cardiomyocyte Function despite Decline in Global Cardiac Function

    Redetzke, Rebecca A.; Gerdes, A. Martin

    2012-01-01

    Thyroid hormones (THs) play a pivotal role in cardiac homeostasis. TH imbalances alter cardiac performance and ultimately cause cardiac dysfunction. Although short-term hyperthyroidism typically leads to heightened left ventricular (LV) contractility and improved hemodynamic parameters, chronic hyperthyroidism is associated with deleterious cardiac consequences including increased risk of arrhythmia, impaired cardiac reserve and exercise capacity, myocardial remodeling, and occasionally heart failure. To evaluate the long-term consequences of chronic hyperthyroidism on LV remodeling and function, we examined LV isolated myocyte function, chamber function, and whole tissue remodeling in a hamster model. Three-month-old F1b hamsters were randomized to control or 10 months TH treatment (0.1% grade I desiccated TH). LV chamber remodeling and function was assessed by echocardiography at 1, 2, 4, 6, 8, and 10 months of treatment. After 10 months, terminal cardiac function was assessed by echocardiography and LV hemodynamics. Hyperthyroid hamsters exhibited significant cardiac hypertrophy and deleterious cardiac remodeling characterized by myocyte lengthening, chamber dilatation, decreased relative wall thickness, increased wall stress, and increased LV interstitial fibrotic deposition. Importantly, hyperthyroid hamsters demonstrated significant LV systolic and diastolic dysfunction. Despite the aforementioned remodeling and global cardiac decline, individual isolated cardiac myocytes from chronically hyperthyroid hamsters had enhanced function when compared with myocytes from untreated age-matched controls. Thus, it appears that long-term hyperthyroidism may impair global LV function, at least in part by increasing interstitial ventricular fibrosis, in spite of normal or enhanced intrinsic cardiomyocyte function. PMID:23056390

  19. Longstanding hyperthyroidism is associated with normal or enhanced intrinsic cardiomyocyte function despite decline in global cardiac function.

    Nathan Y Weltman

    Full Text Available Thyroid hormones (THs play a pivotal role in cardiac homeostasis. TH imbalances alter cardiac performance and ultimately cause cardiac dysfunction. Although short-term hyperthyroidism typically leads to heightened left ventricular (LV contractility and improved hemodynamic parameters, chronic hyperthyroidism is associated with deleterious cardiac consequences including increased risk of arrhythmia, impaired cardiac reserve and exercise capacity, myocardial remodeling, and occasionally heart failure. To evaluate the long-term consequences of chronic hyperthyroidism on LV remodeling and function, we examined LV isolated myocyte function, chamber function, and whole tissue remodeling in a hamster model. Three-month-old F1b hamsters were randomized to control or 10 months TH treatment (0.1% grade I desiccated TH. LV chamber remodeling and function was assessed by echocardiography at 1, 2, 4, 6, 8, and 10 months of treatment. After 10 months, terminal cardiac function was assessed by echocardiography and LV hemodynamics. Hyperthyroid hamsters exhibited significant cardiac hypertrophy and deleterious cardiac remodeling characterized by myocyte lengthening, chamber dilatation, decreased relative wall thickness, increased wall stress, and increased LV interstitial fibrotic deposition. Importantly, hyperthyroid hamsters demonstrated significant LV systolic and diastolic dysfunction. Despite the aforementioned remodeling and global cardiac decline, individual isolated cardiac myocytes from chronically hyperthyroid hamsters had enhanced function when compared with myocytes from untreated age-matched controls. Thus, it appears that long-term hyperthyroidism may impair global LV function, at least in part by increasing interstitial ventricular fibrosis, in spite of normal or enhanced intrinsic cardiomyocyte function.

  20. Recapitulation of Clinical Individual Susceptibility to Drug-Induced QT Prolongation in Healthy Subjects Using iPSC-Derived Cardiomyocytes

    Tadahiro Shinozawa

    2017-02-01

    Full Text Available To predict drug-induced serious adverse events (SAE in clinical trials, a model using a panel of cells derived from human induced pluripotent stem cells (hiPSCs of individuals with different susceptibilities could facilitate major advancements in translational research in terms of safety and pharmaco-economics. However, it is unclear whether hiPSC-derived cells can recapitulate interindividual differences in drug-induced SAE susceptibility in populations not having genetic disorders such as healthy subjects. Here, we evaluated individual differences in SAE susceptibility based on an in vitro model using hiPSC-derived cardiomyocytes (hiPSC-CMs as a pilot study. hiPSCs were generated from blood samples of ten healthy volunteers with different susceptibilities to moxifloxacin (Mox-induced QT prolongation. Different Mox-induced field potential duration (FPD prolongation values were observed in the hiPSC-CMs from each individual. Interestingly, the QT interval was significantly positively correlated with FPD at clinically relevant concentrations (r > 0.66 in multiple analyses including concentration-QT analysis. Genomic analysis showed no interindividual significant differences in known target-binding sites for Mox and other drugs such as the hERG channel subunit, and baseline QT ranges were normal. The results suggest that hiPSC-CMs from healthy subjects recapitulate susceptibility to Mox-induced QT prolongation and provide proof of concept for in vitro preclinical trials.

  1. 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

    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

  2. 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.

    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.

  3. GSK-3α directly regulates β-adrenergic signaling and the response of the heart to hemodynamic stress in mice

    Zhou, Jibin; Lal, Hind; Chen, Xiongwen; Shang, Xiying; Song, Jianliang; Li, Yingxin; Kerkela, Risto; Doble, Bradley W.; MacAulay, Katrina; DeCaul, Morgan; Koch, Walter J.; Farber, John; Woodgett, James; Gao, Erhe; Force, Thomas

    2010-01-01

    The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases consists of 2 highly related isoforms, α and β. Although GSK-3β has an important role in cardiac development, much remains unknown about the function of either GSK-3 isoform in the postnatal heart. Herein, we present what we believe to be the first studies defining the role of GSK-3α in the mouse heart using gene targeting. Gsk3a–/– mice over 2 months of age developed progressive cardiomyocyte and cardiac hypertrophy and contractile dysfunction. Following thoracic aortic constriction in young mice, we observed enhanced hypertrophy that rapidly transitioned to ventricular dilatation and contractile dysfunction. Surprisingly, markedly impaired β-adrenergic responsiveness was found at both the organ and cellular level. This phenotype was reproduced by acute treatment of WT cardiomyocytes with a small molecule GSK-3 inhibitor, confirming that the response was not due to a chronic adaptation to LV dysfunction. Thus, GSK-3α appears to be the central regulator of a striking range of essential processes, including acute and direct positive regulation of β-adrenergic responsiveness. In the absence of GSK-3α, the heart cannot respond effectively to hemodynamic stress and rapidly fails. Our findings identify what we believe to be a new paradigm of regulation of β-adrenergic signaling and raise concerns given the rapid expansion of drug development targeting GSK-3. PMID:20516643

  4. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes

    Jan David Kijlstra

    2015-12-01

    Full Text Available The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening.

