Azorella compacta Organic Extracts Exacerbate Metabolic Dysfunction-Associated Fatty Liver Disease in Mice Fed a High-Fat Diet.

Azorella compacta (A. compacta) is a shrub of the Andean Altiplano of Bolivia, Chile and Peru, consumed by local communities as a traditional medicine for several maladies such as diabetes, hepatic and inflammatory diseases. A. compacta is rich in mulinane- and azorellane-type diterpenoids. For two of these, acute hypoglycemic effects have been described, but the impact of A. compacta diterpenoids on fatty liver disease has not been investigated. Therefore, A. compacta organic fractions were prepared using petroleum ether, dichloromethane and methanol. Their content was characterized by UHPLC/MS, revealing the presence of ten diterpenoids, mainly mulinic acid, azorellanol and mulin-11,13-diene. Next, mice fed with a high-fat diet (HFD), a model of metabolic dysfunction-associated fatty liver disease (MAFLD), received one of the fractions in drinking water for two weeks. After this treatment, hepatic parameters were evaluated. The A. compacta fractions did not reduce hyperglycemia or body weight in the HFD-fed mice but increased the serum levels of hepatic transaminases (AST and ALT), reduced albumin and increased bilirubin, indicating hepatic damage, while histopathological alterations such as steatosis, inflammation and necrosis generated by the HFD were, overall, not ameliorated by the fractions. These results suggest that organic A. compacta extracts may generate hepatic complications in patients with MAFLD.

Adipose Tissue, Non-Communicable Diseases, and Physical Exercise: An Imperfect Triangle.

The study of adipose tissue has received considerable attention due to its importance not just in maintaining body energy homeostasis but also in playing a role in a number of other physiological processes. Beyond storing energy, adipose tissue is important in endocrine, immunological, and neuromodulatory functions, secreting hormones that participate in the regulation of energy homeostasis. An imbalance of these functions will generate structural and functional changes in the adipose tissue, favoring the secretion of deleterious adipocytokines that induce a pro-inflammatory state, allowing the development of metabolic and cardiovascular diseases and even some types of cancer. A common theme worldwide has been the development of professional guidelines for the control and treatment of obesity, with emphasis on hypocaloric diets and exercise. The aim of this review is to examine the pathophysiological mechanisms of obesity, considering the relationship among adipose tissue and two aspects that contribute positively or negatively to keeping a healthy body homeostasis, namely, exercise and noninfectious diseases. We conclude that the relationship of these aspects does not have homogeneous effects among individuals. Nevertheless, it is possible to establish some common mechanisms, like a decrease in pro-inflammatory markers in the case of exercise, and an increase in chronic inflammation in non-communicable diseases. An accurate diagnosis might consider the particular variables of a patient, namely their molecular profile and how it affects its metabolism, routines, and lifestyle; their underling health conditions; and probably even the constitution of their microbiome. We foresee that the development and accessibility of omics approaches and precision medicine will greatly improve the diagnosis, treatment, and successful outcomes for obese patients.

New NADPH Oxidase 2 Inhibitors Display Potent Activity against Oxidative Stress by Targeting p22phox-p47phox Interactions.

NADPH oxidase (NOX2) is responsible for reactive oxygen species (ROS) production in neutrophils and has been recognized as a key mediator in inflammatory and cardiovascular pathologies. Nevertheless, there is a lack of specific NOX2 pharmacological inhibitors. In medicinal chemistry, heterocyclic compounds are essential scaffolds for drug design, and among them, indole is a very versatile pharmacophore. We tested the hypothesis that indole heteroaryl-acrylonitrile derivatives may serve as NOX2 inhibitors by evaluating the capacity of 19 of these molecules to inhibit NOX2-derived ROS production in human neutrophils (HL-60 cells). Of these compounds, C6 and C14 exhibited concentration-dependent inhibition of NOX2 (IC50~1 µM). These molecules also reduced NOX2-derived oxidative stress in cardiomyocytes and prevented cardiac damage induced by ischemia-reperfusion. Compound C6 significantly reduced the membrane translocation of p47phox, a cytosolic subunit that is required for NOX2 activation. Molecular docking analyses of the binding modes of these molecules with p47phox indicated that C6 and C14 interact with specific residues in the inner part of the groove of p47phox, the binding cavity for p22phox. This combination of methods showed that novel indole heteroaryl acrylonitriles represent interesting lead compounds for developing specific and potent NOX2 inhibitors.

