RESUMEN

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.

RESUMEN

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.

RESUMEN

Cardiovascular diseases (CVDs) remain leading causes of death worldwide. While platelet-mediated thrombus formation following the rupture of an atherosclerotic plaque is one of the key pathophysiologic events in CVDs, the role of platelets in previous or more advanced stages of atherosclerosis is less known. Interestingly, the presence of platelets has been observed at the core of the atherosclerotic plaque.In order to study the conditions necessary for platelets to migrate toward an atherosclerotic lesion, we designed an in vitro co-culture model. Platelets were co-cultured with monocytes in Transwell inserts covered with a confluent endothelium and the number of migrating platelets and/or monocytes was determined under different conditions. Platelets were also exposed to media conditioned obtained from co-cultures prior to migration assays.Here we show that coculturing platelets and monocytes increased platelet transmigration, with a considerable number of transmigrated platelets found not associated to monocytes. Interestingly, conditioned media from platelet-monocyte co-cultures also increased platelet transmigration and aggregation, suggesting the existence of soluble factors secreted by monocytes that enhance the migratory and pro-aggregating capabilities of platelets.We conclude that platelets have the machinery to migrate through an activated endothelium, a response that requires the interaction with secreted factors produce in the context of the interaction with monocytes under atherogenic conditions.

Asunto(s)

Plaquetas/metabolismo , Células Endoteliales/metabolismo , Monocitos/metabolismo , Humanos

RESUMEN

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.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Daño por Reperfusión/prevención & control , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citocinas/sangre , Citocinas/metabolismo , Ácidos Docosahexaenoicos/química , Ácidos Grasos Omega-3/química , Hepatocitos/citología , Hepatocitos/metabolismo , Hígado/irrigación sanguínea , Hígado/fisiopatología , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Sustancias Protectoras/farmacología , Ratas Sprague-Dawley , Daño por Reperfusión/fisiopatología , Transaminasas/metabolismo

RESUMEN

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.

Asunto(s)

Acetofenonas/farmacología , Agonistas Adrenérgicos beta/farmacología , Benzoxazoles/farmacología , Calcio/metabolismo , Corazón/efectos de los fármacos , Isoproterenol/farmacología , Contracción Miocárdica/efectos de los fármacos , NADPH Oxidasas/antagonistas & inhibidores , Triazoles/farmacología , Envejecimiento/efectos de los fármacos , Animales , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Femenino , Corazón/fisiología , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , NADPH Oxidasas/metabolismo , Ratas

RESUMEN

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.

Asunto(s)

Sistema Cardiovascular/metabolismo , Óxido Nítrico Sintasa/metabolismo , Animales , Cardiomegalia/metabolismo , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Células Endoteliales/metabolismo , Insuficiencia Cardíaca/metabolismo , Humanos , Inflamación/metabolismo , Óxido Nítrico/metabolismo , Oxidación-Reducción , Especies de Nitrógeno Reactivo/metabolismo , Células Madre/metabolismo , Remodelación Ventricular

RESUMEN

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.

Asunto(s)

Arritmias Cardíacas/metabolismo , Señalización del Calcio , Cardiomiopatías/metabolismo , Glicoproteínas de Membrana/metabolismo , Contracción Miocárdica , NADPH Oxidasas/metabolismo , Animales , Benzoxazoles/farmacología , Calcio/metabolismo , Cardiomiopatías/fisiopatología , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos mdx , NADPH Oxidasa 2 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Estrés Oxidativo , Retículo Sarcoplasmático/metabolismo , Triazoles/farmacología

RESUMEN

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.