  5. High-speed digital imaging of cytosolic Ca2+ and contraction in single cardiomyocytes.

    O'Rourke, B; Reibel, D K; Thomas, A P

    1990-07-01

    A charge-coupled device (CCD) camera, with the capacity for simultaneous spatially resolved photon counting and rapid frame transfer, was utilized for high-speed digital image collection from an inverted epifluorescence microscope. The unique properties of the CCD detector were applied to an analysis of cell shortening and the Ca2+ transient from fluorescence images of fura-2-loaded [corrected] cardiomyocytes. On electrical stimulation of the cell, a series of sequential subimages was collected and used to create images of Ca2+ within the cell during contraction. The high photosensitivity of the camera, combined with a detector-based frame storage technique, permitted collection of fluorescence images 10 ms apart. This rate of image collection was sufficient to resolve the rapid events of contraction, e.g., the upstroke of the Ca2+ transient (less than 40 ms) and the time to peak shortening (less than 80 ms). The technique was used to examine the effects of beta-adrenoceptor activation, fura-2 load, and stimulus frequency on cytosolic Ca2+ transients and contractions of single cardiomyocytes. beta-Adrenoceptor stimulation resulted in pronounced increases in peak Ca2+, maximal rates of rise and decay of Ca2+, extent of shortening, and maximal velocities of shortening and relaxation. Raising the intracellular load of fura-2 had little effect on the rising phase of Ca2+ or the extent of shortening but extended the duration of the Ca2+ transient and contraction. In related experiments utilizing differential-interference contrast microscopy, the same technique was applied to visualize sarcomere dynamics in contracting cells. This newly developed technique is a versatile tool for analyzing the Ca2+ transient and mechanical events in studies of excitation-contraction coupling in cardiomyocytes.

  6. Anti-aging effects of vitamin C on human pluripotent stem cell-derived cardiomyocytes.

    Kim, Yoon Young; Ku, Seung-Yup; Huh, Yul; Liu, Hung-Ching; Kim, Seok Hyun; Choi, Young Min; Moon, Shin Yong

    2013-10-01

    Human pluripotent stem cells (hPSCs) have arisen as a source of cells for biomedical research due to their developmental potential. Stem cells possess the promise of providing clinicians with novel treatments for disease as well as allowing researchers to generate human-specific cellular metabolism models. Aging is a natural process of living organisms, yet aging in human heart cells is difficult to study due to the ethical considerations regarding human experimentation as well as a current lack of alternative experimental models. hPSC-derived cardiomyocytes (CMs) bear a resemblance to human cardiac cells and thus hPSC-derived CMs are considered to be a viable alternative model to study human heart cell aging. In this study, we used hPSC-derived CMs as an in vitro aging model. We generated cardiomyocytes from hPSCs and demonstrated the process of aging in both human embryonic stem cell (hESC)- and induced pluripotent stem cell (hiPSC)-derived CMs. Aging in hESC-derived CMs correlated with reduced membrane potential in mitochondria, the accumulation of lipofuscin, a slower beating pattern, and the downregulation of human telomerase RNA (hTR) and cell cycle regulating genes. Interestingly, the expression of hTR in hiPSC-derived CMs was not significantly downregulated, unlike in hESC-derived CMs. In order to delay aging, vitamin C was added to the cultured CMs. When cells were treated with 100 μM of vitamin C for 48 h, anti-aging effects, specifically on the expression of telomere-related genes and their functionality in aging cells, were observed. Taken together, these results suggest that hPSC-derived CMs can be used as a unique human cardiomyocyte aging model in vitro and that vitamin C shows anti-aging effects in this model.

  7. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    Jan Pavel Kucera

    2015-09-01

    Full Text Available Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs. However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands.CV was significantly lower in strands composed purely of SCMs (5.5±1.5 cm/s, n=11 as compared to PCMs (34.9±2.9 cm/s, n=21 at similar refractoriness (100% SCMs: 122±25 ms, n=9; 100% PCMs: 139±67 ms, n=14. In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV.These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

  8. Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis.

    Parra, Valentina; Eisner, Veronica; Chiong, Mario; Criollo, Alfredo; Moraga, Francisco; Garcia, Alejandra; Härtel, Steffen; Jaimovich, Enrique; Zorzano, Antonio; Hidalgo, Cecilia; Lavandero, Sergio

    2008-01-15

    In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes. Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation. Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

  9. Role of heterotrimeric G protein and calcium in cardiomyocyte hypertrophy induced by IGF-1.

    Carrasco, Loreto; Cea, Paola; Rocco, Paola; Peña-Oyarzún, Daniel; Rivera-Mejias, Pablo; Sotomayor-Flores, Cristian; Quiroga, Clara; Criollo, Alfredo; Ibarra, Cristian; Chiong, Mario; Lavandero, Sergio

    2014-04-01

    In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and β-myosin heavy chain (β-MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin. © 2013 Wiley Periodicals, Inc.

  10. Generation of electrophysiologically functional cardiomyocytes from mouse induced pluripotent stem cells

    Hongran Wang

    2016-03-01

    Full Text Available Induced pluripotent stem (iPS cells can efficiently differentiate into the three germ layers similar to those formed by differentiated embryonic stem (ES cells. This provides a new source of cells in which to establish preclinical allogeneic transplantation models. Our iPS cells were generated from mouse embryonic fibroblasts (MEFs transfected with the Yamanaka factors, the four transcription factors (Oct4, Sox2, Klf4 and c-Myc, without antibiotic selection or MEF feeders. After the formation of embryoid bodies (EBs, iPS cells spontaneously differentiated into Flk1-positive cardiac progenitors and cardiomyocytes expressing cardiac-specific markers such as alpha sarcomeric actinin (α-actinin, cardiac alpha myosin heavy chain (α-MHC, cardiac troponin T (cTnT, and connexin 43 (CX43, as well as cardiac transcription factors Nk2 homebox 5 (Nkx2.5 and gata binding protein 4 (gata4. The electrophysiological activity of iPS cell-derived cardiomyocytes (iPS-CMs was detected in beating cell clusters with optical mapping and RH237 a voltage-sensitive dye, and in single contracting cells with patch-clamp technology. Incompletely differentiated iPS cells formed teratomas when transplanted into a severe combined immunodeficiency (SCID mouse model of myocardial infarction. Our results show that somatic cells can be reprogrammed into pluripotent stem cells, which in turn spontaneously differentiate into electrophysiologically functional mature cardiomyocytes expressing cardiac-specific makers, and that these cells can potentially be used to repair myocardial infarction (MI in the future.