Tetrahydrobiopterin (BH4) Supplementation Prevents the Cardiorenal Effects of Diabetes in Mice by Reducing Oxidative Stress, Inflammation and Fibrosis.

Background: The effects of diabetes on the cardiovascular system as well as in the kidney are profound, which include hypertrophy and fibrosis. Diabetes also induces oxidative stress, at least in part due to the uncoupling of nitric oxide synthase (NOS); this is a shift in NO production toward superoxide production due to reduced levels of the NOS cofactor tetrahydrobiopterin (BH4). With this in mind, we tested the hypothesis that BH4 supplementation may prevent the development of diabetic cardiomyopathy and nephropathy. Methods: Diabetes was induced in Balb/c mice with streptozotocin. Then, diabetic mice were divided into two groups: one group provided with BH4 (sapropterin) in drinking water (daily doses of 15 mg/kg/day, during eight weeks) and the other that received only water. A third group of normoglycemic mice that received only water were used as the control. Results: Cardiac levels of BH4 were increased in mice treated with BH4 (p = 0.0019). Diabetes induced cardiac hypertrophy, which was prevented in the group that received BH4 (p < 0.05). In addition, hypertrophy was evaluated as cardiomyocyte cross-sectional area. This was reduced in diabetic mice that received BH4 (p = 0.0012). Diabetes induced cardiac interstitial fibrosis that was reduced in mice that received BH4 treatment (p < 0.05). We also evaluated in the kidney the impact of BH4 treatment on glomerular morphology. Diabetes induced glomerular hypertrophy compared with normoglycemic mice and was prevented by BH4 treatment. In addition, diabetic mice presented glomerular fibrosis, which was prevented in mice that received BH4. Conclusions: These results suggest that chronic treatment with BH4 in mice ameliorates the cardiorenal effects of diabetes,, probably by restoring the nitroso−redox balance. This offers a possible new alternative to explore a BH4-based treatment for the organ damage caused by diabetes.

Maresin-1 Prevents Liver Fibrosis by Targeting Nrf2 and NF-κB, Reducing Oxidative Stress and Inflammation.

Liver fibrosis is a complex process characterized by the excessive accumulation of extracellular matrix (ECM) and an alteration in liver architecture, as a result of most types of chronic liver diseases such as cirrhosis, hepatocellular carcinoma (HCC) and liver failure. Maresin-1 (MaR1) is derivative of ω-3 docosahexaenoic acid (DHA), which has been shown to have pro-resolutive and anti-inflammatory effects. We tested the hypothesis that the application of MaR1 could prevent the development of fibrosis in an animal model of chronic hepatic damage. Sprague-Dawley rats were induced with liver fibrosis by injections of diethylnitrosamine (DEN) and treated with or without MaR1 for four weeks. In the MaR1-treated animals, levels of AST and ALT were normalized in comparison with DEN alone, the hepatic architecture was improved, and inflammation and necrotic areas were reduced. Cell proliferation, assessed by the mitotic activity index and the expression of Ki-67, was increased in the MaR1-treated group. MaR1 attenuated liver fibrosis and oxidative stress was induced by DEN. Plasma levels of the pro-inflammatory mediators TNF-α and IL-1ß were reduced in MaR1-treated animals, whereas the levels of IL-10, an anti-inflammatory cytokine, increased. Interestingly, MaR1 inhibited the translocation of the p65 subunit of NF-κB, while increasing the activation of Nrf2, a key regulator of the antioxidant response. Finally, MaR1 treatment reduced the levels of the pro-fibrotic mediator TGF-ß and its receptor, while normalizing the hepatic levels of IGF-1, a proliferative agent. Taken together, these results suggest that MaR1 improves the parameters of DEN-induced liver fibrosis, activating hepatocyte proliferation and decreasing oxidative stress and inflammation. These results open the possibility of MaR1 as a potential therapeutic agent in fibrosis and other liver pathologies.

