Nucleotide depletion in hypoxia experimental models of mouse myocardial slices.

OBJECTIVES: Experimental models to test the effective protection against cardiac ischemia injury are still challenging in pre-clinical studies. The use of myocardial slices creates a special link between testing isolated cardiomyocytes and whole-heart research. In this work, we investigated the effects of oxygen deprivation in a hypoxic chamber and treatment with cobalt chloride (CoCl2) on the nucleotide profile in isolated mouse myocardial slices. METHODS: 200 µm-thick left ventricle myocardial slices were obtained from 3-month-old male C57Bl/6J mice using an oscillatory microtome. Slices were then exposed to 1% O2 atmosphere or 100 µM CoCl2 at 37 °C for 45 min and used for nucleotide measurements using ultra-high-performance liquid chromatography. The effects of two short-term experimental models of hypoxia were compared to 2'-deoxyglucose with oligomycin (2-DG + OLIGO) treatment, which inhibited both glycolysis and mitochondrial ATP synthesis. KEY FINDINGS: A significant effect of hypoxia with 1% O2 was observed on adenosine triphosphate (ATP) and total adenine nucleotide (TAN) concentrations as well as on adenylate energy charge (AEC), ATP/ADP and ATP/AMP ratios. Oxygen deprivation caused changes almost as profound as 2-DG + OLIGO, emphasizing the critical role of mitochondrial oxidative phosphorylation in the energy metabolism of cultured heart slices. CoCl2 treatment that elicits hypoxia-like responses via HIF-1α stabilization only slightly affected nucleotide levels. This suggests that mechanisms induced by cobalt ions require more time to change the cardiac energy metabolism. CONCLUSIONS: A short-term culture of myocardial slices in a hypoxic chamber seems to be an appropriate model of cardiac ischemia for testing new pharmacological approaches based on modulating the energy metabolism of cardiac cells.

Endothelial Effects of Simultaneous Expression of Human HO-1, E5NT, and ENTPD1 in a Mouse.

The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5'-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation.

Immunological and Metabolic Causes of Infertility in Polycystic Ovary Syndrome.

Infertility has been recognized as a civilizational disease. One of the most common causes of infertility is polycystic ovary syndrome (PCOS). Closely interrelated immunometabolic mechanisms underlie the development of this complex syndrome and lead to infertility. The direct cause of infertility in PCOS is ovulation and implantation disorders caused by low-grade inflammation of ovarian tissue and endometrium which, in turn, result from immune and metabolic system disorders. The systemic immune response, in particular the inflammatory response, in conjunction with metabolic disorders, insulin resistance (IR), hyperadrenalism, insufficient secretion of progesterone, and oxidative stress lead not only to cardiovascular diseases, cancer, autoimmunity, and lipid metabolism disorders but also to infertility. Depending on the genetic and environmental conditions as well as certain cultural factors, some diseases may occur immediately, while others may become apparent years after an infertility diagnosis. Each of them alone can be a significant factor contributing to the development of PCOS and infertility. Further research will allow clinical management protocols to be established for PCOS patients experiencing infertility so that a targeted therapy approach can be applied to the factor underlying and driving the "vicious circle" alongside symptomatic treatment and ovulation stimulation. Hence, therapy of fertility for PCOS should be conducted by interdisciplinary teams of specialists as an in-depth understanding of the molecular relationships and clinical implications between the immunological and metabolic factors that trigger reproductive system disorders is necessary to restore the physiology and homeostasis of the body and, thus, fertility, among PCOS patients.

Hidden Pool of Cardiac Adenine Nucleotides That Controls Adenosine Production.

Myocardial ischemic adenosine production decreases in subsequent events that may blunt its protective functions. To test the relation between total or mitochondrial cardiac adenine nucleotide pool (TAN) on the energy status with adenosine production, Langendorff perfused rat hearts were subjected to three protocols: 1 min ischemia at 40 min, 10 min ischemia at 50 min, and 1 min ischemia at 85 min in Group I; additional infusion of adenosine (30 µM) for 15 min after 10 min ischemia in Group I-Ado, and 1 min ischemia at 40 and 85 min in the controls (Group No I). A 31P NMR and an HPLC were used for the analysis of nucleotide and catabolite concentrations in the heart and coronary effluent. Cardiac adenosine production in Group I measured after 1 min ischemia at 85 min decreased to less than 15% of that at 40 min in Group I, accompanied by a decrease in cardiac ATP and TAN to 65% of the initial results. Adenosine production at 85 min was restored to 45% of that at 40 min in Group I-Ado, accompanied by a rebound of ATP and TAN by 10% vs. Group I. Mitochondrial TAN and free AMP concentrations paralleled that of total cardiac TAN. Changes in energy equilibrium or mitochondrial function were minor. This study highlights that only a fraction of the cardiac adenine nucleotide pool is available for adenosine production, but further studies are necessary to clarify its nature.

Nucleotide metabolism during experimental preservation for transplantation with Transmedium Transplant Fluid (TTF) in comparison to Histidine-Tryptophan-Ketoglutarate (HTK).