  11. Experimental research on recombinant human endostatin-induced cardiomyocyte apoptosis in rats

    Jing QIN

    2014-03-01

    Full Text Available Objective To explore the recombinant human endostatin (rh-ES-induced cardiotoxicity in rats and its mechanism. Methods Twenty four female Wistar rats were randomly divided into four groups (6 each. Rats in low, moderate and high dose group received rh-ES with a dosage of 3, 6 and 12mg/(kg·d, respectively, by intraperitoneal injection, and rats in control group received the same amount of normal saline alone. Half of rats in each group were sacrificed by spinal dislocation after 4 weeks and 8 weeks of the treatment. Pathomorphologic and ultrastructural changes in rat's myocardial tissue were evaluated by light microscopy and transmission electron microscopy. Cardiomyocyte apoptosis was detected with TdT-mediated dUTP nick end labeling (TUNEL assay. Microvessel density (MVD in myocardial tissue was measured by immunohistochemically marking endothelial cell with CD34. Results No pathomorphologic and ultrastrucural changes were found under light microscope and transmission electron microscope in the low dose and moderate dose groups, but cardiomyocyte damage were found in the high dose group. TUNEL assay revealed more apoptotic cells in high and moderate (only 8 weeks dose groups than in control group (P=0.033, P=0.000, and the apoptosis index was highest in the high dose group at 8 weeks. In addition, compared with the control group, MVD significantly increased in high dose groups at 4 weeks and 8 weeks (P<0.05. Conclusions rh-ES induces the cardiotoxicity in rats, and cardiomyocyte apoptosis is involved in the pathological course of cardiac toxicity. DOI: 10.11855/j.issn.0577-7402.2014.01.02

  12. Diclofenac induces proteasome and mitochondrial dysfunction in murine cardiomyocytes and hearts.

    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. Propofol ameliorates doxorubicin-induced oxidative stress and cellular apoptosis in rat cardiomyocytes

    Lai, H.C. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Yeh, Y.C. [Graduate Institute of Natural Healing Sciences, Nanhua University, Chiayi, Taiwan (China); Wang, L.C. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Ting, C.T.; Lee, W.L. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Lee, H.W. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Wang, K.Y. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine, Chung-Shan Medical University, Taichung, Taiwan (China); Wu, A. [College of Biological Science, University of California, Davis (United States); Su, C.S. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Liu, T.J., E-mail: trliu@vghtc.gov.tw [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China)

    2011-12-15

    Background: Propofol is an anesthetic with pluripotent cytoprotective properties against various extrinsic insults. This study was designed to examine whether this agent could also ameliorate the infamous toxicity of doxorubicin, a widely-used chemotherapeutic agent against a variety of cancer diseases, on myocardial cells. Methods: Cultured neonatal rat cardiomyocytes were administrated with vehicle, doxorubicin (1 {mu}M), propofol (1 {mu}M), or propofol plus doxorubicin (given 1 h post propofol). After 24 h, cells were harvested and specific analyses regarding oxidative/nitrative stress and cellular apoptosis were conducted. Results: Trypan blue exclusion and MTT assays disclosed that viability of cardiomyocytes was significantly reduced by doxorubicin. Contents of reactive oxygen and nitrogen species were increased and antioxidant enzymes SOD1, SOD2, and GPx were decreased in these doxorubicin-treated cells. Mitochondrial dehydrogenase activity and membrane potential were also depressed, along with activation of key effectors downstream of mitochondrion-dependent apoptotic signaling. Besides, abundance of p53 was elevated and cleavage of PKC-{delta} was induced in these myocardial cells. In contrast, all of the above oxidative, nitrative and pro-apoptotic events could be suppressed by propofol pretreatment. Conclusions: Propofol could extensively counteract oxidative/nitrative and multiple apoptotic effects of doxorubicin in the heart; hence, this anesthetic may serve as an adjuvant agent to assuage the untoward cardiac effects of doxorubicin in clinical application. -- Highlights: Black-Right-Pointing-Pointer We evaluate how propofol prevents doxorubicin-induced toxicity in cardiomyocytes. Black-Right-Pointing-Pointer Propofol reduces doxorubicin-imposed nitrative and oxidative stress. Black-Right-Pointing-Pointer Propofol suppresses mitochondrion-, p53- and PKC-related apoptotic signaling. Black-Right-Pointing-Pointer Propofol ameliorates apoptosis and

  14. Mitochondrial p38β and manganese superoxide dismutase interaction mediated by estrogen in cardiomyocytes.

    Han Liu

    Full Text Available While etiology behind the observed acceleration of ischemic heart disease in postmenopausal women is poorly understood, collective scientific data suggest cardioprotective effects of the endogenous female sex hormone, estrogen. We have previously shown that 17β-estradiol (E2 protects cardiomyocytes exposed to hypoxia-reoxygenation (H/R by inhibiting p38α - p53 signaling in apoptosis and activating pro-survival p38β mitogen activated protein kinase (p38β MAPK, leading to suppression of reactive oxygen species (ROS post H/R. However, little is known about the mechanism behind the antioxidant actions of E2-dependent p38β. The aim of this study is to determine whether the cytoprotection by estrogen involves regulation of manganese superoxide dismutase (MnSOD, a major mitochondrial ROS scavenging enzyme, via cardiac p38β.We identified mitochondrial p38β by immunocytochemistry and by immunoblotting in mitochondria isolated from neonatal cardiomyocytes of Sprague-Dawley rats. E2 facilitated the mitochondrial localization of the active form of the kinase, phosphorylated p38β (p-p38β. E2 also reduced the H/R-induced mitochondrial membrane potential decline, augmented the MnSOD activity and suppressed anion superoxide generation, while the dismutase protein expression remained unaltered. Co-immunoprecipitation studies showed physical association between MnSOD and p38β. p38β phosphorylated MnSOD in an E2-dependent manner in in-vitro kinase assays.This work demonstrates for the first time a mitochondrial pool of active p38β and E2-mediated phosphorylation of MnSOD by the kinase. The results shed light on the mechanism behind the cytoprotective actions of E2 in cardiomyocytes under oxidative stress.

  15. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

    Zhang, Pengpeng; Shan, Tizhong; Liang, Xinrong; Deng, Changyan; Kuang, Shihuan

    2014-01-01

    Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor flox/flox mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor flox/flox mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function

  16. A systemized approach to investigate Ca2+ synchronization in clusters of human induced pluripotent stem-cell derived cardiomyocytes

    Aled R Jones

    2016-01-01

    Full Text Available Induced pluripotent stem cell-derived cardiomyocytes (IPS-CM are considered by many to be the cornerstone of future approaches to repair the diseased heart. However, current methods for producing IPS-CM typically yield highly variable populations with low batch-to-batch reproducibility. The underlying reasons for this are not fully understood. Here we report on a systematized approach to investigate the effect of maturation in embryoid bodies (EB versus ‘on plate’ culture on spontaneous activity and regional Ca2+ synchronization in IPS-CM clusters. A detailed analysis of the temporal and spatial organization of Ca2+ spikes in IPS-CM clusters revealed that the disaggregation of EBs between 0.5 and 2 weeks produced IPS-CM characterized by spontaneous beating and high levels of regional Ca2+ synchronization. These phenomena were typically absent in IPS-CM obtained from older EBs (> 2 weeks. The maintenance of all spontaneously active IPS-CM clusters under ‘on plate’ culture conditions promoted the progressive reduction in regional Ca2+ synchronization and the loss of spontaneous Ca2+ spiking. Raising the extracellular [Ca2+] surrounding these quiescent IPS-CM clusters from approximately 0.4 to 1.8 mM unmasked discrete behaviours typified by either a long-lasting Ca2+ elevation that returned to baseline or b persistent, large-amplitude Ca2+ oscillations around an increased cytoplasmic [Ca2+]. The different responses of IPS-CM to elevated extracellular [Ca2+] could be traced back to their routes of derivation. The data point to the possibility of predictably influencing IPS-CM phenotype and response to external activation via defined interventions at early stages in their maturation.

