A novel role for endothelial tetrahydrobiopterin in mitochondrial redox balance.

The redox co-factor tetrahydrobiopterin (BH) 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 BH is observed in cardiovascular disease (CVD) states and results in decreased NO and increased superoxide (O) generation via eNOS uncoupling. Genetic mouse models of augmented endothelial BH synthesis have shown proof of concept that endothelial BH can alter CVD pathogenesis. However, clinical trials of BH therapy in vascular disease have been limited by systemic oxidation, highlighting the need to explore the wider roles of BH to find novel therapeutic targets. In this study, we aimed to elucidate the effects of BH deficiency on mitochondrial function and bioenergetics using targeted knockdown of the BH synthetic enzyme, GTP Cyclohydrolase I (GTPCH). Knockdown of GTPCH by >90% led to marked loss of cellular BH and a striking induction of O generation in the mitochondria of murine endothelial cells. This effect was likewise observed in BH-depleted fibroblasts devoid of NOS, indicating a novel NOS-independent role for BH in mitochondrial redox signalling. Moreover, this BH-dependent, mitochondria-derived ROS further oxidised mitochondrial BH, 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 BH in the regulation of mitochondrial redox signalling and bioenergetic metabolism.