RESUMEN
OBJECTIVES: Investigate if azilsartan protects against myocardial hypertrophy by upregulating nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated pathways. METHODS: Abdominal aortic constriction (AAC)-induced cardiac hypertrophy in rats was applied. Azilsartan or vehicle was administered daily for 6 weeks in sham or AAC rats. Cardiac morphology and ventricular function were determined. Azilsartan effects upon neonatal rat cardiomyocyte (NRCM) hypertrophy and molecular mechanisms were studied in angiotensin (Ang) II-stimulated NRCMs in vitro. Nrf2-small interfering RNA (siRNA) was used to knockdown Nrf2 expression. Messenger RNA (mRNA)/protein expression of Kelch-like erythroid cell-derived protein (Keap)1 and Nrf2 and its downstream antioxidant enzymes was determined by real-time reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. KEY FINDINGS: Azilsartan treatment ameliorated cardiac hypertrophy/fibrosis significantly in AAC rats. Azilsartan increased expression of Nrf2 protein but decreased expression of Keap1 protein. Upregulation of protein expression of Nrf2's downstream antioxidant enzymes by azilsartan treatment was observed. Azilsartan inhibited Ang II-induced NRCM hypertrophy significantly and similar effects on the Keap1-Nrf2 pathway were observed in vivo. Nrf2 knockdown markedly counteracted the beneficial effects of azilsartan on NRCM hypertrophy and the Keap1-Nrf2 pathway. CONCLUSIONS: Azilsartan restrained pressure overload-induced cardiac remodelling by activating the Keap1-Nrf2 pathway and increasing expression of downstream antioxidant enzymes to alleviate oxidative stress.
Asunto(s)
Antagonistas de Receptores de Angiotensina/farmacología , Bencimidazoles/farmacología , Cardiomegalia/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Miocardio/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Oxadiazoles/farmacología , Estrés Oxidativo/efectos de los fármacos , Angiotensina II/metabolismo , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Cardiomegalia/tratamiento farmacológico , Femenino , Ventrículos Cardíacos/efectos de los fármacos , Masculino , Miocitos Cardíacos/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Transducción de Señal , Regulación hacia ArribaRESUMEN
Estrogen deficiency induces cardiac dysfunction and increases the risk of cardiovascular disease in postmenopausal women and in those who underwent bilateral oophorectomy. Previous evidence suggests that puerarin, a phytoestrogen, exerts beneficial effects on cardiac function in patients with cardiac hypertrophy. In this study, we investigated whether puerarin could prevent cardiac hypertrophy and remodeling in ovariectomized, aortic-banded rats. Female SD rats subjected to bilateral ovariectomy (OVX) plus abdominal aortic constriction (AAC). The rats were treated with puerarin (50 mg·kg-1 ·d-1, ip) for 8 weeks. Then echocardiography was assessed, and the rats were sacrificed, their heart tissues were extracted and allocated for further experiments. We showed that puerarin administration significantly attenuated cardiac hypertrophy and remodeling in AAC-treated OVX rats, which could be attributed to activation of PPARα/PPARγ coactivator-1 (PGC-1) pathway. Puerarin administration significantly increased the expression of estrogen-related receptor α, nuclear respiratory factor 1, and mitochondrial transcription factor A in hearts. Moreover, puerarin administration regulated the expression of metabolic genes in AAC-treated OVX rats. Hypertrophic changes could be induced in neonatal rat cardiomyocytes (NRCM) in vitro by treatment with angiotensin II (Ang II, 1 µM), which was attenuated by co-treatemnt with puerarin (100 µM). We further showed that puerarin decreased Ang II-induced accumulation of non-esterified fatty acids (NEFAs) and deletion of ATP, attenuated the Ang II-induced dissipation of the mitochondrial membrane potential, and improved the mitochondrial dysfunction in NRCM. Furthermore, addition of PPARα antagonist GW6471 (10 µM) partially abolished the anti-hypertrophic effects and metabolic effects of puerarin in NRCM. In conclusion, puerarin prevents cardiac hypertrophy in AAC-treated OVX rats through activation of PPARα/PGC-1 pathway and regulation of energy metabolism remodeling. This may provide a new approach to prevent the development of heart failure in postmenopausal women.
