Increasing tetrahydrobiopterin in cardiomyocytes adversely affects cardiac redox state and mitochondrial function independently of changes in NO production

Abstract

Tetrahydrobiopterin (BH4) represents a potential strategy for the treatment of cardiac remodeling, fibrosis and/or diastolic dysfunction. The effects of oral treatment with BH4 (Sapropterin™ or Kuvan™) are however dose-limiting with high dose negating functional improvements. Cardiomyocyte-specific overexpression of GTP cyclohydrolase I (mGCH) increases BH4 several-fold in the heart. Using this model, we aimed to establish the cardiomyocyte-specific responses to high levels of BH4. Quantification of BH4 and BH2 in mGCH transgenic hearts showed age-based variations in BH4:BH2 ratios. Hearts of mice (<6 months) have lower BH4:BH2 ratios than hearts of older mice while both GTPCH activity and tissue ascorbate levels were higher in hearts of young than older mice. No evident changes in nitric oxide (NO) production assessed by nitrite and endogenous iron-nitrosyl complexes were detected in any of the age groups. Increased BH4 production in cardiomyocytes resulted in a significant loss of mitochondrial function. Diminished oxygen consumption and reserve capacity was verified in mitochondria isolated from hearts of 12-month old compared to 3-month old mice, even though at 12 months an improved BH4:BH2 ratio is established. Accumulation of 4-hydroxynonenal (4-HNE) and decreased glutathione levels were found in the mGCH hearts and isolated mitochondria. Taken together, our results indicate that the ratio of BH4:BH2 does not predict changes in neither NO levels nor cellular redox state in the heart. The BH4 oxidation essentially limits the capacity of cardiomyocytes to reduce oxidant stress. Cardiomyocyte with chronically high levels of BH4 show a significant decline in redox state and mitochondrial function.

Keywords: 7,8-Dihydrobiopterin; Biopterin; Electron paramagnetic resonance; GTP cyclohydrolase I; Neopterin.

Figure 1. Cardiac 6R-tetrahydrobiopterin (BH₄), 7,8-dihydrobiopterin (BH₂) and ascorbate HPLC-ECD analysis in cardiomyocyte-specific transgenic (Tg) hearts overexpressing GTPCH (mGCH)

(A and B) Quantification of BH₄, 7,8-dihydropterin (BH₂) in left ventricular tissue of increasing age. (C) Calculated ratio of BH₄ to BH₂ in left ventricular heart tissue from hearts at different ages. (D) Ratio of reduced ascorbate to BH4 in left ventricular tissue form mGCH animals. Assays of BH₄, BH₂ and ascorbate were performed by HPLC-ECD as described in methods. WT, indicate cardiac tissue from C57Bl6 mice hearts. All concentrations were normalized to protein content and results are presented as mean±S.D (n>5) samples per group. (^#,**)p<0.05.

Figure 2. GTP cyclohydrolase (GTPCH) activity in transgenic hearts and liver

Enzyme activity was measured in tissue homogenates (A) heart (C) liver by analyzing the rate of hydrolysis of GTP to 7,8-dihydroneopterin phosphate as described in methods. The oxidation product neopterin was quantified by HPLC with fluorescence detection. Biopterin, the BH₄ and BH₂ metabolite in tissue homogenates (B) heart and (D) liver was directly measured by analyzing supernatant of tissue homogenates clarified by filtration. All values were normalized by protein content in tissue homogenates. Results are presented as mean±SD (n=4). (**, ^¶) p<0.05, ^{(#, ##})p<0.02.

Figure 3. Nitrite analysis in cardiac tissue from C57Bl6 hearts (wild type) and cardiomyocyte targeted GTPCH transgenic hearts (mGCH)

Samples from cardiac tissue were analyzed using nitric oxide analyzer (NOA) with chemiluminescence detection. Results represent the mean±SD (n=6). No differences were established.

Figure 4. Neurotransmitter levels in cardiac issue of C57Bl6 hearts (wild type) and cardiomyocyte targeted GTPCH transgenic hearts (mGCH)

HPLC-ECD analysis of dopamine, norepinephrine, epinephrine and 5-hydroxytryptamine in cardiac tissue were analyzed as described in materials and methods. Results are expressed as mean±S.D. (n=4). No differences were established.