Pharmacological Inhibition of S-Nitrosoglutathione Reductase Reduces Cardiac Damage Induced by Ischemia-Reperfusion.

The cardioprotective effects of nitric oxide (NO) have been described through S-nitrosylation of several important proteins in the mitochondria of the cardiomyocyte. S-nitrosoglutathione reductase (GSNOR) is an enzyme involved in the metabolism of S-nitrosothiols by producing denitrosylation, thus limiting the cardioprotective effect of NO. The effect of GSNOR inhibition on the damage by cardiac ischemia-reperfusion is still unclear. We tested the hypothesis that pharmacological inhibition of GSNOR promotes cardioprotection by increasing the levels of protein S-nitrosylation. In a model of ischemia-reperfusion in isolated rat heart, the effect of a GSNOR inhibitor, 5-chloro-3-(2-[4-ethoxyphenyl) (ethyl) amino]-2-oxoethyl)-1H-indole-2-carboxylic acid (C2), was investigated. Ventricular function and hemodynamics were determined, in addition to tissue damage and S-nitrosylation of mitochondrial proteins. Hearts treated with C2 showed a lower release of myocardial damage marker creatine kinase and a reduction in the infarcted area. It also improved post-ischemia ventricular function compared to controls. These results were associated with increasing protein S-nitrosylation, specifically of the mitochondrial complexes III and V. The pharmacological inhibition of GSNOR showed a concentration-dependent cardioprotective effect, being observed in functional parameters and myocardial damage, which was maximal at 1 µmol/L, associated with increased S-nitrosylation of mitochondrial proteins. These data suggest that GSNOR is an interesting pharmacological target for cardiac reperfusion injury.

Pro-Resolving Lipid Mediator Resolvin E1 Mitigates the Progress of Diethylnitrosamine-Induced Liver Fibrosis in Sprague-Dawley Rats by Attenuating Fibrogenesis and Restricting Proliferation.

Liver fibrosis is a complex process associated to most types of chronic liver disease, which is characterized by a disturbance of hepatic tissue architecture and the excessive accumulation of extracellular matrix. Resolvin E1 (RvE1) is a representative member of the eicosapentaenoic omega-3 lipid derivatives, and is a drug candidate of the growing family of endogenous resolvins. Considering the aforementioned, the main objective of this study was to analyze the hepatoprotective effect of RvE1 in a rat model of liver fibrosis. Male Sprague-Dawley rats received diethylnitrosamine (DEN, 70 mg/mg body weight intraperitoneally (i.p)) as an inductor of liver fibrosis once weekly and RvE1(100 ng/body weight i.p) twice weekly for four weeks. RvE1 suppressed the alterations induced by DEN, normalizing the levels of alanine aminotransferase (ALT), albumin, and lactate dehydrogenase (LDH), and ameliorated DEN injury by decreasing the architecture distortion, inflammatory infiltration, necrotic areas, and microsteatosis. RvE1 also limited DEN-induced proliferation through a decrease in Ki67-positive cells and cyclin D1 protein expression, which is related to an increase of the levels of cleaved caspase-3. Interestingly, we found that RvE1 promotes higher nuclear translocation of nuclear factor κB (NF-κB)p65 than DEN. RvE1 also increased the levels of nuclear the nuclear factor erythroid 2-related factor 2 (Nrf2), but with no antioxidant effect, measured as an increase in glutathione disulfide (GSSG) and a decrease in the ratio of glutathione (GSH)/GSSG. Taken together, these results suggest that RvE1 modulates the fibrogenesis, steatosis, and cell proliferation in a model of DEN induced fibrosis.