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Cardiomegalia/prevención & control , Cardiotónicos/uso terapéutico , Isoflavonas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Angiotensina II/farmacología , Animales , Aorta Abdominal/patología , Cardiomegalia/etiología , Cardiomegalia/patología , Constricción Patológica/complicaciones , Metabolismo Energético/efectos de los fármacos , Femenino , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Ovariectomía , PPAR alfa/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Ratas Sprague-DawleyRESUMEN
Puerarin is an isoflavone isolated from Radix puerariae. Emerging evidence shown that puerarin possesses therapeutic benefits that aid in the prevention of cardiovascular diseases. In this study, we evaluated the effects of puerarin on oxidative stress and cardiac fibrosis induced by abdominal aortic banding (AB) and angiotensin II (AngII). We also investigated the mechanisms underlying this phenomenon. The results of histopathological analysis, as well as measurements of collagen expression and cardiac fibroblast proliferation indicated that puerarin administration significantly inhibited cardiac fibrosis induced by AB and AngII. These effects of puerarin may reflect activation of Nrf2/ROS pathway. This hypothesis is supported by observed decreases of reactive oxygen species (ROS), decreases Keap 1, increases Nrf2 expression and nuclear translocation, and decreases of collagen expressions in cardiac fibroblasts treated with a combination of puerarin and AngII. Inhibition of Nrf2 with specific Nrf2 siRNA or Nrf2 inhibitor brusatol attenuated anti-fibrotic and anti-oxidant effects of puerarin. The metabolic effects of puerarin were mediated by Nrf2 through upregulation of UDP-glucuronosyltransferase (UGT) 1A1. The Nrf2 agonist tBHQ upregulated protein expression of UGT1A1 over time in cardiac fibroblasts. Treatment with Nrf2 siRNA or brusatol dramatically decreased UGT1A1 expression in puerarin-treated fibroblasts. The results of chromatin immunoprecipitation-qPCR further confirmed that puerarin significantly increased binding of Nrf2 to the promoter region of Ugt1a1. Western blot analysis showed that puerarin significantly inhibited AngII-induced phosphorylation of p38-MAPK. A specific inhibitor of p38-MAPK, SB203580, decreased collagen expression, and ROS generation induced by AngII in cardiac fibroblast. Together, these results suggest that puerarin prevents cardiac fibrosis via activation of Nrf2 and inactivation of p38-MAPK. Nrf2 is the key regulator of anti-fibrotic effects and upregulates metabolic enzymes UGT1A1. Autoregulatory circuits between puerarin and Nrf2-regulated UGT1A1 attenuates side effects associated with treatment, but it does not weaken puerarin's pharmacological effects.
RESUMEN
Previous evidence has suggested that puerarin may attenuate cardiac hypertrophy; however, the potential mechanisms have not been determined. Moreover, the use of puerarin is limited by severe adverse events, including intravascular hemolysis. This study used a rat model of abdominal aortic constriction (AAC)-induced cardiac hypertrophy to evaluate the potential mechanisms underlying the attenuating efficacy of puerarin on cardiac hypertrophy, as well as the metabolic mechanisms of puerarin involved. We confirmed that puerarin (50 mg/kg per day) significantly attenuated cardiac hypertrophy, upregulated Nrf2, and decreased Keap1 in the myocardium. Moreover, puerarin significantly promoted Nrf2 nuclear accumulation in parallel with the upregulated downstream proteins, including heme oxygenase 1, glutathione transferase P1, and NAD(P)H:quinone oxidoreductase 1. Similar results were obtained in neonatal rat cardiomyocytes (NRCMs) treated with angiotensin II (Ang II; 1 µM) and puerarin (100 µM), whereas the silencing of Nrf2 abolished the antihypertrophic effects of puerarin. The mRNA and protein levels of UGT1A1 and UGT1A9, enzymes for puerarin metabolism, were significantly increased in the liver and heart tissues of AAC rats and Ang II-treated NRCMs. Interestingly, the silencing of Nrf2 attenuated the puerarin-induced upregulation of UGT1A1 and UGT1A9. The results of chromatin immunoprecipitation-quantitative polymerase chain reaction indicated that the binding of Nrf2 to the promoter region of Ugt1a1 or Ugt1a9 was significantly enhanced in puerarin-treated cardiomyocytes. These results suggest that Nrf2 is the key regulator of antihypertrophic effects and upregulation of the metabolic enzymes UGT1A1 and UGT1A9 of puerarin. The autoregulatory circuits between puerarin and Nrf2-induced UGT1A1/1A9 are beneficial to attenuate adverse effects and maintain the pharmacologic effects of puerarin.