  17. Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy

    Cho, Gun-Sik; Lee, Dong I.; Tampakakis, Emmanouil; Murphy, Sean; Andersen, Peter; Uosaki, Hideki; Chelko, Stephen; Chakir, Khalid; Hong, Ingie; Seo, Kinya; Vincent Chen, Huei-Sheng; Chen, Xiongwen; Basso, Cristina; Houser, Steven R.; Tomaselli, Gordon F.

    2017-01-01

    Summary: Pluripotent stem cells (PSCs) offer unprecedented opportunities for disease modeling and personalized medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs). Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonata...

  18. β-Adrenergic receptor stimulation inhibits proarrhythmic alternans in postinfarction border zone cardiomyocytes: a computational analysis.

    Tomek, Jakub; Rodriguez, Blanca; Bub, Gil; Heijman, Jordi

    2017-08-01

    The border zone (BZ) of the viable myocardium adjacent to an infarct undergoes extensive autonomic and electrical remodeling and is prone to repolarization alternans-induced cardiac arrhythmias. BZ remodeling processes may promote or inhibit Ca 2+ and/or repolarization alternans and may differentially affect ventricular arrhythmogenesis. Here, we used a detailed computational model of the canine ventricular cardiomyocyte to study the determinants of alternans in the BZ and their regulation by β-adrenergic receptor (β-AR) stimulation. The BZ model developed Ca 2+ transient alternans at slower pacing cycle lengths than the control model, suggesting that the BZ may promote spatially heterogeneous alternans formation in an infarcted heart. β-AR stimulation abolished alternans. By evaluating all combinations of downstream β-AR stimulation targets, we identified both direct (via ryanodine receptor channels) and indirect [via sarcoplasmic reticulum (SR) Ca 2+ load] modulation of SR Ca 2+ release as critical determinants of Ca 2+ transient alternans. These findings were confirmed in a human ventricular cardiomyocyte model. Cell-to-cell coupling indirectly modulated the likelihood of alternans by affecting the action potential upstroke, reducing the trigger for SR Ca 2+ release in one-dimensional strand simulations. However, β-AR stimulation inhibited alternans in both single and multicellular simulations. Taken together, these data highlight a potential antiarrhythmic role of sympathetic hyperinnervation in the BZ by reducing the likelihood of alternans and provide new insights into the underlying mechanisms controlling Ca 2+ transient and repolarization alternans. NEW & NOTEWORTHY We integrated, for the first time, postmyocardial infarction electrical and autonomic remodeling in a detailed, validated computer model of β-adrenergic stimulation in ventricular cardiomyocytes. Here, we show that β-adrenergic stimulation inhibits alternans and provide novel insights

  19. Zinc-induced cardiomyocyte relaxation in a rat model of hyperglycemia is independent of myosin isoform

    Yi Ting

    2012-11-01

    Full Text Available Abstract It has been reported previously that diabetic cardiomyopathy can be inhibited or reverted with chronic zinc supplementation. In the current study, we hypothesized that total cardiac calcium and zinc content is altered in early onset diabetes mellitus characterized in part as hyperglycemia (HG and that exposure of zinc ion (Zn2+ to isolated cardiomyocytes would enhance contraction-relaxation function in HG more so than in nonHG controls. To better control for differential cardiac myosin isoform expression as occurs in rodents after β-islet cell necrosis, hypothyroidism was induced in 16 rats resulting in 100% β-myosin heavy chain expression in the heart. β-Islet cell necrosis was induced in half of the rats by streptozocin administration. After 6 wks of HG, both HG and nonHG controls rats demonstrated similar myofilament performance measured as thin filament calcium sensitivity, native thin filament velocity in the myosin motility assay and contractile velocity and power. Extracellular Zn2+ reduced cardiomyocyte contractile function in both groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application. This is a novel finding implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Among the many post-translational modifications examined, only phosphorylation of ryanodine receptor (RyR at S-2808 was significantly higher in HG compared to nonHG. We did not find in our hypothyroid rats any differentiating effects of HG on myofibrillar protein phosphorylation, lysine acetylation, O-linked N-acetylglucosamine and advanced glycated end-products, which are often implicated as complicating factors in cardiac performance due to HG. Our

  20. Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

    Li, Xiao-Li; Zeng, Di; Chen, Yan; Ding, Lu; Li, Wen-Ju; Wei, Ting; Ou, Dong-Bo; Yan, Song; Wang, Bin; Zheng, Qiang-Sun

    2017-02-01

    Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although β-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on α-AR signalling than β-AR signalling. In addition, selective activation of α 1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α 2 - or β-AR signalling induced no or less differentiation, respectively. EPI- and α 1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. These results demonstrate that activation of ARs, particularly of α 1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling. © 2016 John Wiley & Sons Ltd.

  1. HIV-1 subtype C unproductively infects human cardiomyocytes in vitro and induces apoptosis mitigated by an anti-Gp120 aptamer.

    Lopes de Campos, Walter R; Chirwa, Nthato; London, Grace; Rotherham, Lia S; Morris, Lynn; Mayosi, Bongani M; Khati, Makobetsa

    2014-01-01

    HIV-associated cardiomyopathy (HIVCM) is of clinical concern in developing countries because of a high HIV-1 prevalence, especially subtype C, and limited access to highly active antiretroviral therapy (HAART). For these reasons, we investigated the direct and indirect effects of HIV-1 subtype C infection of cultured human cardiomyocytes and the mechanisms leading to cardiomyocytes damage; as well as a way to mitigate the damage. We evaluated a novel approach to mitigate HIVCM using a previously reported gp120 binding and HIV-1 neutralizing aptamer called UCLA1. We established a cell-based model of HIVCM by infecting human cardiomyocytes with cell-free HIV-1 or co-culturing human cardiomyocytes with HIV-infected monocyte derived macrophages (MDM). We discovered that HIV-1 subtype C unproductively (i.e. its life cycle is arrested after reverse transcription) infects cardiomyocytes. Furthermore, we found that HIV-1 initiates apoptosis of cardiomyocytes through caspase-9 activation, preferentially via the intrinsic or mitochondrial initiated pathway. CXCR4 receptor-using viruses were stronger inducers of apoptosis than CCR5 utilizing variants. Importantly, we discovered that HIV-1 induced apoptosis of cardiomyocytes was mitigated by UCLA1. However, UCLA1 had no protective effective on cardiomyocytes when apoptosis was triggered by HIV-infected MDM. When HIV-1 was treated with UCLA1 prior to infection of MDM, it failed to induce apoptosis of cardiomyocytes. These data suggest that HIV-1 causes a mitochondrial initiated apoptotic cascade, which signal through caspase-9, whereas HIV-1 infected MDM causes apoptosis predominantly via the death-receptor pathway, mediated by caspase-8. Furthermore the data suggest that UCLA1 protects cardiomyocytes from caspase-mediated apoptosis, directly by binding to HIV-1 and indirectly by preventing infection of MDM.

  2. Establishment and optimization of NMR-based cell metabonomics study protocols for neonatal Sprague-Dawley rat cardiomyocytes.

