Increasing Tetrahydrobiopterin in Cardiomyocytes Adversely Affects Cardiac Redox State and Mitochondrial Function Independently of Changes in NO Production

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 (\u3c6 \u3emonths) 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

Tetrahydrobiopterin in antenatal brain hypoxia-ischemia-induced motor impairments and cerebral palsy

Antenatal brain hypoxia-ischemia, which occurs in cerebral palsy, is considered a significant cause of motor impairments in children. The mechanisms by which antenatal hypoxia-ischemia causes brain injury and motor deficits still need to be elucidated. Tetrahydrobiopterin is an important enzyme cofactor that is necessary to produce neurotransmitters and to maintain the redox status of the brain. A genetic deficiency of this cofactor from mutations of biosynthetic or recycling enzymes is a well-recognized factor in the development of childhood neurological disorders characterized by motor impairments, developmental delay, and encephalopathy. Experimental hypoxia-ischemia causes a decline in the availability of tetrahydrobiopterin in the immature brain. This decline coincides with the loss of brain function, suggesting this occurrence contributes to neuronal dysfunction and motor impairments. One possible mechanism linking tetrahydrobiopterin deficiency, hypoxia-ischemia, and neuronal injury is oxidative injury. Evidence of the central role of the developmental biology of tetrahydrobiopterin in response to hypoxic ischemic brain injury, especially the development of motor deficits, is discussed

Abstract 17098: Cardiac overexpression of GTP cyclohydrolase 1 attenuates cardiac remodeling after myocardial infarction by a neuronal nitric oxide synthase-mediated mechanism

Introduction: Cardiac remodeling after myocardial infarction is associated with dysregulation of nitric oxide synthase and sarcoplasmic reticulum Ca2+ handling proteins. Emerging evidence suggests that GTP cyclohydrolase 1 (GCH1) regulates the function and expression of nitric oxide synthase and sarcoplasmic reticulum Ca2+ handling proteins. Hypothesis: We hypothesized that cardiac overexpression of GCH1 will benefit postinfarct cardiac remodeling. Methods: Myocardial infarction was produced in transgenic mice with cardiomyocyte-specific overexpression of human GCH1 and in control C57BL/6 mice by ligating the left coronary artery. Sham control mice underwent the same procedures except coronary artery ligation. The left ventricular geometry and function were measured with echocardiography, in Masson’s trichrome-stained heart sections, or in isolated Langendorff-perfused hearts. The expression of GCH1, dimeric and monomeric nitric oxide synthases, sarcoplasmic reticulum Ca2+ handling proteins were determined by Western blot analyses. Results: Compared with sham-operated mice, C57BL/6 mice undergoing coronary artery ligation showed significant decreases in cardiac GCH1 proteins (GCH1/GAPDH: 0.14±0.04 in infarction vs 0.50±0.08 in sham, n=6 mice/group, P\u3c0.05), anterior wall thickness at end-diastole (0.67±0.04 mm vs. 0.92±0.07 mm, n=8-12 mice/group, P\u3c0.05) and at end-systole, fractional shortening, +dP/dt, and the ratios of dimers/monomers of neuronal nitric oxide synthase, ryanodine receptors/GAPDH, and sarcoplasmic reticulum Ca2+ ATPase/GAPDH; and significant increases in infarct size, left ventricular internal diameters, and interstitial fibrosis 4 weeks after surgery. These adverse changes in the left ventricle were significantly attenuated in GCH1 overexpressing mice. Treatment of GCH1 overexpressing mice with 7-nitroindazole (an inhibitor for neuronal nitric oxide synthase) for 4 weeks blocked the beneficial effects of GCH1 overexpression on the heart after myocardial infarction. Conclusions: Cardiac overexpression of GCH1 ameliorates postinfarct cardiac remodeling by elevating the dimerization of neuronal nitric oxide synthase and expression of sarcoplasmic reticulum Ca2+ handling proteins

Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications

International audienceMitochondria are recognized as one of the most important targets for new drug design in cancer, cardiovascular, and neurological diseases. Currently, the most effective way to deliver drugs specifically to mitochondria is by covalent linking a lipophilic cation such as an alkyltriphenylphosphonium moiety to a pharmacophore of interest. Other delocalized lipophilic cations, such as rhodamine, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles, have, also been used for mitochondrial delivery of small molecules. Depending on the approach used, and the cell and mitochondrial membrane potentials, more than 1000-fold higher mitochondrial concentration can be achieved. Mitochondrial targeting has been developed to study mitochondrial physiology and dysfunction and the interaction between mitochondria and other subcellular organelles and for treatment of a variety of diseases such as neurodegeneration and cancer. In this Review, we discuss efforts to target small-molecule compounds to mitochondria for probing mitochondria function, as. diagnostic tools and potential therapeutics. We describe the physicochemical basis for mitochondrial accumulation of lipophilic cations, synthetic chemistry strategies to target compounds to mitochondria, mitochondrial probes, and sensors, and examples of mitochondrial targeting of bioactive compounds. Finally, we review published attempts to apply mitochondria-targeted agents for the treatment of cancer and neurodegenerative diseases

Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium

Small GTPase Rap1 plays a prominent role in endothelial cell (EC) homeostasis by promoting NO release. Endothelial deletion of the two highly homologous Rap1 isoforms, Rap1A and Rap1B, leads to endothelial dysfunction ex vivo and hypertension in vivo. Mechanistically, we showed that Rap1B promotes NO release in response to shear flow by promoting mechanosensing complex formation involving VEGFR2 and Akt activation. However, the specific contribution of the Rap1A isoform to NO release and the underlying molecular mechanisms through which the two Rap1 isoforms control endothelial function are unknown. Here, we demonstrate that endothelial dysfunction resulting from knockout of both Rap1A and Rap1B isoforms is ameliorated by exogenous L-Arg administration to rescue NO-dependent vasorelaxation and blood pressure. We confirmed that Rap1B is rapidly activated in response to agonists that trigger eNOS activation, and its deletion in ECs attenuates eNOS activation, as detected by decreased Ser1177 phosphorylation. Somewhat surprising was the finding that EC deletion of Rap1A does not lead to impaired agonist-induced vasorelaxation ex vivo. Mechanistically, the deletion of Rap1A led to elevated eNOS phosphorylation both at the inhibitory, T495, and the activating Ser1177 residues. These findings indicate that the two Rap1 isoforms act via distinct signaling pathways: while Rap1B directly positively regulates eNOS activation, Rap1A prevents negative regulation of eNOS. Notably, the combined deficiency of Rap1A and Rap1B has a severe effect on eNOS activity and NO release with an in vivo impact on endothelial function and vascular homeostasis

Treatment of Cells and Tissues with Chromate Maximizes Mitochondrial 2Fe2S EPR Signals

In a previous study on chromate toxicity, an increase in the 2Fe2S electron paramagnetic resonance (EPR) signal from mitochondria was found upon addition of chromate to human bronchial epithelial cells and bovine airway tissue ex vivo. This study was undertaken to show that a chromate-induced increase in the 2Fe2S EPR signal is a general phenomenon that can be used as a low-temperature EPR method to determine the maximum concentration of 2Fe2S centers in mitochondria. First, the low-temperature EPR method to determine the concentration of 2Fe2S clusters in cells and tissues is fully developed for other cells and tissues. The EPR signal for the 2Fe2S clusters N1b in Complex I and/or S1 in Complex II and the 2Fe2S cluster in xanthine oxidoreductase in rat liver tissue do not change in intensity because these clusters are already reduced; however, the EPR signals for N2, the terminal cluster in Complex I, and N4, the cluster preceding the terminal cluster, decrease upon adding chromate. More surprising to us, the EPR signals for N3, the cluster preceding the 2Fe2S cluster in Complex I, also decrease upon adding chromate. Moreover, this method is used to obtain the concentration of the 2Fe2S clusters in white blood cells where the 2Fe2S signal is mostly oxidized before treatment with chromate and becomes reduced and EPR detectable after treatment with chromate. The increase of the g = 1.94 2Fe2S EPR signal upon the addition of chromate can thus be used to obtain the relative steady-state concentration of the 2Fe2S clusters and steady-state concentration of Complex I and/or Complex II in mitochondria

Detection and Characterization of Reactive Oxygen and Nitrogen Species in Biological Systems by Monitoring Species-Specific Products

WOS:000417088100001Significance: Since the discovery of the superoxide dismutase enzyme, the generation and fate of short-lived oxidizing, nitrosating, nitrating, and halogenating species in biological systems has been of great interest. Despite the significance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in numerous diseases and intracellular signaling, the rigorous detection of ROS and RNS has remained a challenge. Recent Advances: Chemical characterization of the reactions of selected ROS and RNS with electron paramagnetic resonance (EPR) spin traps and fluorescent probes led to the establishment of species-specific products, which can be used for specific detection of several forms of ROS and RNS in cell-free systems and in cultured cells in vitro and in animals in vivo. Profiling oxidation products from the ROS and RNS probes provides a rigorous method for detection of those species in biological systems. Critical Issues: Formation and detection of species-specific products from the probes enables accurate characterization of the oxidative environment in cells. Measurement of the total signal (fluorescence, chemiluminescence, etc.) intensity does not allow for identification of the ROS/RNS formed. It is critical to identify the products formed by using chromatographic or other rigorous techniques. Product analyses should be accompanied by monitoring of the intracellular probe level, another factor controlling the yield of the product(s) formed. Future Directions: More work is required to characterize the chemical reactivity of the ROS/RNS probes, and to develop new probes/detection approaches enabling real-time, selective monitoring of the specific products formed from the probes