title: A novel role for endothelial tetrahydrobiopterin in mitochondrial redox balance.
abstract: The redox co-factor tetrahydrobiopterin (BH4) regulates nitric oxide (NO) and reactive oxygen species (ROS) production by endothelial NOS (eNOS) and is an important redox-dependent signalling molecule in the endothelium. Loss of endothelial BH4is observed in cardiovascular disease (CVD) states and results in decreased NO and increased superoxide (O2-) generation via eNOS uncoupling. Genetic mouse models of augmented endothelial BH4synthesis have shown proof of concept that endothelial BH4can alter CVD pathogenesis. However, clinical trials of BH4therapy in vascular disease have been limited by systemic oxidation, highlighting the need to explore the wider roles of BH4to find novel therapeutic targets. In this study, we aimed to elucidate the effects of BH4deficiency on mitochondrial function and bioenergetics using targeted knockdown of the BH4synthetic enzyme, GTP Cyclohydrolase I (GTPCH). Knockdown of GTPCH by >90% led to marked loss of cellular BH4and a striking induction of O2-generation in the mitochondria of murine endothelial cells. This effect was likewise observed in BH4-depleted fibroblasts devoid of NOS, indicating a novel NOS-independent role for BH4in mitochondrial redox signalling. Moreover, this BH4-dependent, mitochondria-derived ROS further oxidised mitochondrial BH4, concomitant with changes in the thioredoxin and glutathione antioxidant pathways. These changes were accompanied by a modest increase in mitochondrial size, mildly attenuated basal respiratory function, and marked changes in the mitochondrial proteome and cellular metabolome, including the accumulation of the TCA intermediate succinate. Taken together, these data reveal a novel NOS-independent role for BH4in the regulation of mitochondrial redox signalling and bioenergetic metabolism.
authors.affiliation: BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, United Kingdom.
authors.forename: Jade
authors.initials: J
authors.lastname: Bailey
authors.affiliation: BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Shaw
authors.affiliation: Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, United Kingdom.
authors.forename: Roman
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Oxford Parkinson's Disease Centre, Department of Physiology, Anatomy and Genetics, South Parks Road, Oxford OX1 3QX, United Kingdom.
authors.forename: Brent J
authors.initials: BJ
authors.lastname: Ryan
authors.affiliation: Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, United Kingdom.
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Kessler
authors.affiliation: Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: McCullagh
authors.affiliation: Oxford Parkinson's Disease Centre, Department of Physiology, Anatomy and Genetics, South Parks Road, Oxford OX1 3QX, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Wade-Martins
authors.affiliation: BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, United Kingdom. Electronic address: keith.channon@cardiov.ox.ac.uk.
authors.forename: Keith M
authors.initials: KM
authors.lastname: Channon
authors.affiliation: BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, United Kingdom.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Crabtree
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D002318:Cardiovascular Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004727:Endothelium / Q000378:metabolism / Q000473:pathology; D004734:Energy Metabolism / Q000235:genetics*; D006136:GTP Cyclohydrolase / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D001708:Biopterins; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D006136:GTP Cyclohydrolase; C003402:sapropterin
keywords: Mitochondria; Nitric oxide synthase; Redox state; Superoxide; Tetrahydrobiopterin