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 BH4 is 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 BH4 synthesis have shown proof of concept that endothelial BH4 can alter CVD pathogenesis. However, clinical trials of BH4 therapy in vascular disease have been limited by systemic oxidation, highlighting the need to explore the wider roles of BH4 to find novel therapeutic targets. In this study, we aimed to elucidate the effects of BH4 deficiency on mitochondrial function and bioenergetics using targeted knockdown of the BH4 synthetic enzyme, GTP Cyclohydrolase I (GTPCH). Knockdown of GTPCH by >90% led to marked loss of cellular BH4 and 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 BH4 in 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 BH4 in the regulation of mitochondrial redox signalling and bioenergetic metabolism.

The Nitric Oxide System in Peripheral Artery Disease: Connection with Oxidative Stress and Biopterins

“…Treatment of a BH4 deficiency can be accomplished either by BH4 supplementation or with the use of antioxidants. Use of the mitochondrial-targeted antioxidant MitoTEMPO has been shown to increase the BH4/BH2 ratio in vitro [65]. In other studies in PAD patients, certain antioxidants targeting NADPH oxidase, a source of ROS, including propionyl-L-carnitine (PLC) and dark chocolate, have been shown to decrease serum markers of oxidative stress and increase serum NOx, the marker of NO biosynthesis [66,67].…”

ROS Signaling in the Pathogenesis of Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS)

“…It is thought that BH 4 couples L-arginine oxidation to NADPH reduction by preventing the disassociation of the ferrous-dioxygen complex of heme [63], suggesting that BH 4 can be used as pharmacological agent to treat vascular diseases. BH 4 has also been implicated in the maintenance of mitochondrial redox balance [64]. The mechanism by which eNOS becomes uncoupled appears to be through increases in the endogenous NOS uncoupler, asymmetric dimethylarginine (ADMA) and the generation of peroxynitrite [65,66].…”

Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation

“…We have recently determined that the impact of BH4 on cellular redox signalling in endothelial cells and animal models, where abolition of BH4 leads to increased cellular generation of superoxide 16 – 19 . Moreover, we also demonstrated that a proportion of this superoxide is produced directly from uncoupled eNOS, and that BH4 plays an important role in the mitochondria 20 . While these studies show that the molecular and cellular mechanisms through which BH4 exerts its effects on eNOS are now well established, the downstream impact of these regulatory mechanisms on eNOS-dependent signalling, and the identification of the resulting target proteins remain unknown.…”