title: Stimulation of cardiomyogenesis from mouse embryonic stem cells by nuclear translocation of cardiotrophin-1.
abstract: BACKGROUND
  Cardiotrophin-1 (CT-1) controls cardiomyogenesis of mouse embryonic stem (ES) cells.
  
  
  OBJECTIVE
  To investigate the signaling pathway underlying the action of CT-1 on cardiac cell differentiation.
  
  
  METHODS
  Protein expression was analyzed by western blot technique and cardiac areas by immunohistochemistry. Calcium, reactive oxygen species (ROS) and nitric oxide (NO) were assessed by microfluorometry using fluo-4, H2DCF, and DAF-2DA, respectively. Gene inactivation of CT-1 was achieved by siRNA technology.
  
  
  RESULTS
  CT-1 as well as its receptor gp 130 were transiently upregulated during differentiation of ES cells. Exogenous CT-1 enhanced cardiomyogenesis, increased the cardiac transcription factors MEF2c, Nkx-2.5, TEAD3 and GATA4, the cardiac proteins α-actinin, MLC2a, MYH7, MLC1a, MLC2v and HCN4 as well as vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), fibroblast growth factor-2 (FGF-2) and atrial natriuretic peptide (ANP). CT-1 downregulation by small interfering RNA (siRNA) inhibited cardiomyogenesis and decreased VEGF, PDGF-BB, FGF-2 and ANP expression. CT-1 raised intracellular calcium which was abolished by the intracellular calcium chelator BAPTA, AM and thapsigargin. Moreover, CT-1 treatment increased ROS, followed by NO generation and NOS3 activation. During ES cell differentiation CT-1 was translocated to the cell nucleus. Exogenous CT-1 induced nuclear translocation of endogenous CT-1, which was inhibited by BAPTA, the NOS inhibitor L-N(G)-Nitroarginine methyl ester (l-NAME), the radical scavenger N-(2-mercaptopropionyl)-glycine (NMPG) as well as the janus kinase 2 (JAK2) inhibitor AG490 and the PI3 kinase (PI3K) inhibitor LY294002.
  
  
  CONCLUSIONS
  Nuclear translocation of CT-1 regulates cardiomyogenesis of ES cells and involves calcium, NO, ROS as well as CT-1 regulated signaling pathways.
authors.affiliation: Department of Physiology, Justus Liebig University Gießen, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Mascheck
authors.affiliation: Department of Physiology, Justus Liebig University Gießen, Germany.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Sharifpanah
authors.affiliation: School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China; Partner State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Suk Ying
authors.initials: SY
authors.lastname: Tsang
authors.affiliation: Clinic of Internal Medicine I, Cardiology Division, Friedrich Schiller University Jena, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Wartenberg
authors.affiliation: Department of Physiology, Justus Liebig University Gießen, Germany. Electronic address: heinrich.sauer@physiologie.med.uni-giessen.de.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sauer
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics*; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D007150:Immunohistochemistry; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D012313:RNA / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D016207:Cytokines; D017382:Reactive Oxygen Species; D012313:RNA; C092073:cardiotrophin 1
keywords: Cardiomyogenesis; Cardiotrophin-1; Embryonic stem cell; Nuclear transport