title: NADPH oxidase and eNOS control cardiomyogenesis in mouse embryonic stem cells on ascorbic acid treatment.
abstract: Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1(+)) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4-7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-ß and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis.
authors.affiliation: Department of Internal Medicine I, Cardiology Division, Friedrich Schiller University, Jena, Germany.
authors.forename: Caroline
authors.initials: C
authors.lastname: Bartsch
authors.forename: Mohamed M
authors.initials: MM
authors.lastname: Bekhite
authors.forename: Anne
authors.initials: A
authors.lastname: Wolheim
authors.forename: Madeleine
authors.initials: M
authors.lastname: Richter
authors.forename: Carola
authors.initials: C
authors.lastname: Ruhe
authors.forename: Bianka
authors.initials: B
authors.lastname: Wissuwa
authors.forename: Anja
authors.initials: A
authors.lastname: Marciniak
authors.forename: Jörg
authors.initials: J
authors.lastname: Müller
authors.forename: Regine
authors.initials: R
authors.lastname: Heller
authors.forename: Hans-Reiner
authors.initials: HR
authors.lastname: Figulla
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sauer
authors.forename: Maria
authors.initials: M
authors.lastname: Wartenberg
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D002454:Cell Differentiation; D019070:Cell Lineage; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D019255:NADPH Oxidases / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D017382:Reactive Oxygen Species; D052250:Nitric Oxide Synthase Type III; D019255:NADPH Oxidases; D001205:Ascorbic Acid