    Zhang, Ming; Sun, Bo; Zhang, Qi; Gao, Rong; Liu, Qiao; Dong, Fangting; Fang, Haiqin; Peng, Shuangqing; Li, Famei; Yan, Xianzhong

    2017-01-15

    A quenching, harvesting, and extraction protocol was optimized for cardiomyocytes NMR metabonomics analysis in this study. Trypsin treatment and direct scraping cells in acetonitrile were compared for sample harvesting. The results showed trypsin treatment cause normalized concentration increasing of phosphocholine and metabolites leakage, since the trypsin-induced membrane broken and long term harvesting procedures. Then the intracellular metabolite extraction efficiency of methanol and acetonitrile were compared. As a result, washing twice with phosphate buffer, direct scraping cells and extracting with acetonitrile were chosen to prepare cardiomyocytes extracts samples for metabonomics studies. This optimized protocol is rapid, effective, and exhibits greater metabolite retention. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Signal transduction in cultered cardiomyocytes : alpha1-adrenergic and endothelin receptor mediated responses

    H.W. de Jonge (Jet)

    1996-01-01

    textabstractAlready in ancient times the Greek were aware of the heart in the human body and they gave it the name kardia, which is still in use in words as cardiac, myocardial, tachycardia and bradycardia. In those times the importance of the heart was appraised by Aristotle (384-322 B.C.), who

  4. Dystrophin is required for the normal function of the cardio-protective K(ATP channel in cardiomyocytes.

    Laura Graciotti

    Full Text Available Duchenne and Becker muscular dystrophy patients often develop a cardiomyopathy for which the pathogenesis is still unknown. We have employed the murine animal model of Duchenne muscular dystrophy (mdx, which develops a cardiomyopathy that includes some characteristics of the human disease, to study the molecular basis of this pathology. Here we show that the mdx mouse heart has defects consistent with alteration in compounds that regulate energy homeostasis including a marked decrease in creatine-phosphate (PC. In addition, the mdx heart is more susceptible to anoxia than controls. Since the cardio-protective ATP sensitive potassium channel (K(ATP complex and PC have been shown to interact we investigated whether deficits in PC levels correlate with other molecular events including K(ATP ion channel complex presence, its functionality and interaction with dystrophin. We found that this channel complex is present in the dystrophic cardiac cell membrane but its ability to sense a drop in the intracellular ATP concentration and consequently open is compromised by the absence of dystrophin. We further demonstrate that the creatine kinase muscle isoform (CKm is displaced from the plasma membrane of the mdx cardiac cells. Considering that CKm is a determinant of K(ATP channel complex function we hypothesize that dystrophin acts as a scaffolding protein organizing the K(ATP channel complex and the enzymes necessary for its correct functioning. Therefore, the lack of proper functioning of the cardio-protective K(ATP system in the mdx cardiomyocytes may be part of the mechanism contributing to development of cardiac disease in dystrophic patients.

  5. ALDH2 Inhibition Potentiates High Glucose Stress-Induced Injury in Cultured Cardiomyocytes

    Guodong Pan

    2016-01-01

    Full Text Available Aldehyde dehydrogenase (ALDH gene superfamily consists of 19 isozymes. They are present in various organs and involved in metabolizing aldehydes that are biologically generated. For instance, ALDH2, a cardiac mitochondrial ALDH isozyme, is known to detoxify 4-hydroxy-2-nonenal, a reactive aldehyde produced upon lipid peroxidation in diabetic conditions. We hypothesized that inhibition of ALDH leads to the accumulation of unmetabolized 4HNE and consequently exacerbates injury in cells subjected to high glucose stress. H9C2 cardiomyocyte cell lines were pretreated with 10 μM disulfiram (DSF, an inhibitor of ALDH2 or vehicle (DMSO for 2 hours, and then subjected to high glucose stress {33 mM D-glucose (HG or 33 mM D-mannitol as an osmotic control (Ctrl} for 24 hrs. The decrease in ALDH2 activity with DSF pretreatment was higher in HG group when compared to Ctrl group. Increased 4HNE adduct formation with DSF pretreatment was higher in HG group compared to Ctrl group. Pretreatment with DSF leads to potentiated HG-induced cell death in cultured H9C2 cardiomyocytes by lowering mitochondrial membrane potential. Our results indicate that ALDH2 activity is important in preventing high glucose induced cellular dysfunction.

  6. Generation of Cardiomyocytes in Pipe-Based Microbioreactor Under Segmented Flow

    Dimitry Spitkovsky

    2016-05-01

    Full Text Available Background/Aims: Embryonic stem (ES cells have got a broad range differentiation potential. The differentiation is initiated via aggregation of non-differentiated ES cells into embryoid body (EB capable of multi-lineage development. However experimental variables present in standard differentiation techniques lead to high EB heterogeneity, affecting development into the cells of desired lineage, and do not support the process automatization and scalability. Methods: Here we present a novel pipe based microbioreactor (PBM setup based on segmented flow, designed for spatial maintenance of temperature, nutrition supply, gas supply and sterility. Results: We verified PBM feasibility for continuous process generating cardiac cells starting from single ES cell suspension followed by EB formation for up to 10 days. The ES cells used in the study were genetically modified for cardiac-specific EGFP expression allowing optical monitoring of cardiomyocytes while EBs remained within PBM for up to 10 days. Efficiency of cardiac cells formation within PBM was similar compared to a standard hanging drop based protocol. Conclusion: Our findings ensure further development of microfluidic bioreactor technology to enable robust cardiomyocytes production for needs of drug screening, tissue engineering and other applications.

  7. Exogenous cathepsin V protein protects human cardiomyocytes HCM from angiotensin Ⅱ-Induced hypertrophy.

    Huang, Kun; Gao, Lu; Yang, Ming; Wang, Jiliang; Wang, Zheng; Wang, Lin; Wang, Guobin; Li, Huili

    2017-08-01

    Angiotensin (Ang) Ⅱ-induced cardiac hypertrophy can deteriorate to heart failure, a leading cause of mortality. Endogenous Cathepsin V (CTSV) has been reported to be cardioprotective against hypertrophy. However, little is known about the effect of exogenous CTSV on cardiac hypertrophy. We used the human cardiomyocytes HCM as a cell model to investigate the effects of exogenous CTSV on Ang Ⅱ-induced cardiac cell hypertrophy. Cell surface area and expression of classical markers of hypertrophy were analyzed. We further explored the mechanism of CTSV cardioprotective by assessing the levels and activities of PI3K/Akt/mTOR and MAPK signaling pathway proteins. We found that pre-treating cardiomyocytes with CTSV could significantly inhibit Ang Ⅱ-induced hypertrophy. The mRNA expression of hypertrophy markers ANP, BNP and β-MHC was obviously elevated in Ang Ⅱ-treated cardiac cells. Whereas, exogenous CTSV effectively halted this elevation. Further study revealed that the protective effects of exogenous CTSV might be mediated by repressing the phosphorylation of proteins in the PI3K/Akt/mTOR and MAPK pathways. Based on our results, we concluded that exogenous CTSV inhibited Ang Ⅱ-induced hypertrophy in HCM cells by inhibiting PI3K/Akt/mTOR. This study provides experimental evidence for the application of CTSV protein for the treatment of cardiac hypertrophy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes.