RESUMEN

Cardiovascular disease (CVD) is the leading cause of death worldwide. Healthy eating is among its safeguards, especially the daily intake of fruits and vegetables. In this context it has been shown that tomato (Solanum lycopersicum) presents antiplatelet activity. In the present study, we evaluated in vitro antiplatelet activity of fresh hybrid tomato process (nine hybrids: Apt 410, H 9888, Bos 8066, Sun 6366, AB3, HMX 7883, H 9665, H 7709, and H 9997), paste and its by-product of industrial processes (pomace). We assessed antiplatelet activity ex vivo and bleeding time in rats that ingested 0.1 and 1.0 g/kg of pomace each day. In studies in vitro, no significant differences in antiplatelet activity was observed in fresh tomato hybrids. Furthermore, the agro-industrial process did not affect the antiplatelet activity of paste and pomace. Likewise, pomace intake of 1.0 g/kg per day prolonged bleeding time and reduced ex vivo platelet aggregation in rats. The data obtained indicate that tomato has one or more compounds that caused antiplatelet activity. Regular consumption of tomato and its industrial derivatives could be part of a CVD prevention regimen.

Asunto(s)

Plaquetas/fisiología , Enfermedades Cardiovasculares/fisiopatología , Extractos Vegetales/metabolismo , Solanum lycopersicum/metabolismo , Animales , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/prevención & control , Femenino , Manipulación de Alimentos , Humanos , Técnicas In Vitro , Solanum lycopersicum/química , Solanum lycopersicum/genética , Masculino , Agregación Plaquetaria , Ratas , Ratas Wistar

RESUMEN

Whereas cardiac-derived c-kit(+) stem cells (CSCs) and bone marrow-derived mesenchymal stem cells (MSCs) are undergoing clinical trials testing safety and efficacy as a cell-based therapy, the relative therapeutic and biologic efficacy of these two cell types is unknown. We hypothesized that human CSCs have greater ability than MSCs to engraft, differentiate, and improve cardiac function. We compared intramyocardial injection of human fetal CSCs (36,000) with two doses of adult MSCs (36,000 and 1,000,000) or control (phosphate buffered saline) in nonobese diabetic/severe combined immune deficiency mice after coronary artery ligation. The myocardial infarction-induced enlargement in left ventricular chamber dimensions was ameliorated by CSCs (p < .05 for diastolic and systolic volumes), as was the decline in ejection fraction (EF; p < .05). Whereas 1 × 10(6) MSCs partially ameliorated ventricular remodeling and improved EF to a similar degree as CSCs, 36,000 MSCs did not influence chamber architecture or function. All cell therapies improved myocardial contractility, but CSCs preferentially reduced scar size and reduced vascular afterload. Engraftment and trilineage differentiation was substantially greater with CSCs than with MSCs. Adult-cultured c-kit(+)CSCs were less effective than fetal, but were still more potent than high-dose MSCs. These data demonstrate enhanced CSC engraftment, differentiation, and improved cardiac remodeling and function in ischemic heart failure. MSCs required a 30-fold greater dose than CSCs to improve cardiac function and anatomy. Together, these findings demonstrate a greater potency of CSCs than bone marrow MSCs in cardiac repair.

Asunto(s)

Células Madre Fetales/metabolismo , Células Madre Mesenquimatosas/metabolismo , Infarto del Miocardio/terapia , Proteínas Proto-Oncogénicas c-kit/metabolismo , Trasplante de Células Madre , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Células Cultivadas , Cicatriz/metabolismo , Cicatriz/patología , Cicatriz/terapia , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Células Madre Fetales/fisiología , Pruebas de Función Cardíaca , Hemodinámica , Humanos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Contracción Miocárdica , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/citología , Miocardio/metabolismo , Miocardio/patología , Remodelación Ventricular

RESUMEN

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.

Asunto(s)

Humanos , Aterosclerosis/enzimología , Especies Reactivas de Oxígeno , NADPH Oxidasas/metabolismo , Plaquetas/enzimología , Activación Plaquetaria/fisiología , Antioxidantes , Isoprostanos , Polifenoles

RESUMEN

Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and ß-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed ß-adrenergic inotropic responses that were associated with impaired ß-agonist-induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation.