Apocynin Treatment Prevents Cardiac Connexin 43 Hemichannels Hyperactivity by Reducing Nitroso-Redox Stress in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a fatal disease that causes cardiomyopathy and is associated with oxidative stress. In the heart, oxidative stress interferes with the location of connexin 43 (Cx43) to the intercalated discs causing its lateralization to the plasma membrane where Cx43 forms hemichannels. We tested the hypothesis that in DMD cardiomyopathy, increased oxidative stress is associated with the formation and activation of Cx43 hemichannels. For this, we used mdx mice as a DMD model and evaluated cardiac function, nitroso-redox changes and Cx43 hemichannels permeability. Mdx hearts presented increased NADPH oxidase-derived oxidative stress and increased Cx43 S-nitrosylation compared to controls. These redox changes were associated with increased Cx43 lateralization, decreased cardiac contractility and increased arrhythmic events. Pharmacological inhibition of NADPH oxidase using apocynin (one month) reduced systemic oxidative stress and reversed the aforementioned changes towards normal, except Cx43 lateralization. Opening of Cx43 hemichannels was blocked by apocynin treatment and by acute hemichannel blockade with carbenoxolone. NADPH oxidase inhibition also prevented the occurrence of apoptosis in mdx hearts and reversed the ventricular remodeling. These results show that NADPH oxidase activity in DMD is associated with S-nitrosylation and opening of Cx43 hemichannels. These changes lead to apoptosis and cardiac dysfunction and were prevented by NADPH oxidase inhibition.

Maresin 1, a Proresolving Lipid Mediator, Ameliorates Liver Ischemia-Reperfusion Injury and Stimulates Hepatocyte Proliferation in Sprague-Dawley Rats.

Maresin-1 (MaR1) is a specialized pro-resolving mediator, derived from omega-3 fatty acids, whose functions are to decrease the pro-inflammatory and oxidative mediators, and also to stimulate cell division. We investigated the hepatoprotective actions of MaR1 in a rat model of liver ischemia-reperfusion (IR) injury. MaR1 (4 ng/gr body weight) was administered prior to ischemia (1 h) and reperfusion (3 h), and controls received isovolumetric vehicle solution. To analyze liver function, transaminases levels and tissue architecture were assayed, and serum cytokines TNF-α, IL-6, and IL-10, mitotic activity index, and differential levels of NF-κB and Nrf-2 transcription factors, were analyzed. Transaminase, TNF-α levels, and cytoarchitecture were normalized with the administration of MaR1 and associated with changes in NF-κB. IL-6, mitotic activity index, and nuclear translocation of Nrf-2 increased in the MaR1-IR group, which would be associated with hepatoprotection and cell proliferation. Taken together, these results suggest that MaR1 alleviated IR liver injury, facilitated by the activation of hepatocyte cell division, increased IL-6 cytokine levels, and the nuclear localization of Nrf-2, with a decrease of NF-κB activity. All of them were related to an improvement of liver injury parameters. These results open the possibility of MaR1 as a potential therapeutic tool in IR and other hepatic pathologies.

Effects of Phaseolus vulgaris Extract on Lipolytic Activity and Differentiation of 3T3-L1 Preadipocytes into Mature Adipocytes: A Strategy to Prevent Obesity.

BACKGROUND: Overweight and obesity are defined as abnormal or excessive fat accumulation that may be harmful for health. A global trend in this area is the search for natural compounds that have a proven beneficial effect and no clinical complications. Phaseolus vulgaris (bean) is a vegetable highly consumed worldwide. One of its effects, the most reported, is weight reduction in overweight individuals. OBJECTIVE: The objective of this study was to investigate the antiobesity activity of this legume in mature 3T3-L1 adipocytes and in rat white adipose tissue in an ex vivo model. DESIGN: Mature adipocytes 3T3-L1 and rat adipose tissue were treated with bean extracts. We quantified lipolysis in mature 3T3-L1 adipocytes and in rat white adipose tissue in an ex vivo model. RESULTS: In an ex vivo assay with adipose tissue, methanolic and aqueous green bean extracts increased glycerol release to the medium compared to control (p < 0.05 and p < 0.001 respectively). Treatment of 3T3-L1 adipocytes with green bean extracts (800 and 1000 µg/mL) increased glycerol release significantly (p < 0.0001). Extracts at concentrations between 500 and 1000 µg/mL reduced intracellular triglyceride accumulation by 34.4% and 47.1% compared to control (p < 0.0001). DISCUSSION: Our results propose that bioactive compounds of green beans exert a direct mechanism on adipocytes through lipolysis. CONCLUSION: We have identified a novel capacity of bean extracts related to lipolytic activity both in vitro and ex vivo, resulting in a powerful lipolytic effect. Moreover, we also found that bean extracts has an antiadipogenic effect during the differentiation of 3T3-L1 preadipocytes. These results suggest that bean is a good candidate for the development of functional ingredients that can help reduce the high rates of death from cardiovascular diseases associated with obesity.

