A colorimetric assay method for measuring d-glutamate cyclase activity.

In mammals, metabolism of free d-glutamate is regulated by d-glutamate cyclase (DGLUCY), which reversibly converts d-glutamate to 5-oxo-d-proline and H2O. Metabolism of these d-amino acids by DGLUCY is thought to regulate cardiac function. In this study, we established a simple, accurate, and sensitive colorimetric assay method for measuring DGLUCY activity. To this end, we optimized experimental procedures for derivatizing 5-oxo-d-proline with 2-nitrophenylhydrazine hydrochloride. 5-Oxo-d-proline was derivatized with 2-nitrophenylhydrazine hydrochloride in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a catalyst to generate the acid hydrazides, whose levels were then determined using a colorimetric method. Under optimized conditions, we examined the sensitivity and accuracy of the colorimetric method and compared our technique with other methods by high-performance liquid chromatography with ultraviolet-visible or fluorescence detection. Moreover, we assessed the suitability of this colorimetric method for measuring DGLUCY activity in biological samples. Our colorimetric method could determine DGLUCY activity with adequate validity and reliability. This method will help to elucidate the relationship among DGLUCY activity, the physiological and pathological roles of d-glutamate and 5-oxo-d-proline, and cardiac function.

Ablation of cardiac TIGAR preserves myocardial energetics and cardiac function in the pressure overload heart failure model.

Despite the advances in medical therapy, the morbidity and mortality of heart failure (HF) remain unacceptably high. HF results from reduced metabolism-contraction coupling efficiency, so the modulation of cardiac metabolism may be an effective strategy for therapeutic interventions. Tumor suppressor p53 (TP53) and its downstream target TP53-induced glycolysis and apoptosis regulator (TIGAR) are known to modulate cardiac metabolism and cell fate. To investigate TIGAR's function in HF, we compared myocardial, metabolic, and functional outcomes between TIGAR knockout (TIGAR-/-) mice and wild-type (TIGAR+/+) mice subjected to chronic thoracic transverse aortic constriction (TAC), a pressure-overload HF model. In wild-type mice hearts, p53 and TIGAR increased markedly during HF development. Eight weeks after TAC surgery, the left ventricular (LV) dysfunction, fibrosis, oxidative damage, and myocyte apoptosis were significantly advanced in wild-type than in TIGAR-/- mouse heart. Further, myocardial high-energy phosphates in wild-type hearts were significantly decreased compared with those of TIGAR-/- mouse heart. Glucose oxidation and glycolysis rates were also reduced in isolated perfused wild-type hearts following TAC than those in TIGAR-/- hearts, which suggest that the upregulation of TIGAR in HF causes impaired myocardial energetics and function. The effects of TIGAR knockout on LV function were also replicated in tamoxifen (TAM)-inducible cardiac-specific TIGAR knockout mice (TIGARflox/flox/Tg(Myh6-cre/Esr1) mice). The ablation of TIGAR during pressure-overload HF preserves myocardial function and energetics. Thus, cardiac TIGAR-targeted therapy to increase glucose metabolism will be a novel strategy for HF. NEW & NOTEWORTHY The present study is the first to demonstrate that TP53-induced glycolysis and apoptosis regulator (TIGAR) is upregulated in the myocardium during experimental heart failure (HF) in mice and that TIGAR knockout can preserve the heart function and myocardial energetics during HF. Reducing TIGAR to enhance myocardial glycolytic energy production is a promising therapeutic strategy for HF.

Structural and enzymatic properties of mammalian d-glutamate cyclase.

d-Glutamate cyclase (DGLUCY) is a unique enzyme that reversibly converts free d-glutamate to 5-oxo-d-proline and H2O. Mammalian DGLUCY is highly expressed in the mitochondrial matrix in the heart, and its downregulation disrupts d-glutamate and/or 5-oxo-d-proline levels, contributing to the onset and/or exacerbation of heart failure. However, detailed characterisation of DGLUCY has not yet been performed. Herein, the structural and enzymatic properties of purified recombinant mouse DGLUCY were examined. The results revealed a dimeric oligomerisation state, and both d-glutamate-to-5-oxo-d-proline and 5-oxo-d-proline-to-d-glutamate reactions were catalysed in a stereospecific manner. Catalytic activity is modulated by divalent cations and nucleotides including ATP and ADP. Interestingly, the presence of Mn2+ completely abolished the 5-oxo-d-proline-to-d-glutamate reaction but stimulated the d-glutamate-to-5-oxo-d-proline reaction. The optimum pH is ∼8.0, similar to that in the mitochondrial matrix, and the catalytic efficiency for d-glutamate is markedly higher than that for 5-oxo-d-proline. These findings suggest that DGLUCY functions as a metalloenzyme that degrades d-glutamate in the mitochondrial matrix in mammalian cells. The results also provide insight into the correlation between DGLUCY enzyme activity and the physiological and pathological roles of d-glutamate and 5-oxo-d-proline in cardiac function, which is of relevance to the risk of onset of heart failure.

Cardiac-Specific Bdh1 Overexpression Ameliorates Oxidative Stress and Cardiac Remodeling in Pressure Overload-Induced Heart Failure.