Organ preservation solutions are essential to diminish ischemic/hypoxic injury during cold storage and to improve graft survival. In our experiments, we investigated novel solutions that target such mechanisms as Transmedium Transplant Fluid (TTF) in comparison to PlegiStore solution (HTK). Rat hearts were infused with TTF or HTK and then subjected to 4 hours of 4 °C preservation followed by 25 minutes of reperfusion in the Langendorff system. Assessment of purine release from the heart, mechanical function, and cardiac nucleotide content in the heart homogenates was done. A significant increase in the uric acid, hypoxanthine, inosine, and total purine metabolite concentrations were observed in the HTK hearts when compared to TTF. The TTF group had lower left ventricular systolic pressure and left ventricular end-diastolic pressure when compared to the HTK. Left ventricular diastolic pressure, minimal dp/dt, and maximal dp/dt in both groups were similar. The concentration of ADP in the heart homogenates of the HTK group was increased when compared to the TTF group. ATP and GTP concentration showed a tendency to increase in the homogenates of TTF hearts when NAD, AMP, GDP, GMP, and ADPR were similar in both groups of rats. TTF provided enhanced cardioprotection as evidenced by inhibiting the purine nucleotide metabolites released from the rat hearts during reperfusion and enhanced systolic and diastolic mechanical function recovery. In particular, better preservation of GTP and ATP concentrations may translate into enhanced protection of endothelium and the cytoskeleton, which are not adequately protected with current preservation techniques.

Cardiac Mitochondria Dysfunction in Dyslipidemic Mice.

Dyslipidemia triggers many severe pathologies, including atherosclerosis and chronic inflammation. Several lines of evidence, including our studies, have suggested direct effects of dyslipidemia on cardiac energy metabolism, but details of these effects are not clear. This study aimed to investigate how mild dyslipidemia affects cardiac mitochondria function and vascular nucleotide metabolism. The analyses were performed in 3- and 6-month-old knock-out mice for low-density lipoprotein receptor (Ldlr-/-) and compared to wild-type C57Bl/6J mice (WT). Cardiac isolated mitochondria function was analyzed using Seahorse metabolic flux analyzer. The mechanical function of the heart was measured using echocardiography. The levels of fusion, fission, and mitochondrial biogenesis proteins were determined by ELISA kits, while the cardiac intracellular nucleotide concentration and vascular pattern of nucleotide metabolism ecto-enzymes were analyzed using reverse-phase high-performance liquid chromatography. We revealed the downregulation of mitochondrial complex I, together with a decreased activity of citrate synthase (CS), reduced levels of nuclear respiratory factor 1 and mitochondrial fission 1 protein, as well as lower intracellular adenosine and guanosine triphosphates' pool in the hearts of 6-month Ldlr-/- mice vs. age-matched WT. The analysis of vascular ecto-enzyme pattern revealed decreased rate of extracellular adenosine monophosphate hydrolysis and increased ecto-adenosine deaminase activity (eADA) in 6-month Ldlr-/- vs. WT mice. No changes were observed in echocardiography parameters in both age groups of Ldlr-/- mice. Younger hyperlipidemic mice revealed no differences in cardiac mitochondria function, CS activity, intracellular nucleotides, mitochondrial biogenesis, and dynamics but exhibited minor changes in vascular eADA activity vs. WT. This study revealed that dysfunction of cardiac mitochondria develops during prolonged mild hyperlipidemia at the time point corresponding to the formation of early vascular alterations.

An unusual nicotinamide derivative, 4-pyridone-3-carboxamide ribonucleoside (4PYR), is a novel endothelial toxin and oncometabolite.

Our recent studies identified a novel pathway of nicotinamide metabolism that involves 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) and demonstrated its endothelial cytotoxic effect. This study tested the effects of 4PYR and its metabolites in experimental models of breast cancer. Mice were divided into groups: 4T1 (injected with mammary 4T1 cancer cells), 4T1 + 4PYR (4PYR-treated 4T1 mice), and control, maintained for 2 or 21 days. Lung metastasis and endothelial function were analyzed together with blood nucleotides (including 4PYR), plasma amino acids, nicotinamide metabolites, and vascular ectoenzymes of nucleotide catabolism. 4PYR metabolism was also evaluated in cultured 4T1, MDA-MB-231, MCF-7, and T47D cells. An increase in blood 4PYR in 4T1 mice was observed at 2 days. 4PYR and its metabolites were noticed after 21 days in 4T1 only. Higher blood 4PYR was linked with more lung metastases in 4T1 + 4PYR vs. 4T1. Decreased L-arginine, higher asymmetric dimethyl-L-arginine, and higher vascular ecto-adenosine deaminase were observed in 4T1 + 4PYR vs. 4T1 and control. Vascular relaxation caused by flow-dependent endothelial activation in 4PYR-treated mice was significantly lower than in control. The permeability of 4PYR-treated endothelial cells was increased. Decreased nicotinamide but enhanced nicotinamide metabolites were noticed in 4T1 vs. control. Reduced N-methylnicotinamide and a further increase in Met2PY were observed in 4T1 + 4PYR vs. 4T1 and control. In cultured breast cancer cells, estrogen and progesterone receptor antagonists inhibited the production of 4PYR metabolites. 4PYR formation is accelerated in cancer and induces metabolic disturbances that may affect cancer progression and, especially, metastasis, probably through impaired endothelial homeostasis. 4PYR may be considered a new oncometabolite.