NOX Inhibition Improves ß-Adrenergic Stimulated Contractility and Intracellular Calcium Handling in the Aged Rat Heart.

Cardiac aging is characterized by alterations in contractility and intracellular calcium ([Ca2+]i) homeostasis. It has been suggested that oxidative stress may be involved in this process. We and others have reported that in cardiomyopathies the NADPH oxidase (NOX)-derived superoxide is increased, with a negative impact on [Ca2+]i and contractility. We tested the hypothesis that in the aged heart, [Ca2+]i handling and contractility are disturbed by NOX-derived superoxide. For this we used adults (≈5 month-old) and aged (20⁻24 month-old) rats. Contractility was evaluated in isolated hearts, challenged with isoproterenol. To assess [Ca2+]i, isolated cardiac myocytes were field-stimulated and [Ca2+]i was monitored with fura-2. Cardiac concentration-response to isoproterenol was depressed in aged compared to adults hearts (p < 0.005), but was restored by NOX inhibitors apocynin and VAS2870. In isolated cardiomyocytes, apocynin increased the amplitude of [Ca2+]i in aged myocytes (p < 0.05). Time-50 [Ca2+]i decay was increased in aged myocytes (p < 0.05) and reduced towards normal by NOX inhibition. In addition, we found that myofilaments Ca2+ sensitivity was reduced in aged myocytes (p < 0.05), and was further reduced by apocynin. NOX2 expression along with NADPH oxidase activity was increased in aged hearts. Phospholamban phosphorylation (Ser16/Thr17) after isoproterenol treatment was reduced in aged hearts compared to adults and was restored by apocynin treatment (p < 0.05). In conclusion, ß-adrenergic-induced contractility was depressed in aged hearts, and NOX inhibition restored back to normal. Moreover, altered Ca2+ handling in aged myocytes was also improved by NOX inhibition. These results suggest a NOX-dependent effect in aged myocytes at the level of Ca2+ handling proteins and myofilaments.

High-Intensity Exercise Reduces Cardiac Fibrosis and Hypertrophy but Does Not Restore the Nitroso-Redox Imbalance in Diabetic Cardiomyopathy.

Diabetic cardiomyopathy refers to the manifestations in the heart as a result of altered glucose homeostasis, reflected as fibrosis, cellular hypertrophy, increased oxidative stress, and apoptosis, leading to ventricular dysfunction. Since physical exercise has been indicated as cardioprotective, we tested the hypothesis that high-intensity exercise training could reverse the cardiac maladaptations produced by diabetes. For this, diabetes was induced in rats by a single dose of alloxan. Diabetic rats were randomly assigned to a sedentary group or submitted to a program of exercise on a treadmill for 4 weeks at 80% of maximal performance. Another group of normoglycemic rats was used as control. Diabetic rat hearts presented cardiomyocyte hypertrophy and interstitial fibrosis. Chronic exercise reduced both parameters but increased apoptosis. Diabetes increased the myocardial levels of the mRNA and proteins of NADPH oxidases NOX2 and NOX4. These altered levels were not reduced by exercise. Diabetes also increased the level of uncoupled endothelial nitric oxide synthase (eNOS) that was not reversed by exercise. Finally, diabetic rats showed a lower degree of phosphorylated phospholamban and reduced levels of SERCA2 that were not restored by high-intensity exercise. These results suggest that high-intensity chronic exercise was able to reverse remodeling in the diabetic heart but was unable to restore the nitroso-redox imbalance imposed by diabetes.