BACKGROUND: Energy starvation and the shift of energy substrate from fatty acids to glucose is the hallmark of metabolic remodeling during heart failure progression. However, ketone body metabolism in the failing heart has not been fully investigated. METHODS AND RESULTS: Microarray data analysis and mitochondrial isobaric tags for relative and absolute quantification proteomics revealed that the expression of D-ß-hydroxybutyrate dehydrogenase I (Bdh1), an enzyme that catalyzes the NAD+/NADH coupled interconversion of acetoacetate and ß-hydroxybutyrate, was increased 2.5- and 2.8-fold, respectively, in the heart after transverse aortic constriction. In addition, ketone body oxidation was upregulated 2.2-fold in transverse aortic constriction hearts, as determined by the amount of 14CO2 released from the metabolism of [1-14C] ß-hydroxybutyrate in isolated perfused hearts. To investigate the significance of this augmented ketone body oxidation, we generated heart-specific Bdh1-overexpressing transgenic mice to recapitulate the observed increase in basal ketone body oxidation. Bdh1 transgenic mice showed a 1.7-fold increase in ketone body oxidation but did not exhibit any differences in other baseline characteristics. When subjected to transverse aortic constriction, Bdh1 transgenic mice were resistant to fibrosis, contractile dysfunction, and oxidative damage, as determined by the immunochemical detection of carbonylated proteins and histone acetylation. Upregulation of Bdh1 enhanced antioxidant enzyme expression. In our in vitro study, flow cytometry revealed that rotenone-induced reactive oxygen species production was decreased by adenovirus-mediated Bdh1 overexpression. Furthermore, hydrogen peroxide-induced apoptosis was attenuated by Bdh1 overexpression. CONCLUSIONS: We demonstrated that ketone body oxidation increased in failing hearts, and increased ketone body utilization decreased oxidative stress and protected against heart failure.

Late-onset Mitochondrial Cardiomyopathy Triggered by Anticancer Treatment.

We report the case of a 62-year-old woman with a history of bilateral hearing impairment, who developed mitochondrial cardiomyopathy after chemotherapy. The patient underwent postoperative cisplatin chemotherapy after the surgical treatment of cervical cancer. The systolic function of her left ventricle decreased significantly. A tissue examination of the left ventricle revealed mitochondrial cardiomyopathy. Genetic testing revealed mutations in mitochondrial 3,243 A→G. Nine hundred fifty-five individual mutations were identified by next-generation sequencing. Since cardiovascular complications are the second leading cause of morbidity and mortality in patients undergoing cancer treatment, mitochondrial cardiomyopathy should be considered a potential cause of heart failure.

Activation of PPAR-α in the early stage of heart failure maintained myocardial function and energetics in pressure-overload heart failure.

Failing heart loses its metabolic flexibility, relying increasingly on glucose as its preferential substrate and decreasing fatty acid oxidation (FAO). Peroxisome proliferator-activated receptor α (PPAR-α) is a key regulator of this substrate shift. However, its role during heart failure is complex and remains unclear. Recent studies reported that heart failure develops in the heart of myosin heavy chain-PPAR-α transgenic mice in a manner similar to that of diabetic cardiomyopathy, whereas cardiac dysfunction is enhanced in PPAR-α knockout mice in response to chronic pressure overload. We created a pressure-overload heart failure model in mice through transverse aortic constriction (TAC) and activated PPAR-α during heart failure using an inducible transgenic model. After 8 wk of TAC, left ventricular (LV) function had decreased with the reduction of PPAR-α expression in wild-type mice. We examined the effect of PPAR-α induction during heart failure using the Tet-Off system. Eight weeks after the TAC operation, LV construction was preserved significantly by PPAR-α induction with an increase in PPAR-α-targeted genes related to fatty acid metabolism. The increase of expression of fibrosis-related genes was significantly attenuated by PPAR-α induction. Metabolic rates measured by isolated heart perfusions showed a reduction in FAO and glucose oxidation in TAC hearts, but the rate of FAO preserved significantly owing to the induction of PPAR-α. Myocardial high-energy phosphates were significantly preserved by PPAR-α induction. These results suggest that PPAR-α activation during pressure-overloaded heart failure improved myocardial function and energetics. Thus activating PPAR-α and modulation of FAO could be a promising therapeutic strategy for heart failure.NEW & NOTEWORTHY The present study demonstrates the role of PPAR-α activation in the early stage of heart failure using an inducible transgenic mouse model. Induction of PPAR-α preserved heart function, and myocardial energetics. Activating PPAR-α and modulation of fatty acid oxidation could be a promising therapeutic strategy for heart failure.

Pyk2 aggravates hypoxia-induced pulmonary hypertension by activating HIF-1α.

Pulmonary arterial hypertension (PAH) is a refractory disease characterized by uncontrolled vascular remodeling and elevated pulmonary arterial pressure. Although synthetic inhibitors of some tyrosine kinases have been used to treat PAH, their therapeutic efficacies and safeties remain controversial. Thus, the establishment of novel therapeutic targets based on the molecular pathogenesis underlying PAH is a clinically urgent issue. In the present study, we demonstrated that proline-rich tyrosine kinase 2 (Pyk2), a nonreceptor type protein tyrosine kinase, plays a crucial role in the pathogenesis of pulmonary hypertension (PH) using an animal model of hypoxia-induced PH. Resistance to hypoxia-induced PH was markedly higher in Pyk2-deficient mice than in wild-type mice. Pathological investigations revealed that medial thickening of the pulmonary arterioles, which is a characteristic of hypoxia-induced PH, was absent in Pyk2-deficient mice, suggesting that Pyk2 is involved in the hypoxia-induced aberrant proliferation of vascular smooth muscle cells in hypoxia-induced PH. In vitro experiments using human pulmonary smooth muscle cells showed that hypoxic stress increased the proliferation and migration of cells in a Pyk2-dependent manner. We also demonstrated that Pyk2 plays a crucial role in ROS generation during hypoxic stress and that this Pyk2-dependent generation of ROS is necessary for the activation of hypoxia-inducible factor-1α, a key molecule in the pathogenesis of hypoxia-induced PH. In summary, the results of the present study reveal that Pyk2 plays an important role in the pathogenesis of hypoxia-induced PH. Therefore, Pyk2 may represent a promising therapeutic target for PAH in a clinical setting.