Asunto(s)

Fenómenos Fisiológicos Cardiovasculares , Glutatión Reductasa/metabolismo , Alcohol Deshidrogenasa , Animales , Calcio/metabolismo , Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , Diástole/efectos de los fármacos , Femenino , Glutatión Reductasa/deficiencia , Hemodinámica/efectos de los fármacos , Isoproterenol/farmacología , Ratones , Ratones Endogámicos C57BL , Contracción Miocárdica/efectos de los fármacos , Miocardio/citología , Miocardio/enzimología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Óxido Nítrico Sintasa/metabolismo , Nitrosación , Transporte de Proteínas/efectos de los fármacos , Receptores Adrenérgicos beta/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Vasodilatación/efectos de los fármacos

RESUMEN

Both cardiac myocytes and cardiac stem cells (CSCs) express the receptor of growth hormone releasing hormone (GHRH), activation of which improves injury responses after myocardial infarction (MI). Here we show that a GHRH-agonist (GHRH-A; JI-38) reverses ventricular remodeling and enhances functional recovery in the setting of chronic MI. This response is mediated entirely by activation of GHRH receptor (GHRHR), as demonstrated by the use of a highly selective GHRH antagonist (MIA-602). One month after MI, animals were randomly assigned to receive: placebo, GHRH-A (JI-38), rat recombinant GH, MIA-602, or a combination of GHRH-A and MIA-602, for a 4-wk period. We assessed cardiac performance and hemodynamics by using echocardiography and micromanometry derived pressure-volume loops. Morphometric measurements were carried out to determine MI size and capillary density, and the expression of GHRHR was assessed by immunofluorescence and quantitative RT-PCR. GHRH-A markedly improved cardiac function as shown by echocardiographic and hemodynamic parameters. MI size was substantially reduced, whereas myocyte and nonmyocyte mitosis was markedly increased by GHRH-A. These effects occurred without increases in circulating levels of growth hormone and insulin-like growth factor I and were, at least partially, nullified by GHRH antagonism, confirming a receptor-mediated mechanism. GHRH-A stimulated CSCs proliferation ex vivo, in a manner offset by MIA-602. Collectively, our findings reveal the importance of the GHRH signaling pathway within the heart. Therapy with GHRH-A although initiated 1 mo after MI substantially improved cardiac performance and reduced infarct size, suggesting a regenerative process. Therefore, activation of GHRHR provides a unique therapeutic approach to reverse remodeling after MI.

Asunto(s)

Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Infarto del Miocardio/metabolismo , Receptores de Neuropéptido/metabolismo , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Sermorelina/análogos & derivados , Transducción de Señal/fisiología , Remodelación Ventricular/efectos de los fármacos , Análisis de Varianza , Animales , Proliferación Celular/efectos de los fármacos , Ecocardiografía , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Hormona del Crecimiento/administración & dosificación , Hormona Liberadora de Hormona del Crecimiento/administración & dosificación , Hormona Liberadora de Hormona del Crecimiento/agonistas , Hormona Liberadora de Hormona del Crecimiento/antagonistas & inhibidores , Hormona Liberadora de Hormona del Crecimiento/farmacología , Hemodinámica/efectos de los fármacos , Técnicas Histológicas , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Manometría , Infarto del Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Sermorelina/administración & dosificación , Sermorelina/farmacología