Nitric Oxide Synthase 1 Modulates Basal and ß-Adrenergic-Stimulated Contractility by Rapid and Reversible Redox-Dependent S-Nitrosylation of the Heart.

S-nitrosylation of several Ca2+ regulating proteins in response to ß-adrenergic stimulation was recently described in the heart; however the specific nitric oxide synthase (NOS) isoform and signaling pathways responsible for this modification have not been elucidated. NOS-1 activity increases inotropism, therefore, we tested whether ß-adrenergic stimulation induces NOS-1-dependent S-nitrosylation of total proteins, the ryanodine receptor (RyR2), SERCA2 and the L-Type Ca2+ channel (LTCC). In the isolated rat heart, isoproterenol (10 nM, 3-min) increased S-nitrosylation of total cardiac proteins (+46±14%) and RyR2 (+146±77%), without affecting S-nitrosylation of SERCA2 and LTCC. Selective NOS-1 blockade with S-methyl-L-thiocitrulline (SMTC) and Nω-propyl-l-arginine decreased basal contractility and relaxation (-25-30%) and basal S-nitrosylation of total proteins (-25-60%), RyR2, SERCA2 and LTCC (-60-75%). NOS-1 inhibition reduced (-25-40%) the inotropic response and protein S-nitrosylation induced by isoproterenol, particularly that of RyR2 (-85±7%). Tempol, a superoxide scavenger, mimicked the effects of NOS-1 inhibition on inotropism and protein S-nitrosylation; whereas selective NOS-3 inhibitor L-N5-(1-Iminoethyl)ornithine had no effect. Inhibition of NOS-1 did not affect phospholamban phosphorylation, but reduced its oligomerization. Attenuation of contractility was abolished by PKA blockade and unaffected by guanylate cyclase inhibition. Additionally, in isolated mouse cardiomyocytes, NOS-1 inhibition or removal reduced the Ca2+-transient amplitude and sarcomere shortening induced by isoproterenol or by direct PKA activation. We conclude that 1) normal cardiac performance requires basal NOS-1 activity and S-nitrosylation of the calcium-cycling machinery; 2) ß-adrenergic stimulation induces rapid and reversible NOS-1 dependent, PKA and ROS-dependent, S-nitrosylation of RyR2 and other proteins, accounting for about one third of its inotropic effect.

Nitric oxide synthases, S-nitrosylation and cardiovascular health: from molecular mechanisms to therapeutic opportunities (review).

The understanding of nitric oxide (NO) signaling has grown substantially since the identification of endothelial derived relaxing factor (EDRF). NO has emerged as a ubiquitous signaling molecule involved in diverse physiological and pathological processes. Perhaps the most significant function, independent of EDRF, is that of NO signaling mediated locally in signaling modules rather than relying upon diffusion. In this context, NO modulates protein function via direct post­translational modification of cysteine residues. This review explores NO signaling and related reactive nitrogen species involved in the regulation of the cardiovascular system. A critical concept in the understanding of NO signaling is that of the nitroso­redox balance. Reactive nitrogen species bioactivity is fundamentally linked to the production of reactive oxygen species. This interaction occurs at the chemical, enzymatic and signaling effector levels. Furthermore, the nitroso­redox equilibrium is in a delicate balance, involving the cross­talk between NO and oxygen­derived species signaling systems, including NADPH oxidases and xanthine oxidase.

Vascular hypercontractility and endothelial dysfunction before development of atherosclerosis in moderate dyslipidemia: role for nitric oxide and interleukin-6.