Asunto(s)
Cardiomegalia/metabolismo , Cardiomegalia/prevención & control , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Isoflavonas/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Cardiomegalia/genética , Cardiomegalia/patología , Femenino , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Estrés Oxidativo/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/efectos de los fármacosRESUMEN
This study aimed to investigate the anti-oxidant and anti-hypertrophic effects of puerarin-7-O-glucuronide, a water-soluble puerarin metabolite. The anti-oxidant effects of puerarin-7-O-glucuronide were assessed by measurement of intracellular superoxide levels, total superoxide dismutase (SOD) activity, total anti-oxidant capacity, and glutathione (GSH)/glutathione disulfide (GSSG) ratio in cultured neonatal rat cardiomyocytes (NRCMs) stimulated with the xanthine oxidase (XO)/xanthine (X) system or angiotensin II. The activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and expression of NADPH oxidase subunits p22phox and p47phox were determined. The anti-hypertrophic effects of puerarin-7-O-glucuronide in angiotensin II-challenged NRCMs were characterized by changes in cell morphology and expression of hypertrophic genes. In the pharmacokinetic study, the plasma concentration of puerarin-7-O-glucuronide was determined by rapid resolution-liquid chromatography-tandem mass spectrometry (RR-LC-MS/MS). Puerarin-7-O-glucuronide prevented XO/X-induced increase in intracellular superoxide production and decreases in total SOD activity, GSH/GSSG ratio, and total anti-oxidant capacity. Puerarin-7-O-glucuronide also reversed angiotensin II-induced increases in intracellular superoxide production and NADPH oxidase activity and decreases in total SOD activity. These anti-oxidant effects of puerarin-7-O-glucuronide were accompanied by downregulation of p22phox and p47phox. Furthermore, puerarin-7-O-glucuronide prevented angiotensin II-induced increases in cell surface area and perimeter, as well as changes in Nppa, Myh7, and Myh6. In the pharmacokinetic study, puerarin-7-O-glucuronide was cleared with a half-life of 0.94 h after intravenous administration. Puerarin could be detected in rat plasma, albeit in low concentration, as early as 5 min after intravenous administration of puerarin-7-O-glucuronide. These anti-oxidant and anti-hypertrophic properties of puerarin-7-O-glucuronide were similar to those of its parent compound puerarin. These results support the development of puerarin-7-O-glucuronide as a novel pharmaceutical agent for therapeutic application.
