Cardioprotective role of IGF-1 in the hypertrophied myocardium of the spontaneously hypertensive rats: A key effect on NHE-1 activity

Aim: Myocardial Na+/H+ exchanger-1 (NHE-1) hyperactivity and oxidative stress are interrelated phenomena playing pivotal roles in the development of pathological cardiac hypertrophy and heart failure. Exercise training is effective to convert pathological into physiological hypertrophy in the spontaneously hypertensive rats (SHR), and IGF-1—key humoral mediator of exercise training—inhibits myocardial NHE-1, at least in normotensive rats. Therefore, we hypothesize that IGF-1 by hampering NHE-1 hyperactivity and oxidative stress should exert a cardioprotective effect in the SHR. Methods: NHE-1 activity [proton efflux (JH+) mmol L-1 min-1], expression and phosphorylation; H2O2 production; superoxide dismutase (SOD) activity; contractility and calcium transients were measured in SHR hearts in the presence/absence of IGF-1. Results: IGF-1 significantly decreased NHE-1 activity (JH+ at pHi 6.95: 1.39 ± 0.32, n = 9 vs C 3.27 ± 0.3, n = 20, P 2O2 production accompanied by an increase in SOD activity. In addition, IGF-1 improved cardiomyocyte contractility as evidenced by an increase in sarcomere shortening and a decrease in the relaxation constant, underlined by an increase in the amplitude and rate of decay of the calcium transients. Conclusion: IGF-1 exerts a cardioprotective role on the hypertrophied hearts of the SHR, in which the inhibition of NHE-1 hyperactivity, as well as the positive inotropic and antioxidant effects, emerges as key players.Centro de Investigaciones Cardiovasculare

Efecto de los ácidos grasos W3 sobre la estructura de membranas y actividad del NHE-1 en ratas normotensas y espontáneamente hipertensas (SHR)

Los ácidos grasos de la serie w-3 han sido asociados experimental y epidemiológicamente a diversos estados fisiopatológicos, como inflamación, diabetes, enfermedad cardiovascular y cáncer. Uno de los mecanismos moleculares mediante los cuales estos ácidos grasos ejercen su efecto biológico consiste en su incorporación en membranas celulares, afectando las propiedades biofísicas y bioquímicas de las mismas, impactando específicamente sobre la estructura y organización de lipid rafts, la localización de proteínas de vías de señalización y, en definitiva, sobre numerosas funciones celulares. Los lipid rafts se definen como dominios especializados de membranas biológicas, enriquecidos en esfingolípidos (SLs) y colesterol (Col), que reclutan proteínas específicas. Las caveolas son un tipo especializado de rafts que se caracteriza por su estructura invaginada de 50-100 nm de diámetro, revestida y estabilizada por caveolinas y cavinas, entre otras proteínas. El intercambiador Na+/H+ (NHE-1), una proteína integral de membrana, está involucrado en el mantenimiento del pH intracelular (pH ). Su actividad está regulada por la sensibilidad del sitio alostérico i para el H+, fosforilación y por la unión de ATP, lípidos y factores de crecimiento. Diversos estudios en corazón de ratas SHR han demostrado la hiperactividad de dicho transportador, y que la inhibición del mismo revierte la hipertrofia que éstos presentan. El objetivo es estudiar la composición lipídica y contenidos de PUFAs de la serie w3 en sangre de ratas normotensas (Wistar) y espontáneamente hipertensas (SHR), en relación con la organización de membrana plasmática –lipid rafts/caveolas- y actividad del intercambiador Na+ /H+ miocárdico (NHE-1)

Aldosterone stimulates the cardiac Na +/H + exchanger via transactivation of the epidermal growth factor receptor

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na/H exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90 RSK and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.Facultad de Ciencias Médica

Aldosterone stimulates the cardiac Na +/H + exchanger via transactivation of the epidermal growth factor receptor

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na/H exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90 RSK and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.Facultad de Ciencias Médica

Aldosterone stimulates the cardiac Na +/H + exchanger via transactivation of the epidermal growth factor receptor

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na/H exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90 RSK and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.Facultad de Ciencias Médica

The positive inotropic effect of angiotensin II : Role of endothelin-1 and reactive oxygen species

