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.

Keywords: Mitochondria; Nitric oxide synthase; Redox state; Superoxide; Tetrahydrobiopterin.

Fig. 1

Model of endothelial BH₄deficiency through knock down of GTPCH leads to increased mitochondrial superoxide production. sEnd.1 murine endothelial cells were transfected with an siRNA pool targeted to Gch1 (siRNA) or a non-targeting (NS) scrambled control siRNA. The cells were then harvested and analysed for GTPCH protein expression by Western blotting, or biopterin levels using HPLC with electrochemical and fluorescent detection. A) Western blot analysis shows that cellular GTPCH protein was diminished by over 90% following exposure to Gch1-specific siRNA, compared to untreated cells, non-specific (NS) siRNA, and DNA only transfected control cells. B) Gch1-specific siRNA significantly decreased the detectable levels of cellular biopterins and C) the ratio of BH₄ to BH₂ + B, and D) increased the proportion of oxidised biopterins compared with NS siRNA controls. E) Gch1 siRNA lead to significantly decreased NOS activity and F) NOx accumulation using a NO Analyser. n =3 (*, p<0.05) (**, p<0.01). A combination of methods was used to elucidate the source of superoxide induced by modified redox state in response to BH₄ deficiency. G) sEnd.1 endothelial cells were transfected with Gch1-specific and control siRNA, incubated for 72 h and then exposed to MitoSOX™ Red, MitoTracker® Green and/or Hoechst nuclear stain or dihydroethidium (DHE). Confocal microscopy shows an increase in mitochondria-derived superoxide from BH₄-deficient cells exhibited by increased MitoSOX™ fluorescence and MitoTracker® Green co-localisation. H) Quantitative measurements were obtained following exposure of these GCH-specific and NS control cells to dihydroethidium. The specific product, 2-hydroxyethidium, was measured by HPLC as outlined in the ‘Experimental Procedures’; increased accumulation of 2-hydroyethidium, indicative of superoxide formation, was observed in cells with diminished levels of BH₄. This signal was attenuated by pre-treatment with MitoTEMPO. I) MitoTEMPO also partially restores the attenuated BH₄:BH₂+B ratio following transfection of Gch1-specific siRNA. n =4 (*, p<0.05) (**, p<0.01) (†, p<0.05).

Fig. 2

Elevated superoxide production is attenuated by rotenone, or supplementation with sepiapterin. Gch1-specific siRNA and NS treated control were incubated with DHE, and the accumulation of 2-hydoxyethidium was measured by HPLC with fluorescent detection. A) Pre-treatment of cells with L-NAME (200 µmol/litre), apocynin (100 µmol/litre), oxypurinol (100 µmol/litre), rotenone (2 µmol/litre) or TTFA (5 µmol/litre) reveals a significant attenuation of superoxide in BH4 deficient Gch1-siRNA cells treated with L-NAME (*p<0.05) or rotenone (***, p<0.001), compared to control cells. (Inset: i, L-NAME and ii, Rotenone inhibitable fractions) B) Similarly, GCH-tet cells demonstrate decreased 2-hydroxyethidium accumulation compares to TET cells that do not express GCH (***p<0.001). The increase in superoxide levels in GCH-tet cells following 10 days exposure to doxycycline is also significantly reduced by rotenone treatment (***p<0.001). C and D) Treatment of Gch1-siRNA and GCH-tet cells with sepiapterin restored BH₄ levels to at least basal levels, and E and F) abolished the superoxide production that was elevated by BH₄ depletion. n =4/6 (*, p<0.05) (***, p<0.001) (†, p<0.01).

Fig. 3

Cellular BH₄deficiency alters mitochondrial biopterin balance. A) Enriched/isolated fractions of mitochondria were obtained from sEnd.1 cells as outlined in ‘Experimental Procedures’, and their purity confirmed by Western blot analysis using anti-SDH, -VDAC, -cytochrome C, and -GAPDH antibodies. Importantly, these proteins were observed in the mitochondrial fraction, and GAPDH in the cytosolic-, without contamination of the mitochondrial fraction. B) Mitochondrial biopterin levels were determined using HPLC with electrochemical and fluorescent detectors as previously. BH₄, BH₂ and biopterin levels were all significantly attenuated in mitochondria isolated from BH₄ deficient vs. NS control cells, together with C) a lower BH₄:BH₂+B ratio, and D) a higher percentage of oxidised biopterins (BH₂+B). n =3 (*, p<0.05).