    Zhang, Xiao-Dong; Coulibaly, Zana A; Chen, Wei Chun; Ledford, Hannah A; Lee, Jeong Han; Sirish, Padmini; Dai, Gu; Jian, Zhong; Chuang, Frank; Brust-Mascher, Ingrid; Yamoah, Ebenezer N; Chen-Izu, Ye; Izu, Leighton T; Chiamvimonvat, Nipavan

    2018-03-16

    Small-conductance Ca 2+ -activated K + (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca 2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca 2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca 2+ microdomains necessary for SK channel activation. SK currents coupled with Ca 2+ influx via L-type Ca 2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca 2+ store, suggesting that LTCCs provide the immediate Ca 2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Ca v 1.2 Ca 2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Ca v 1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca 2+ signaling.

  9. Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes

    Greenberg, Cynthia C.; Connelly, Patricia S.; Daniels, Mathew P.; Horowits, Robert

    2008-01-01

    Krp1, also called sarcosin, is a cardiac and skeletal muscle kelch repeat protein hypothesized to promote the assembly of myofibrils, the contractile organelles of striated muscles, through interaction with N-RAP and actin. To elucidate its role, endogenous Krp1 was studied in primary embryonic mouse cardiomyocytes. While immunofluorescence showed punctate Krp1 distribution throughout the cell, detergent extraction revealed a significant pool of Krp1 associated with cytoskeletal elements. Reduction of Krp1 expression with siRNA resulted in specific inhibition of myofibril accumulation with no effect on cell spreading. Immunostaining analysis and electron microscopy revealed that cardiomyocytes lacking Krp1 contained sarcomeric proteins with longitudinal periodicities similar to mature myofibrils, but fibrils remained thin and separated. These thin myofibrils were degraded by a scission mechanism distinct from the myofibril disassembly pathway observed during cell division in the developing heart. The data are consistent with a model in which Krp1 promotes lateral fusion of adjacent thin fibrils into mature, wide myofibrils and contribute insight into mechanisms of myofibrillogenesis and disassembly

  10. Tampering with springs: phosphorylation of titin affecting the mechanical function of cardiomyocytes.

    Hamdani, Nazha; Herwig, Melissa; Linke, Wolfgang A

    2017-06-01

    Reversible post-translational modifications of various cardiac proteins regulate the mechanical properties of the cardiomyocytes and thus modulate the contractile performance of the heart. The giant protein titin forms a continuous filament network in the sarcomeres of striated muscle cells, where it determines passive tension development and modulates active contraction. These mechanical properties of titin are altered through post-translational modifications, particularly phosphorylation. Titin contains hundreds of potential phosphorylation sites, the functional relevance of which is only beginning to emerge. Here, we provide a state-of-the-art summary of the phosphorylation sites in titin, with a particular focus on the elastic titin spring segment. We discuss how phosphorylation at specific amino acids can reduce or increase the stretch-induced spring force of titin, depending on where the spring region is phosphorylated. We also review which protein kinases phosphorylate titin and how this phosphorylation affects titin-based passive tension in cardiomyocytes. A comprehensive overview is provided of studies that have measured altered titin phosphorylation and titin-based passive tension in myocardial samples from human heart failure patients and animal models of heart disease. As our understanding of the broader implications of phosphorylation in titin progresses, this knowledge could be used to design targeted interventions aimed at reducing pathologically increased titin stiffness in patients with stiff hearts.

  11. Prostanoid Receptors Involved in Regulation of the Beating Rate of Neonatal Rat Cardiomyocytes

    Mechiche, Hakima; Grassin-Delyle, Stanislas; Robinet, Arnaud; Nazeyrollas, Pierre; Devillier, Philippe

    2012-01-01

    Although prostanoids are known to be involved in regulation of the spontaneous beating rate of cultured neonatal rat cardiomyocytes, the various subtypes of prostanoid receptors have not been investigated in detail. In our experiments, prostaglandin (PG)F2α and prostanoid FP receptor agonists (fluprostenol, latanoprost and cloprostenol) produced a decrease in the beating rate. Two prostanoid IP receptor agonists (iloprost and beraprost) induced first a marked drop in the beating rate and then definitive abrogation of beating. In contrast, the prostanoid DP receptor agonists (PGD2 and BW245C) and TP receptor agonists (U-46619) produced increases in the beating rate. Sulprostone (a prostanoid EP1 and EP3 receptor agonist) induced marked increases in the beating rate, which were suppressed by SC-19220 (a selective prostanoid EP1 antagonist). Butaprost (a selective prostanoid EP2 receptor agonist), misoprostol (a prostanoid EP2 and EP3 receptor agonist), 11-deoxy-PGE1 (a prostanoid EP2, EP3 and EP4 receptor agonist) did not alter the beating rate. Our results strongly suggest that prostanoid EP1 receptors are involved in positive regulation of the beating rate. Prostanoid EP1 receptor expression was confirmed by western blotting with a selective antibody. Hence, neonatal rat cardiomyocytes express both prostanoid IP and FP receptors (which negatively regulate the spontaneous beating rate) and prostanoid TP, DP1 and EP1 receptors (which positively regulate the spontaneous beating rate). PMID:22984630

  12. Evidence for the Role of BAG3 in Mitochondrial Quality Control in Cardiomyocytes.

    Tahrir, Farzaneh G; Knezevic, Tijana; Gupta, Manish K; Gordon, Jennifer; Cheung, Joseph Y; Feldman, Arthur M; Khalili, Kamel

    2017-04-01

    Mitochondrial abnormalities impact the development of myofibrillar myopathies. Therefore, understanding the mechanisms underlying the removal of dysfunctional mitochondria from cells is of great importance toward understanding the molecular events involved in the genesis of cardiomyopathy. Earlier studies have ascribed a role for BAG3 in the development of cardiomyopathy in experimental animals leading to the identification of BAG3 mutations in patients with heart failure which may play a part in the onset of disease development and progression. BAG3 is co-chaperone of heat shock protein 70 (HSP70), which has been shown to modulate apoptosis and autophagy, in several cell models. In this study, we explore the potential role of BAG3 in mitochondrial quality control. We demonstrate that siRNA mediated suppression of BAG3 production in neonatal rat ventricular cardiomyocytes (NRVCs) significantly elevates the level of Parkin, a key component of mitophagy. We found that both BAG3 and Parkin are recruited to depolarized mitochondria and promote mitophagy. Suppression of BAG3 in NRVCs significantly reduces autophagy flux and eliminates clearance of Tom20, an essential import receptor for mitochondria proteins, after induction of mitophagy. These observations suggest that BAG3 is critical for the maintenance of mitochondrial homeostasis under stress conditions, and disruptions in BAG3 expression impact cardiomyocyte function. J. Cell. Physiol. 232: 797-805, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Alignment of human cardiomyocytes on laser patterned biphasic core/shell nanowire assemblies

    Kiefer, Karin; Haidar, Ayman; Abdul-Khaliq, Hashim; Lee, Juseok; Martinez Miró, Marina; Kaan Akkan, Cagri; Cenk Aktas, Oral; Veith, Michael