Figure 5. Low-temperature EPR analysis (40°K) of cardiac tissue form C57Bl6 hearts (wild type) and cardiomyocyte targeted GTPCH transgenic hearts (mGCH)

(A) Standard samples of 5- and 6-coordinated complexes of hemoglobin(Fe-NO). (B) mGCH and wild type cardiac samples: a) mGCH- 4 months; b) mGCH-6 months; c) mGCH-2 months; d) C57Bl6-12 months. Single samples were inserted in quartz EPR tubes and snap frozen in liquid nitrogen as soon as isolated. Instrument setting are as described in methods. (▼) ubiquinone radical; (Fe-S) mitochondrial 2FeS2 centers.

Figure 6. Low-temperature EPR analysis (12°K) of cardiac tissue from C57Bl6 hearts (wild type) and GTPCH transgenic cardiomyocyt

e. (A) Wide field EPR scanning of wild type and mGCH-samples (ages: 2,4, and 6 month) revealed a high spin Fe(III)-porphyrin signal g~6 that shows a higher intensity in young transgenic than wild type hearts (inset: zoom of g~6 signal) (B) EPR signal of cardiac mitochondrial complexes showing non-significant variations between transgenic than wild type tissue. EPR spectra display the sum of the various redox centers of protein complexes such as: (◊) cI-N4 4Fe4S, (○) cI-N2 4Fe4S, (▼) aconitase 3Fe4S, cI-N1b 2Fe2S, cII-S1 2Fe2S, cII-S3 3Fe4S, (◘) ubiquinone radical, (●) cI-N1b 2Fe2S, cII-S1 2Fe2S, (◘) cII-S1 2Fe2S, cI-N1b 2Fe2S, (◊)cI-N4 4Fe4S, Rieske 2Fe2S, (■) cI-N3 4Fe4S.

Figure 7. Bioenergetics characterization of isolated cardiac mitochondria from C57Bl6 (WT) and cardiomyocyte-targeted GTPCH transgenic hearts (Tg)

(A) Isolated mitochondria (3 μg) attached to the 96 well plate (Seahorse) used for analysis; (B) Mitochondrial oxygen consumption of 3-month old WT-mitochondria (red traces), MGCH-Tg (green traces) before (State 2) or after stimulation state-3 respiration with ADP (1 mM), and followed by inhibition of ATP-dependent oxygen consumption with complex V inhibitor oligomycin (2.5 μg/ml). Maximal oxygen consumption capacity was assessed by addition of mitochondrial uncoupler FCCP (0.75 μM) (state 3 uncoupled) and complete inhibition of oxygen consumption was tested with antimycin A (4 μM), (lower panel); (C) as B but mitochondria were isolated from 12-month old mice. (D) Quantification of State 3 respiration stimulated by ADP in mitochondria from wild type and Tg hearts 3-months and 12-moths old; (E) same as D except the state 3 uncoupled stimulated by FCCP. Results are represented as mean±S.D of triplicate experiments. (***) p<0.05.

Figure 8. Accumulation of reactive lipid species and reduced glutathione depletion in GTPCH transgenic hearts

(A) Low levels of positive staining for 4-HNE in cardiomyocytes of young wild type and GTPCH transgenic hearts (Top panels A–B); 4-HNE positive staining is higher in old GTPCH transgenic than wild type hearts (Bottom panels C–D); (B) Analysis of 4-HNE immunostaining score in wild type (WT) young (3 months) and old (12 months) and age-matched GTPCH transgenic (Tg) hearts; results are representative of at least 3 different samples. (***) p˂0.05 and (***(***) p<0.01. (C) western blotting of 4-HNE-modified proteins from isolated mitochondria from 2-month old and 4-month old hearts; (D) cardiac levels of reduced glutathione in C57Bl6 (wild type, WT) and GTPCH transgenic (Tg) hearts analyzed directly by HPLC-ECD. Results are the mean±S.D (n=3 each group age); (**) p<0.05.