RESUMEN

UNLABELLED: Erythropoietin (EPO) has the potential to improve ischemic tissue by mobilizing endothelial progenitor cells and enhancing neovascularization. We hypothesized that combining EPO with human chorionic gonadotrophin (hCG) would improve post-myocardial infarction (MI) effects synergistically. METHODS: After MI, five to seven animals were randomly assigned to each of the following treatments: control; hCG; EPO; hCG + EPO, and prolactin (PRL) + EPO. Follow-up echocardiograms were performed to assess cardiac structure and function. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and western blot analysis for apoptosis-related proteins, and cell proliferation by immunostaining for Ki67 and c-kit cells. RESULTS: The MI-mediated increased chamber systolic dimension (p < 0.05 in controls) was attenuated by hCG, EPO, and hCG + EPO (p < 0.05 vs. control) but not PRL + EPO. Similarly all treatment groups, except PRL + EPO, reduced MI-induced increases (p < 0.05 vs. control) in ejection fraction (EF). The functional improvement in the EPO-treated groups was accompanied by increased capillary density. Apoptosis was markedly reduced in all treated groups. Significantly more cardiac c-kit(+) cells were found in the hCG + EPO group. CONCLUSION: Our findings revealed that EPO, hCG, or their combination ameliorate cardiac remodeling post-MI. Whereas EPO stimulates neovascularization only and hCG + EPO stimulates c-kit+ cell proliferation. These data suggest that combining mobilizing and proliferative agents adds to the durability and sustainability of cytokine-based therapies for remodeling post-MI.

Asunto(s)

Eritropoyetina/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Remodelación Ventricular , Animales , Apoptosis , Proliferación Celular , Gonadotropina Coriónica/metabolismo , Ecocardiografía/métodos , Humanos , Etiquetado Corte-Fin in Situ , Antígeno Ki-67/biosíntesis , Masculino , Proteínas Proto-Oncogénicas c-kit/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar

RESUMEN

BACKGROUND: The susceptibility to fibrosis and progression of renal disease is mitigated by inhibition of the renin-angiotensin system (RAS). We hypothesized that activation of the intrarenal RAS predisposes to renal fibrosis in aging. METHODS: Intrarenal expression of angiotensin II type 1 (AT(1)R), type 2 (AT(2)R), and (pro)renin receptors, ACE and ACE-2, as well as pro- and antioxidant enzymes were measured in 3-month-old (young), 14-month-old (middle-aged), and 24-month-old (old) male Sprague-Dawley rats. RESULTS: Old rats manifested glomerulosclerosis and severe tubulointerstitial fibrosis with increased fibronectin and TGF-ß expression (7-fold). AT(1)R /AT(2)R ratios were increased in middle-aged (cortical 1.6-fold, medullary 5-fold) and old rats (cortical 2-fold, medullary 4-fold). Similarly, (pro)renin receptor expression was increased in middle-aged (cortical 2-fold, medullary 3-fold) and old (cortical 5-fold, medullary 3-fold) rats. Cortical ACE was increased (+35%) in old rats, whereas ACE-2 was decreased (-50%) in middle-aged and old rats. NADPH oxidase activity was increased (2-fold), whereas antioxidant capacity and expression of the mitochondrial enzyme manganese superoxide dismutase (cortical -40%, medullary -53%) and medullary endothelial nitric oxide synthase (-48%) were decreased in old rats. CONCLUSION: Age-related intrarenal activation of the RAS preceded the development of severe renal fibrosis, suggesting that it contributes to the increased susceptibility to renal injury observed in the elderly.

Asunto(s)

Envejecimiento/fisiología , Corteza Renal/patología , Enfermedades Renales/metabolismo , Médula Renal/patología , Peptidil-Dipeptidasa A/metabolismo , Receptores de Angiotensina/metabolismo , Sistema Renina-Angiotensina/fisiología , Enzima Convertidora de Angiotensina 2 , Animales , Susceptibilidad a Enfermedades , Fibrosis , Corteza Renal/metabolismo , Enfermedades Renales/etiología , Médula Renal/metabolismo , Masculino , Óxido Nítrico/metabolismo , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Renina/metabolismo , Superóxido Dismutasa/metabolismo , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