Many effects believed to be because of angiotensin II (Ang II) are attributable to the action of endothelin (ET)-1, which is released/produced by Ang II. We investigated whether Ang II elicits its positive inotropic effect (PIE) by the action of endogenous ET-1, in addition to the role played by reactive oxygen species (ROS) in this mechanism. Cat cardiomyocytes were used for: (1) sarcomere shortening measurements; (2) ROS measurements by epifluorescence; (3) immunohistochemical staining for preproET-1, BigET-1, and ET-1; and (4) measurement of preproET-1 mRNA by RT-PCR. Cells were exposed to 1 nmol/L Ang II for 15 minutes. This low concentration of Ang II increases sarcomere shortening by 29.2±3.7% (P<0.05). This PIE was abrogated by Na+/H+ exchanger or Na+/Ca2+ exchanger reverse mode inhibition. The production of ROS increased in response to Ang II treatment (ΔROS respect to control: 68±15 fluorescence units; P<0.05). The Ang II-induced PIE and ROS production were blocked by the Ang II type 1 receptor blocker losartan, the nonselective ET-1 receptor blocker TAK044, the selective ETA receptor blocker BQ-123, or the ROS scavenger N-(2-mercapto-propionyl)glycine. Exogenous ET-1 (0.4 nmol/L) induced a similar PIE and increase in ROS production to those caused by Ang II. Immunostaining for preproET-1, BigET-1, and ET-I was positive in cardiomyocytes. The preproET-1 mRNA abundance increased from 100±4.6% in control to 241.9±39.9% in Ang II-treated cells (P<0.05). We conclude that the PIE after exposure to 1 nmol/L Ang II is due to endogenous ET-1 acting through the ETA receptor and triggering ROS production, Na+/H+ exchanger stimulation, and Na+/Ca2+ exchanger reverse mode activation.Facultad de Ciencias Médica

Estrés oxidativo: nexo entre aldosterona y el daño del miocardio mediado por el NHE1

En los estudios clínicos RALES, EPHESUS y EMPHASIS se demostraron los efectos beneficiosos del antagonismo de la aldosterona (Aldo) en la insuficiencia cardíaca (IC), aunque no se conocen los mecanismos implicados. Por otro lado, el estrés oxidativo, aumentado en la IC, resulta deletéreo por diferentes mecanismos, siendo uno de ellos la activación de quinasas redox-sensibles que fosforilan y estimulan al NHE1. Además, la inhibición del NHE1 es beneficiosa en modelos experimentales de hipertrofia e IC.Facultad de Ciencias Médica

The positive inotropic effect of angiotensin II : Role of endothelin-1 and reactive oxygen species

Many effects believed to be because of angiotensin II (Ang II) are attributable to the action of endothelin (ET)-1, which is released/produced by Ang II. We investigated whether Ang II elicits its positive inotropic effect (PIE) by the action of endogenous ET-1, in addition to the role played by reactive oxygen species (ROS) in this mechanism. Cat cardiomyocytes were used for: (1) sarcomere shortening measurements; (2) ROS measurements by epifluorescence; (3) immunohistochemical staining for preproET-1, BigET-1, and ET-1; and (4) measurement of preproET-1 mRNA by RT-PCR. Cells were exposed to 1 nmol/L Ang II for 15 minutes. This low concentration of Ang II increases sarcomere shortening by 29.2±3.7% (P<0.05). This PIE was abrogated by Na+/H+ exchanger or Na+/Ca2+ exchanger reverse mode inhibition. The production of ROS increased in response to Ang II treatment (ΔROS respect to control: 68±15 fluorescence units; P<0.05). The Ang II-induced PIE and ROS production were blocked by the Ang II type 1 receptor blocker losartan, the nonselective ET-1 receptor blocker TAK044, the selective ETA receptor blocker BQ-123, or the ROS scavenger N-(2-mercapto-propionyl)glycine. Exogenous ET-1 (0.4 nmol/L) induced a similar PIE and increase in ROS production to those caused by Ang II. Immunostaining for preproET-1, BigET-1, and ET-I was positive in cardiomyocytes. The preproET-1 mRNA abundance increased from 100±4.6% in control to 241.9±39.9% in Ang II-treated cells (P<0.05). We conclude that the PIE after exposure to 1 nmol/L Ang II is due to endogenous ET-1 acting through the ETA receptor and triggering ROS production, Na+/H+ exchanger stimulation, and Na+/Ca2+ exchanger reverse mode activation.Facultad de Ciencias Médica

Papel del intercambiador Na<SUP>+</SUP>/H<SUP>+</SUP> en la hipertrofia cardíaca fisiológica