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Angiotensina II/toxicidad , Antioxidantes/farmacología , Cardiomegalia/prevención & control , Glucurónidos/farmacología , Isoflavonas/farmacología , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Solventes/química , Agua/química , Animales , Animales Recién Nacidos , Antioxidantes/administración & dosificación , Antioxidantes/química , Antioxidantes/farmacocinética , Cardiomegalia/inducido químicamente , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patología , Células Cultivadas , Femenino , Glucurónidos/administración & dosificación , Glucurónidos/química , Glucurónidos/farmacocinética , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Inyecciones Intravenosas , Isoflavonas/administración & dosificación , Isoflavonas/química , Isoflavonas/farmacocinética , Masculino , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Ratas Sprague-Dawley , Solubilidad , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Xantina/farmacología , Xantina Oxidasa/farmacologíaRESUMEN
Cluster of differentiation 73 (CD73) is an ecto-5' nucleotidase which catalyzes the conversion of AMP to adenosine. One of the many functions of adenosine is to suppress the activity of tissue nonspecific alkaline phosphatase (TNAP), an enzyme important in regulating intracellular calcification. Since myocardial calcification is associated with various cardiac disease states, we studied the individual roles and crosstalk between CD73 and TNAP in regulating myocyte responses to the α1 adrenoceptor agonist phenylephrine in terms of calcification and hypertrophy. Cultured neonatal rat cardiomyocytes were treated with 10 µM phenylephrine for 24 h in the absence or presence of the stable adenosine analog 2-chloro-adenosine, the TNAP inhibitor tetramisole or the CD73 inhibitor α,ß-methylene ADP. Phenylephrine produced marked hypertrophy as evidenced by significant increases in myocyte surface area and ANP gene expression, as well as calcification determined by Alizarin Red S staining. These responses were associated with reduced CD73 gene and protein expression and CD73 activity. Conversely, TNAP expression and activity were significantly increased although both were suppressed by 2-chloro-adenosine. CD73 inhibition alone significantly reduced myocyte-derived adenosine levels by >50 %, and directly induced hypertrophy and calcification in the absence of phenylephrine. These responses and those to phenylephrine were abrogated by TNAP inhibition. We conclude that TNAP contributes to the hypertrophic effect of phenylephrine, as well as its ability to produce cardiomyocyte calcification. These responses are minimized by CD73-dependent endogenously produced adenosine.
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5'-Nucleotidasa/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/toxicidad , Fosfatasa Alcalina/metabolismo , Cardiomegalia/inducido químicamente , Miocitos Cardíacos/efectos de los fármacos , Fenilefrina/toxicidad , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Calcificación Vascular/inducido químicamente , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/genética , Adenosina/metabolismo , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/genética , Animales , Animales Recién Nacidos , Factor Natriurético Atrial/metabolismo , Cardiomegalia/enzimología , Cardiomegalia/genética , Cardiomegalia/patología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación de la Expresión Génica , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 1/metabolismo , Transducción de Señal , Factores de Tiempo , Calcificación Vascular/enzimología , Calcificación Vascular/genética , Calcificación Vascular/patologíaRESUMEN
The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation. Ubiquitously expressed, FTO was identified in heart homogenates although its function is unknown. We studied whether FTO is specifically expressed within the cardiac myocyte and its potential role pertaining to the hypertrophic effect of the adipokine leptin. Most experiments were performed using cultured neonatal rat cardiomyocytes which showed nuclei-specific FTO expression. Leptin significantly increased FTO expression which was associated with myocyte hypertrophy although both events were abrogated by FTO knockdown with siRNA. Administration of a leptin receptor antibody to either normal or obese rats significant reduced myocardial FTO protein expression. Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects. Expression of the cut-like homeobox 1(CUX1) transcriptional factor was significantly increased by leptin although this was restricted to the cathepsin L-dependent, proteolytically-derived shorter p110CUX1 isoform whereas the longer p200CUX1 protein was not significantly affected. Cathepsin L expression and activity were both significantly increased by leptin whereas a cathepsin L peptide inhibitor or siRNA specific for CUX1 completely prevented the leptin-induced increase in FTO expression. The cathepsin L peptide inhibitor or siRNA-induced knockdown of either CUX1 or FTO abrogated the hypertrophic response to leptin. Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent. This study demonstrates leptin-induced FTO upregulation in cardiomyocytes via JAK2/STAT3- dependent CUX1 upregulation and suggests an FTO regulatory function of leptin. It also demonstrates for the first time a functional role of FTO in the cardiomyocyte.