Atherosclerosis is a chronic disease that affects peripheral arteries and the aorta. Several inflammatory processes are required until the production of an atheroma. Before the atheroma appears, endothelial dysfunction is a key event. We hypothesized that endothelial dysfunction occurs in a mouse model of mild dyslipidemia, the mouse deficient in apolipoprotein E (apoE(+/-)). Using aortic rings preparation, we found that apoE(+/-) mice showed increased developed tension in response to KCl 60 mM when using a range a pre-loads from 0.5 to 2.0 grams (p = 0.038). Next, we tested the vasorelaxant capacity of apoE(+/-) aortas (pre-contracted with phenylephrine) in response to acetylcholine, an endothelium-dependent vasodilator. ApoE(+/-) aortas showed diminished vasorelaxation in a range of Ach concentrations (p = 0.0032). Next we assessed the levels of plasma NO metabolites, nitrite plus nitrate. These were significantly reduced, along with a significant decrease of the endothelial nitric oxide synthase in ApoE(+/-) mice. When we analyzed the morphology of the aortas in apoE(+/-) mice, these showed no signs of atheroma. In addition, we analyzed the levels of inflammatory cytokines, TNF-alpha, MCP-1 and interleukin 6 (Il-6). While TNF-alpha was similar in both groups, (18.3 ± 2 pg/mL in wild type vs. 17.5 ± 2 pg/mL in apoE(+/-)), MCP-1 was increased in ApoE deficient mice (71.5 ± 0.8 pg/mL in wild type vs. 85.1 ± 7.4 pg/mL in ApoE(+/-) mice, p = 0.006), along with IL-6 (24.7 ± 1.7 pg/ml in wild type vs. 47.1 ± 12.5 in ApoE mice, p = 0.0055). These results suggest that mild dyslipidemia produces a pro-inflammatory state, associated with diminished NOS and NO production, which produces endothelial dysfunction.

NADPH oxidase-2 inhibition restores contractility and intracellular calcium handling and reduces arrhythmogenicity in dystrophic cardiomyopathy.

Duchenne muscular dystrophy may affect cardiac muscle, producing a dystrophic cardiomyopathy in humans and the mdx mouse. We tested the hypothesis that oxidative stress participates in disrupting calcium handling and contractility in the mdx mouse with established cardiomyopathy. We found increased expression (fivefold) of the NADPH oxidase (NOX) 2 in the mdx hearts compared with wild type, along with increased superoxide production. Next, we tested the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility was decreased in mdx myocytes compared with wild type, and this was restored toward normal by pretreating with apocynin. In addition, the amplitude of evoked intracellular Ca(2+) concentration transients that was diminished in mdx myocytes was also restored with NOX2 inhibition. Total sarcoplasmic reticulum (SR) Ca(2+) content was reduced in mdx hearts and normalized by apocynin treatment. Additionally, NOX2 inhibition decreased the production of spontaneous diastolic calcium release events and decreased the SR calcium leak in mdx myocytes. In addition, nitric oxide (NO) synthase 1 (NOS-1) expression was increased eightfold in mdx hearts compared with wild type. Nevertheless, cardiac NO production was reduced. To test whether this paradox implied NOS-1 uncoupling, we treated cardiac myocytes with exogenous tetrahydrobioterin, along with the NOX inhibitor VAS2870. These agents restored NO production and phospholamban phosphorylation in mdx toward normal. Together, these results demonstrate that, in mdx hearts, NOX2 inhibition improves the SR calcium handling and contractility, partially by recoupling NOS-1. These findings reveal a new layer of nitroso-redox imbalance in dystrophic cardiomyopathy.

NOS1AP modulates intracellular Ca(2+) in cardiac myocytes and is up-regulated in dystrophic cardiomyopathy.