S-Nitrosylation is a ubiquitous post-translational modification that regulates diverse biologic processes. In skeletal muscle, hypernitrosylation of the ryanodine receptor (RyR) causes sarcoplasmic reticulum (SR) calcium leak, but whether abnormalities of cardiac RyR nitrosylation contribute to dysfunction of cardiac excitation-contraction coupling remains controversial. In this study, we tested the hypothesis that cardiac RyR2 is hyponitrosylated in heart failure, because of nitroso-redox imbalance. We evaluated excitation-contraction coupling and nitroso-redox balance in spontaneously hypertensive heart failure rats with dilated cardiomyopathy and age-matched Wistar-Kyoto rats. Spontaneously hypertensive heart failure myocytes were characterized by depressed contractility, increased diastolic Ca(2+) leak, hyponitrosylation of RyR2, and enhanced xanthine oxidase derived superoxide. Global S-nitrosylation was decreased in failing hearts compared with nonfailing. Xanthine oxidase inhibition restored global and RyR2 nitrosylation and reversed the diastolic SR Ca(2+) leak, improving Ca(2+) handling and contractility. Together these findings demonstrate that nitroso-redox imbalance causes RyR2 oxidation, hyponitrosylation, and SR Ca(2+) leak, a hallmark of cardiac dysfunction. The reversal of this phenotype by inhibition of xanthine oxidase has important pathophysiologic and therapeutic implications.

Asunto(s)

Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Procesamiento Proteico-Postraduccional , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Xantina Oxidasa/metabolismo , Animales , Humanos , Oxidación-Reducción , Ratas , Ratas Endogámicas WKY , Superóxidos/metabolismo

RESUMEN

Whether the growth hormone (GH)/insulin-like growth factor 1(IGF-1) axis exerts cardioprotective effects remains controversial; and the underlying mechanism(s) for such actions are unclear. Here we tested the hypothesis that growth hormone-releasing hormone (GHRH) directly activates cellular reparative mechanisms within the injured heart, in a GH/IGF-1 independent fashion. After experimental myocardial infarction (MI), rats were randomly assigned to receive, during a 4-week period, either placebo (n = 14), rat recombinant GH (n = 8) or JI-38 (n = 8; 50 microg/kg per day), a potent GHRH agonist. JI-38 did not elevate serum levels of GH or IGF-1, but it markedly attenuated the degree of cardiac functional decline and remodeling after injury. In contrast, GH administration markedly elevated body weight, heart weight, and circulating GH and IGF-1, but it did not offset the decline in cardiac structure and function. Whereas both JI-38 and GH augmented levels of cardiac precursor cell proliferation, only JI-38 increased antiapoptotic gene expression. The receptor for GHRH was detectable on myocytes, supporting direct activation of cardiac signal transduction. Collectively, these findings demonstrate that within the heart, GHRH agonists can activate cardiac repair after MI, suggesting the existence of a potential signaling pathway based on GHRH in the heart. The phenotypic profile of the response to a potent GHRH agonist has therapeutic implications.

Asunto(s)

Cardiotónicos/farmacología , Hormona Liberadora de Hormona del Crecimiento/agonistas , Hormona del Crecimiento/farmacología , Infarto del Miocardio/prevención & control , Animales , Western Blotting , Peso Corporal/efectos de los fármacos , Ecocardiografía , Femenino , Hormona del Crecimiento/sangre , Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Hormona Liberadora de Hormona del Crecimiento/farmacología , Corazón/efectos de los fármacos , Corazón/fisiopatología , Hemodinámica/efectos de los fármacos , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/metabolismo , Infarto del Miocardio/patología , Miocardio/metabolismo , Miocardio/patología , Tamaño de los Órganos/efectos de los fármacos , Distribución Aleatoria , Ratas , Ratas Endogámicas F344 , Receptores de Neuropéptido/metabolismo , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Proteínas Recombinantes/farmacología

