A novel role for endothelial tetrahydrobiopterin in mitochondrial redox balance

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 decreasedNO 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

Tetrahydrobiopterin augments endothelium-dependent dilatation in sedentary but not in habitually exercising older adults

Endothelium-dependent dilatation (EDD) is impaired with ageing in sedentary, but not in regularly exercising adults. We tested the hypotheses that differences in tetrahydrobiopterin (BH(4)) bioactivity are key mechanisms explaining the impairment in EDD with sedentary ageing, and the maintenance of EDD with ageing in regularly exercising adults. Brachial artery flow-mediated dilatation (FMD), normalized for local shear stress, was measured after acute oral placebo or BH(4) in young sedentary (YS) (n = 10; 22 ± 1 years, mean ± s.e.m.), older sedentary (OS) (n = 9; 62 ± 2), and older habitually aerobically trained (OT) (n = 12; 66 ± 1) healthy men. At baseline, FMD was ∼50% lower in OS versus YS (1.12 ± 0.09 versus 0.57 ± 0.09 (Δmm (dyn cm(−2))) × 10(−2), P < 0.001; 1 dyn = 10(−5) N), but was preserved in OT (0.93 ± 0.08 (Δmm (dyn cm(−2))) × 10(−2)). BH(4) administration improved FMD by ∼45% in OS (1.00 ± 0.10 (Δmm (dyn cm(−2))) × 10(−2), P < 0.01 versus baseline), but did not affect FMD in YS or OT. Endothelium-independent dilatation neither differed between groups at baseline nor changed with BH(4) administration. These results suggest that BH(4) bioactivity may be a key mechanism involved in the impairment of conduit artery EDD with sedentary ageing, and the EDD-preserving effect of habitual exercise