NOS1AP gene (nitric oxide synthase 1-adaptor protein) is strongly associated with abnormalities in the QT interval of the electrocardiogram and with sudden cardiac death. To determine the role of NOS1AP in the physiology of the cardiac myocyte, we assessed the impact of silencing NOS1AP, using siRNA, on [Ca(2+)]i transients in neonatal cardiomyocytes. In addition, we examined the co-localization of NOS1AP with cardiac ion channels, and finally, evaluated the expression of NOS1AP in a mouse model of dystrophic cardiomyopathy. Using siRNA, NOS1AP levels were reduced to ~30% of the control levels (p<0.05). NOS1AP silencing in cardiac myocytes reduced significantly the amplitude of electrically evoked calcium transients (p<0.05) and the degree of S-nitrosylation of the cells (p<0.05). Using confocal microscopy, we evaluated NOS1AP subcellular location and interactions with other proteins by co-localization analysis. NOS1AP showed a high degree of co-localization with the L-type calcium channel and the inwardly rectifying potassium channel Kir3.1, a low degree of co-localization with the ryanodine receptor (RyR2) and alfa-sarcomeric actin and no co-localization with connexin 43, suggesting functionally relevant interactions with the ion channels that regulate the action potential duration. Finally, using immunofluorescence and Western blotting, we observed that in mice with dystrophic cardiomyopathy, NOS1AP was significantly up-regulated (p<0.05). These results suggest for a role of NOS1AP on cardiac arrhythmias, acting on the L-type calcium channel, and potassium channels, probably through S-nitrosylation.

Coordinated endothelial nitric oxide synthase activation by translocation and phosphorylation determines flow-induced nitric oxide production in resistance vessels.

BACKGROUND/AIMS: Endothelial nitric oxide synthase (eNOS) is associated with caveolin-1 (Cav-1) in plasma membrane. We tested the hypothesis that eNOS activation by shear stress in resistance vessels depends on synchronized phosphorylation, dissociation from Cav-1 and translocation of the membrane-bound enzyme to Golgi and cytosol. METHODS: In isolated, perfused rat arterial mesenteric beds, we evaluated the effect of changes in flow rate (2-10 ml/min) on nitric oxide (NO) production, eNOS phosphorylation at serine 1177, eNOS subcellular distribution and co-immunoprecipitation with Cav-1, in the presence or absence of extracellular Ca(2+). RESULTS: Increases in flow induced a biphasic rise in NO production: a rapid transient phase (3-5-min) that peaked during the first 15 s, followed by a sustained phase, which lasted until the end of stimulation. Concomitantly, flow caused a rapid translocation of eNOS from the microsomal compartment to the cytosol and Golgi, paralleled by an increase in eNOS phosphorylation and a reduction in eNOS-Cav-1 association. Transient NO production, eNOS translocation and dissociation from Cav-1 depended on extracellular Ca(2+), while sustained NO production was abolished by the PI3K-Akt blocker wortmannin. CONCLUSIONS: In intact resistance vessels, changes in flow induce NO production by transient Ca(2+)-dependent eNOS translocation from membrane to intracellular compartments and sustained Ca(2+)-independent PI3K-Akt-mediated phosphorylation.

Hydralazine and organic nitrates restore impaired excitation-contraction coupling by reducing calcium leak associated with nitroso-redox imbalance.

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1(-/-)) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca(2+) cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1(-/-) compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca(2+) transient (Δ[Ca(2+)]i) responses in NOS1(-/-) cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca(2+)]i across the range of pacing frequencies and increased myofilament Ca(2+) sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca(2+) reuptake, and restored SR Ca(2+) content. HYD and NTG at low concentrations (1 µm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca(2+) leak, HYD improves Ca(2+) cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.

NADPH oxidasa: rol en aterosclerosis y función plaquetaria / NADPH OXIDASE: role in atherosclerosis and platelet function

Reactive oxygen species have emerged as important molecules in cardiovascular function. Recent research has shown that the NADPH oxidases are important sources of superoxide in vascular cells and myocytes. The NADPH oxidases vascular share some, but not all, of the characteristics of the enzyme in neutrophils, both produce superoxide, which is metabolized to hydrogen peroxide, at the same time these reactive oxygen species serve as second messengers activate multiple intracellular signalling pathways. NADPH oxidases are essential in the physiological response of vascular cellsto pathological states such as atherosclerosis, and are functionally relevant in activation and recruitment of platelets. Recent studies suggest a key role for NADPH oxidase in the formation of a specific product from the oxidation of arachidonic acid, and a potential role in the process of recruitment of platelets. Taking into account these characteristics and evidence of the involvement of the NADPH oxidases in cardiovascular diseases as the thrombosis, inhibition of this enzymatic system appears as a promising therapy to treat and prevent these diseases.