Fig. 4

Altered mitochondrial redox state induces changes in mitochondrial structure and function. A and B) The observed changes in mitochondrial redox state also altered mitochondrial bioenergetics, as shown by the significant decrease in basal oxygen consumption rates (OCR) in Gch1-specific siRNA transfected cells compared to their NS treated control cells. OCR was measured using an XFe96 extracellular flux assay. OCR measurements were taken at baseline and then following sequential injections of oligomycin, FCCP and rotenone+antimycin A OCR was significantly decrease in siRNA-treated (red) cells at baseline (vs control, blue). Analysis of electron micrographs from C) NS control cells, and D) Gch1 siRNA transfected cells reveals that cellular mitochondria exhibit E) a trend towards increased area, F) and a significant increase in length, G) independent of a difference in absolute number (averages from NS =11 cells, Gch1 =12 cells). H) Evidence for increased mitochondrial size and mitochondrial fusion was supported by Western blot analysis; TOM20 and COXVI increased in Gch1 siRNA cells compared to NS control cells. I-L) Further Western blotting demonstrates a striking decrease in p(616)Drp1, but not p(637)Drp1, with no difference in mitofusin 2 or OPA1. M and N) No change in the quantity of mtDNA was observed between GCH siRNA and control cells. All data represents 6–8 independent experiments (*, p<0.05). n =4–5 for Western blots.

Fig. 5

Modulation of the mitochondrial proteome by BH₄. Gch1 siRNA and control cells underwent proteomic analysis by mass spectrometry. A) Normalised relative abundance of detected proteins was determined as described in the ‘Experimental Procedures’ (data shown in Supplemental Tables 1 and 2), and significant changes in 46 proteins presented in the heat map. Data presented represent 46 candidate proteins, changed by over 1.5-fold and 1% false discovery rate. Proteins were ranked by fold difference between Gch1 siRNA and NS expressing cells. Higher abundant proteins are expressed in Red, and those of lower abundance, compared to the average of all values, are shown in Blue. B) Ingenuity Pathway Analysis was used to highlight which pathways where experimentally modulated by BH₄ in our cell model. Thioredoxin (downregulated) and glutathione (upregulated) antioxidant systems were found to be down altered in BH₄ deficient cells, as well as several proteins from the ‘Oxidative Phosphorylation’ pathway identified by IPA. C) These proteomic data were confirmed by Western blot analysis, and those ‘hits’ shown to be modified in abundance to the largest extent were validated using specific antibodies. TXNIP, TRX and FH were shown to be decreased upon knockdown of Gch1, and GR and VDAC were demonstrated to be significantly elevated. D) Densitometric analysis of Western blot bands. E) and F) Exposure of NS or Gch1-siRNA treated endothelial cells to either MitoTEMPO or sepiapterin was sufficient to restore the decreased levels of a candidate protein, TRX, induced by BH₄ deficiency. n =3 (*, P<0.05).

Fig. 6

Metabolomic analysis reveals a role for BH₄in metabolism. sEnd.1 endothelial cells were transfected with Gch1-specific siRNA and compared to cells transfected with NS scrambled control siRNA. Cell lysates were harvested and underwent metabolomics analysis as outlined in the ‘Experimental Procedures’. A) Glycolysis (red) and TCA cycle (blue) metabolites were detected and quantification established by comparison with known standards. Succinate, fumarate, phosphoenolpyruvate and Fructose 1-,6-bisphosphate were significantly increased in response to Gch1-specific siRNA transfection, while isocitrate levels were sown to be markedly diminished compared to NS control cells. B) The impact of these changes in mitochondrial electron transport chain protein expression, glycolytic and TCA cycle metabolites, was to significantly decrease ATP/ADP ratio induced following knockdown of Gch1 by targeted siRNA, and the resulting BH₄ deficiency. n =3 (*, p<0.05) (**, p<0.01) (***, p<0.001).

Fig. 7

Schematic representation of the interaction and crosstalk between BH4, ROS generation and mitochondrial signalling. A feed forward cascade of ROS generation from the mitochondria and BH4 oxidation results in deleterious effects on mitochondrial metabolism, protein signalling and function.