title: Mitochondrial reactive oxygen species mediate cardiomyocyte formation from embryonic stem cells in high glucose.
abstract: Accumulating evidence points to reactive oxygen species (ROS) as important signaling molecules for cardiomyocyte differentiation in embryonic stem (ES) cells. Given that ES cells are normally maintained and differentiated in medium containing supraphysiological levels of glucose (25 mM), a condition which is known to result in enhanced cellular ROS formation, we questioned whether this high glucose concentration was necessary for cardiomyocyte lineage potential. We show here that ES cells cultured in physiological glucose (5 mM), maintained their general stemness qualities but displayed an altered mitochondrial metabolism, which resulted in decreased ROS production. Furthermore, ES and induced pluripotent stem (iPS) cells differentiated in lower glucose concentrations failed to generate cardiomyocyte structures; an effect mimicked with antioxidant treatments using catalase, N-acetyl cysteine and mitoubiquinone, under high glucose conditions in ES cells. Molecular analysis revealed that ES cells differentiated in 5 mM glucose had reduced expression of the pro-cardiac NOX4 gene and diminished phosphorylation of p38 mitogen-activated protein kinase (MAPK), together with specific changes in the cardiac transcriptional network. These outcomes could be reversed by supplementation of low glucose cultures with ascorbic acid, paradoxically acting as a pro-oxidant. Furthermore, forced expression of an upstream p38 MAPK kinase (MKK6) could bypass the requirement for ROS during differentiation to cardiomyocytes under low glucose conditions, illustrating a key role for p38 in the cardiac differentiation program. Together these data demonstrate that endogenous ROS control is important for cardiomyocyte formation from ES cells, and furthermore that supraphysiological glucose, by supplying ROS, is absolutely required.
authors.affiliation: Laboratory of Cellular Signaling, Department of Regenerative Cardiology, Centro Nacional de Investigaciones Cardiovasculares, (CNIC) Carlos III, Madrid, Spain.
authors.forename: Francisco Luna
authors.initials: FL
authors.lastname: Crespo
authors.forename: Veronica R
authors.initials: VR
authors.lastname: Sobrado
authors.forename: Laura
authors.initials: L
authors.lastname: Gomez
authors.forename: Ana M
authors.initials: AM
authors.lastname: Cervera
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: McCreath
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D005947:Glucose / Q000494:pharmacology*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D048051:p38 Mitogen-Activated Protein Kinases; D005947:Glucose