RESUMEN

Nitric oxide (NO) exerts ubiquitous signaling via posttranslational modification of cysteine residues, a reaction termed S-nitrosylation. Important substrates of S-nitrosylation that influence cardiac function include receptors, enzymes, ion channels, transcription factors, and structural proteins. Cardiac ion channels subserving excitation-contraction coupling are potentially regulated by S-nitrosylation. Specificity is achieved in part by spatial colocalization of ion channels with nitric oxide synthases (NOSs), enzymatic sources of NO in biologic systems, and by coupling of NOS activity to localized calcium/second messenger concentrations. Ion channels regulate cardiac excitability and contractility in millisecond timescales, raising the possibility that NO-related species modulate heart function on a beat-to-beat basis. This review focuses on recent advances in understanding of NO regulation of the cardiac action potential and of the calcium release channel ryanodine receptor, which is crucial for the generation of force. S-Nitrosylation signaling is disrupted in pathological states in which the redox state of the cell is dysregulated, including ischemia, heart failure, and atrial fibrillation.

Asunto(s)

Canales Iónicos/metabolismo , Miocardio/metabolismo , Procesamiento Proteico-Postraduccional , Regulación Alostérica , Animales , Fibrilación Atrial/metabolismo , Acoplamiento Excitación-Contracción , Corazón/fisiología , Corazón/fisiopatología , Insuficiencia Cardíaca/metabolismo , Humanos , Canales Iónicos/química , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Contracción Miocárdica , Óxido Nítrico/metabolismo , Nitrosación , Transducción de Señal

Asunto(s)

Mitocondrias Cardíacas/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Gatos , Citosol/fisiología , Transporte de Electrón/fisiología , Metabolismo Energético/fisiología , Corazón/fisiología , Óxido Nítrico/fisiología , Permeabilidad , Canales de Potasio/fisiología , Proteína Quinasa C-epsilon/fisiología , Urea/metabolismo

RESUMEN

The role of nitric oxide (NO) in cardiac contractility is complex and controversial. Several NO donors have been reported to cause positive or negative inotropism. NO can bind to guanylate cyclase, increasing cGMP production and activating PKG. NO may also directly S-nitrosylate cysteine residues of specific proteins. We used the isolated rat heart preparation to test the hypothesis that the differential inotropic effects depend on the degree of NO production and the signaling recruited. SNAP (S-nitroso-N-acetylpenicillamine), a NO donor, increased contractility at 0.1, 1 and 10 microM. This effect was independent of phospholamban phosphorylation, was not affected by PKA inhibition with H-89 (N-[2((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide), but it was abolished by the radical scavenger Tempol (4-hydroxy-[2,2,4,4]-tetramethyl-piperidine-1-oxyl). However, at 100 microM SNAP reduced contractility, effect reversed to positive inotropism by guanylyl cyclase blockade with ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), and abolished by PKG inhibition with KT5823, but not affected by Tempol. SNAP increased tissue cGMP at 100 microM, but not at lower concentrations. Consistently, a cGMP analog also reduced cardiac contractility. Finally, SNAP at 1 microM increased the level of S-nitrosylation of various cardiac proteins, including the ryanodine receptor. This study demonstrates the biphasic role for NO in cardiac contractility in a given preparation; furthermore, the differential effect is clearly ascribed to the signaling pathways involved. We conclude that although NO is highly diffusible, its output determines the fate of the messenger: low NO concentrations activate redox processes (S-nitrosylation), increasing contractility; while the cGMP-PKG pathway is activated at high NO concentrations, reducing contractility.

Asunto(s)

Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Contracción Miocárdica/fisiología , Óxido Nítrico/metabolismo , S-Nitroso-N-Acetilpenicilamina/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Carbazoles/farmacología , GMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Óxidos N-Cíclicos/farmacología , Masculino , Contracción Miocárdica/efectos de los fármacos , Óxido Nítrico/biosíntesis , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , S-Nitroso-N-Acetilpenicilamina/antagonistas & inhibidores , S-Nitroso-N-Acetilpenicilamina/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Marcadores de Spin