    2014-01-01

    The management of end stage heart failure patients is only possible by heart transplantation or by the implantation of artificial hearts as a bridge for later transplantation. However, these therapeutic strategies are limited by a lack of donor hearts and by the associated complications, such as coagulation and infection, due to the used artificial mechanical circulatory assist devices. Therefore, new strategies for myocardial regenerative approaches are under extensive research to produce contractile myocardial tissue in the future to replace non-contractile myocardial ischemic and scarred tissue. Different approaches, such as cell transplantation, have been studied intensively. Although successful approaches have been observed, there are still limitations to the application. It is envisaged that myocardial tissue engineering can be used to help replace infarcted non-contractile tissue. The developed tissue should later mimic the aligned fibrillar structure of the extracellular matrix and provide important guidance cues for the survival, function and the needed orientation of cardiomyocytes. Nanostructured surfaces have been tested to provide a guided direction that cells can follow. In the present study, the cellular adhesion/alignment of human cardiomyocytes and the biocompatibility have been investigated after cultivation on different laser-patterned nanowires compared with unmodified nanowires. As a result, the nanostructured surfaces possessed good biocompatibility before and after laser modification. The laser-induced scalability of the pattern enabled the growth and orientation of the adhered myocardial tissue. Such approaches may be used to modify the surface of potential scaffolds to develop myocardial contractile tissue in the future. (paper)

  14. High levels of glucose induce "metabolic memory" in cardiomyocyte via epigenetic histone H3 lysine 9 methylation.

    Yu, Xi-Yong; Geng, Yong-Jian; Liang, Jia-Liang; Zhang, Saidan; Lei, He-Ping; Zhong, Shi-Long; Lin, Qiu-Xiong; Shan, Zhi-Xin; Lin, Shu-Guang; Li, Yangxin

    2012-09-01

    Diabetic patients continue to develop inflammation and cardiovascular complication even after achieving glycemic control, suggesting a "metabolic memory". Metabolic memory is a major challenge in the treatment of diabetic complication, and the mechanisms underlying metabolic memory are not clear. Recent studies suggest a link between chromatin histone methylation and metabolic memory. In this study, we tested whether histone 3 lysine-9 tri-methylation (H3K9me3), a key epigenetic chromatin marker, was involved in high glucose (HG)-induced inflammation and metabolic memory. Incubating cardiomyocyte cells in HG resulted in increased levels of inflammatory cytokine IL-6 mRNA when compared with myocytes incubated in normal culture media, whereas mannitol (osmotic control) has no effect. Chromatin immunoprecipitation (ChIP) assays showed that H3K9me3 levels were significantly decreased at the promoters of IL-6. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase, Suv39h1, were also reduced after HG treatment. HG-induced apoptosis, mitochondrial dysfunction and cytochrome-c release were reversible. However, the effects of HG on the expression of IL-6 and the levels of H3K9me3 were irreversible after the removal of HG from the culture. These results suggest that HG-induced sustained inflammatory phenotype and epigenetic histone modification, rather than HG-induced mitochondrial dysfunction and apoptosis, are main mechanisms responsible for metabolic memory. In conclusion, our data demonstrate that HG increases expression of inflammatory cytokine and decreases the levels of histone-3 methylation at the cytokine promoter, and suggest that modulating histone 3 methylation and inflammatory cytokine expression may be a useful strategy to prevent metabolic memory and cardiomyopathy in diabetic patients.

  15. Depletion of Tip60 from In Vivo Cardiomyocytes Increases Myocyte Density, Followed by Cardiac Dysfunction, Myocyte Fallout and Lethality.

    Joseph B Fisher

    Full Text Available Tat-interactive protein 60 (Tip60, encoded by the Kat5 gene, is a member of the MYST family of acetyltransferases. Cancer biology studies have shown that Tip60 induces the DNA damage response, apoptosis, and cell-cycle inhibition. Although Tip60 is expressed in the myocardium, its role in cardiomyocytes (CMs is unclear. Earlier studies here showed that application of cardiac stress to globally targeted Kat5+/-haploinsufficient mice resulted in inhibition of apoptosis and activation of the CM cell-cycle, despite only modest reduction of Tip60 protein levels. It was therefore of interest to ascertain the effects of specifically and substantially depleting Tip60 from CMs using Kat5LoxP/-;Myh6-Cre mice in the absence of stress. We report initial findings using this model, in which the effects of specifically depleting Tip60 protein from ventricular CMs, beginning at early neonatal stages, were assessed in 2-12 week-old mice. Although 5'-bromodeoxyuridine immunostaining indicated that CM proliferation was not altered at any of these stages, CM density was increased in 2 week-old ventricles, which persisted in 4 week-old hearts when TUNEL staining revealed inhibition of apoptosis. By week 4, levels of connexin-43 were depleted, and its patterning was dysmorphic, concomitant with an increase in cardiac hypertrophy marker expression and interstitial fibrosis. This was followed by systolic dysfunction at 8 weeks, after which extensive apoptosis and CM fallout occurred, followed by lethality as mice approached 12 weeks of age. In summary, chronic depletion of Tip60 from the ventricular myocardium beginning at early stages of neonatal heart development causes CM death after 8 weeks; hence, Tip60 protein has a crucial function in the heart.

  16. Divergent effects of adrenaline in human induced pluripotent stem cell-derived cardiomyocytes obtained from hypertrophic cardiomyopathy

    Chandra Prajapati

    2018-02-01

    Full Text Available Hypertrophic cardiomyopathy (HCM is a common inherited cardiac disease that affects the heart muscle with diverse clinical outcomes. HCM can cause sudden cardiac death (SCD during or immediately after mild to rigorous physical activity in young patients. However, the mechanism causing SCD as a result of exercise remains unknown, but exercise-induced ventricular arrhythmias are thought to be responsible for this fatal consequence. To understand the disease mechanism behind HCM in a better way, we generated patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs from HCM patients carrying either the MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. We extensively investigated the effects of low to high concentrations of adrenaline on action potential characteristics, and the occurrence of arrhythmias in the presence of various concentrations of adrenaline and in wash-out condition. We classified and quantified different types of arrhythmias observed in hiPSC-CMs, and found that the occurrence of arrhythmias was dependent on concentrations of adrenaline and positions of mutations in genes causing HCM. In addition, we observed ventricular tachycardia types of arrhythmias in hiPSC-CMs carrying the TPM1-Asp175Asn mutation. We additionally examined the antiarrhythmic potency of bisoprolol in HCM-specific hiPSC-CMs. However, bisoprolol could not reduce the occurrence of arrhythmias during administration or during the wash-out condition of adrenaline in HCM-specific hiPSC-CMs. Our study demonstrates hiPSC-CMs as a promising tool for studying HCM. The experimental design used in this study could be suitable and beneficial for studying other components and drugs related to cardiac disease in general.

  17. Embryonic template-based generation and purification of pluripotent stem cell-derived cardiomyocytes for heart repair

    Dierickx, P.; Doevendans, P.A.; Geijsen, N.; van Laake, L.W.

    2012-01-01

    Cardiovascular disease remains a leading cause of death in Western countries. Many types of cardiovascular diseases are due to a loss of functional cardiomyocytes, which can result in irreversible cardiac failure. Since the adult human heart has limited regenerative potential, cardiac

  18. In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing

    Nicolas, J.A.Y.; Hendriksen, P.J.M.; Haan, de L.H.J.; Koning, R.; Rietjens, I.M.C.M.; Bovee, T.F.H.