Ante situaciones de sobrecarga hemodinámica el corazón responde aumentando su masa, condición denominada hipertrofia cardíaca (HC). La HC puede ser fisiológica, inducida por entrenamiento aeróbico intenso o embarazo; o patológica cuando se asocia a enfermedad cardiovascular, por ejemplo hipertensión arterial o post infarto agudo de miocardio. Mientras que la HC fisiológica constituye una respuesta adaptativa del miocardio que preserva o mejora la función contráctil, la HC patológica es uno de los principales predictores de insuficiencia cardíaca y mortalidad cardiovascular.Facultad de Ciencias Médica

Physiological cardiac hypertrophy: Critical role of AKT in the prevention of NHE-1 hyperactivity

Background: The involvement of NHE-1 hyperactivity, critical for pathological cardiac hypertrophy (CH), in physiological CH has not been elucidated yet. Stimulation of NHE-1 increases intracellular Na+ and Ca2 + favouring calcineurin activation. Since myocardial stretch, an activator of NHE-1, is common to both types of CH, we speculate that NHE-1 hyperactivity may also happen in physiological CH. However, calcineurin activation is characteristic only for pathological hypertrophy. We hypothesize that an inhibitory AKT-dependent mechanism prevents NHE-1 hyperactivity in the setup of physiological CH. Methods: Physiological CH was induced in rats by swimming (90 min/day, 12 weeks) or in cultured isolated cardiomyocytes with IGF-1 (10 nmol/L). Results: Training induced eccentric CH development (left ventricular weight/tibial length: 22.0 ± 0.3 vs. 24.3 ± 0.7 mg/mm; myocyte cross sectional area: 100 ± 3.2 vs. 117 ± 4.1 %; sedentary (Sed) and swim-trained (Swim) respectively; p < 0.05] with decreased myocardial stiffness and collagen deposition [1.7 ± 0.05 % (Sed) vs. 1.4 ± 0.09 % (Swim); p < 0.05]. Increased phosphorylation of AKT, ERK1/2, p90RSK and NHE-1 at the consensus site for ERK1/2-p90RSK were detected in the hypertrophied hearts (P-AKT: 134 ± 10 vs. 100 ± 5; P-ERK1/2: 164 ± 17 vs. 100 ± 18; P-p90RSK: 160 ± 18 vs. 100 ± 9; P-NHE-1 134 ± 10 vs. 100 ± 10; % in Swim vs. Sed respectively; p < 0.05). No significant changes were detected neither in calcineurin activation [calcineurin Aβ 100 ± 10 (Sed) vs. 96 ± 12 (Swim)], nor NFAT nuclear translocation [100 ± 3.11 (Sed) vs. 95 ± 9.81 % (Swim)] nor NHE-1 expression [100 ± 8.5 (Sed) vs. 95 ± 6.7 % (Swim)]. Interestingly, the inhibitory phosphorylation of the NHE-1 consensus site for AKT was increased in the hypertrophied myocardium (151.6 ± 19.4 (Swim) vs. 100 ± 9.5 % (Sed); p < 0.05). In isolated cardiomyocytes 24 hours IGF-1 increased cell area (114 ± 1.3 %; p < 0.05) and protein/DNA content (115 ± 3.9 %, p < 0.05), effects not abolished by NHE-1 inhibition with cariporide (114 ± 3 and 117 ± 4.4 %, respectively). IGF-1 significantly decreased NHE-1 activity during pHi recovery from sustained intracellular acidosis (JH + at pHi 6.8: 4.08 ± 0.74 and 9.09 ± 1.21 mmol/L/min, IGF-1 vs. control; p < 0.05), and abolished myocardial slow force response, the mechanical counterpart of stretch-induced NHE-1 activation. Conclusions: NHE-1 hyperactivity seems not to be involved in physiological CH development, contrary to what characterizes pathological CH. We propose that AKT, through an inhibitory phosphorylation of the NHE-1, prevents its stretch-induced activation. This posttranslational modification emerges as an adaptive mechanism that avoids NHE-1 hyperactivity preserving its housekeeping functioning.Fil: Yeves, Alejandra del Milagro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; ArgentinaFil: Villa-Abrille, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; ArgentinaFil: Perez, Nestor Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; ArgentinaFil: Medina, Andrés Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; ArgentinaFil: Escudero, Eduardo M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; ArgentinaFil: Ennis, Irene Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Centro de Investigaciones Cardiovasculares ; Argentin