    2015-01-01

    The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on

  19. Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

    Desy S. Lee

    2015-09-01

    Full Text Available Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs. We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo, to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

  20. Matrix production and remodeling capacity of cardiomyocyte progenitor cells produce and remodel matrix during in vitro differentiation

    Bax, N.A.M.; Marion, van M.H.; Shah, B.; Goumans, M.J.T.H; Bouten, C.V.C.; Schaft, van der D.W.J.

    2012-01-01

    Cell-based therapy has emerged as a treatment modality for myocardial repair. Especially cardiac resident stem cells are considered a potential cell source since they are able to differentiate into cardiomyocytes and have improved heart function after injury in a preclinical model for myocardial

  1. Bioinspired onion epithelium-like structure promotes the maturation of cardiomyocytes derived from human pluripotent stem cells.

    Xu, Cong; Wang, Li; Yu, Yue; Yin, Fangchao; Zhang, Xiaoqing; Jiang, Lei; Qin, Jianhua

    2017-08-22

    Organized cardiomyocyte alignment is critical to maintain the mechanical properties of the heart. In this study, we present a new and simple strategy to fabricate a biomimetic microchip designed with an onion epithelium-like structure and investigate the guided behavior of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) on the substrate. The hiPSC-CMs were observed to be confined by the three dimensional surficial features morphologically, analogous to the in vivo microenvironment, and exhibited an organized anisotropic alignment on the onion epithelium-like structure with good beating function. The calcium imaging of hiPSC-CMs demonstrated a more mature Ca 2+ spark pattern as well. Furthermore, the expression of sarcomere genes (TNNI3, MYH6 and MYH7), potassium channel genes (KCNE1 and KCNH2), and calcium channel genes (RYR2) was significantly up-regulated on the substrate with an onion epithelium-like structure instead of the surface without the structure, indicating a more matured status of cardiomyocytes induced by this structure. It appears that the biomimetic micropatterned structure, analogous to in vivo cellular organization, is an important factor that might promote the maturation of hiPSC-CMs, providing new biological insights to guide hiPSC-CM maturation by biophysical factors. The established approach may offer an effective in vitro model for investigating cardiomyocyte differentiation, maturation and tissue engineering applications.

  2. miR-31a-5p promotes postnatal cardiomyocyte proliferation by targeting RhoBTB1

    Xiao, Junjie; Liu, Hui; Cretoiu, Dragos; Toader, Daniela Oana; Suciu, Nicolae; Shi, Jing; Shen, Shutong; Bei, Yihua; Sluijter, Joost Pg; Das, Saumya; Kong, Xiangqing; Li, Xinli

    2017-01-01

    A limited number of microRNAs (miRNAs, miRs) have been reported to control postnatal cardiomyocyte proliferation, but their strong regulatory effects suggest a possible therapeutic approach to stimulate regenerative capacity in the diseased myocardium. This study aimed to investigate the miRNAs

  3. Silymarin Component 2,3-dehydrosilybin Attenuates Cardiomyocyte Damage Following Hypoxia/Reoxygenation by Limiting Oxidative Stress

    Gabrielová, E.; Křen, Vladimír; Jabůrek, Martin; Modriansky, M.

    2015-01-01

    Roč. 64, č. 1 (2015), s. 79-91 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP301/11/0662 Institutional support: RVO:61388971 ; RVO:67985823 Keywords : Silymarin * Dehydrosilybin * Neonatal rat cardiomyocytes Subject RIV: ED - Physiology Impact factor: 1.643, year: 2015

  4. MUSCLEMOTION : A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo

    Sala, Luca; van Meer, Berend J; Tertoolen, Leon T; Bakkers, Jeroen; Bellin, Milena; Davis, Richard P; Denning, Chris N; Dieben, Michel A; Eschenhagen, Thomas; Giacomelli, Elisa; Grandela, Catarina; Hansen, Arne; Holman, Eduard; Jongbloed, Monique R; Kamel, Sarah M; Koopman, Charlotte D; Lachaud, Quentin; Mannhardt, Ingra; Mol, Mervyn P; Mosqueira, Diogo; Orlova, Valeria V; Passier, Robert; Ribeiro, Marcelo C; Saleem, Umber; Smith, Godfrey; Burton, Francis L L; Mummery, Christine L

    2017-01-01

    Rationale: There are several methods to measure cardiomyocyte (CM) and muscle contraction but these require customized hardware, expensive apparatus and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across

  5. Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function

    M.J. Birket (Matthew J.); M.C. Ribeiro (Marcelo C.); G. Kosmidis (Georgios); D. Ward (Dorien); A.R. Leitoguinho (Ana Rita); V. van de Pol (Vera); C. Dambrot (Cheryl); H.D. Devalla (Harsha D.); R.P. Davis (Richard P.); P.G. Mastroberardino (Pier); D.E. Atsma (Douwe); R. Passier (Robert); C.L. Mummery (Christine)

    2015-01-01

    textabstractMaximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust

  6. Sequestration of fatty acids in triglycerides prevents endoplasmic reticulum stress in an in vitro model of cardiomyocyte lipotoxicity

    Bosma, M.; Dapito, D.H.; Drosatos-Tampakaki, Z.; Huiping-Son, N.; Huang, L.S.; Kersten, A.H.; Drosatos, K.; Goldberg, I.J.

    2014-01-01

    We used human cardiomyocyte-derived cells to create an in vitro model to study lipid metabolism and explored the effects of PPAR gamma, ACSL1 and ATGL on fatty acid-induced ER stress. Compared to oleate, palmitate treatment resulted in less intracellular accumulation of lipid droplets and more ER

  7. Engineered Biomaterials Control Differentiation and Proliferation of Human-Embryonic-Stem-Cell-Derived Cardiomyocytes via Timed Notch Activation

    Jason C. Tung

    2014-03-01

    Full Text Available For cell-based treatments of myocardial infarction, a better understanding of key developmental signaling pathways and more robust techniques for producing cardiomyocytes are required. Manipulation of Notch signaling has promise as it plays an important role during cardiovascular development, but previous studies presented conflicting results that Notch activation both positively and negatively regulates cardiogenesis. We developed surface- and microparticle-based Notch-signaling biomaterials that function in a time-specific activation-tunable manner, enabling precise investigation of Notch activation at specific developmental stages. Using our technologies, a biphasic effect of Notch activation on cardiac differentiation was found: early activation in undifferentiated human embryonic stem cells (hESCs promotes ectodermal differentiation, activation in specified cardiovascular progenitor cells increases cardiac differentiation. Signaling also induces cardiomyocyte proliferation, and repeated doses of Notch-signaling microparticles further enhance cardiomyocyte population size. These results highlight the diverse effects of Notch activation during cardiac development and provide approaches for generating large quantities of cardiomyocytes.

  8. β3-adrenergic receptor activation induces TGFβ1 expression in cardiomyocytes via the PKG/JNK/c-Jun pathway.

    Xu, Zhongcheng; Wu, Jimin; Xin, Junzhou; Feng, Yenan; Hu, Guomin; Shen, Jing; Li, Mingzhe; Zhang, Youyi; Xiao, Han; Wang, Li