RESUMEN
BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na+/K+ pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized. METHODS: Effects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects). RESULTS: As expected from SERCA2a stimulation, compound 8 induced SR Ca2+ compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity. CONCLUSIONS: The pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity.
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Etiocolanolona/análogos & derivados , Insuficiencia Cardíaca , Ratas , Ratones , Animales , Cobayas , Insuficiencia Cardíaca/metabolismo , Enfermedad Crónica , Inhibidores Enzimáticos , Cardiotónicos/uso terapéutico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Miocitos Cardíacos/metabolismo , Calcio/metabolismoRESUMEN
Heart failure (HF) therapeutic toolkit would strongly benefit from the availability of ino-lusitropic agents with a favorable pharmacodynamics and safety profile. Istaroxime is a promising agent, which combines Na+/K+ pump inhibition with sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) stimulation; however, it has a very short half-life and extensive metabolism to a molecule named PST3093. The present work aims to investigate whether PST3093 still retains the pharmacodynamic and pharmacokinetic properties of its parent compound. We studied PST3093 for its effects on SERCA2a and Na+/K+ ATPase activities, Ca2+ dynamics in isolated myocytes, and hemodynamic effects in an in vivo rat model of diabetic [streptozotocin (STZ)-induced] cardiomyopathy. Istaroxime infusion in HF patients led to accumulation of PST3093 in the plasma; clearance was substantially slower for PST3093 than for istaroxime. In cardiac rat preparations, PST3093 did not inhibit the Na+/K+ ATPase activity but retained SERCA2a stimulatory activity. In in vivo echocardiographic assessment, PST3093 improved overall cardiac performance and reversed most STZ-induced abnormalities. PST3093 intravenous toxicity was considerably lower than that of istaroxime, and it failed to significantly interact with 50 off-targets. Overall, PST3093 is a "selective" SERCA2a activator, the prototype of a novel pharmacodynamic category with a potential in the ino-lusitropic approach to HF with prevailing diastolic dysfunction. Its pharmacodynamics are peculiar, and its pharmacokinetics are suitable to prolong the cardiac beneficial effect of istaroxime infusion. SIGNIFICANCE STATEMENT: Heart failure (HF) treatment would benefit from the availability of ino-lusitropic agents with favourable profiles. PST3093 is the main metabolite of istaroxime, a promising agent combining Na+/K+ pump inhibition and sarcoplasmic reticulum Ca2+ ATPase2a (SERCA2a) stimulation. PST3093 shows a longer half-life in human circulation compared to istaroxime, selectively activates SERCA2a, and improves cardiac performance in a model of diabetic cardiomyopathy. Overall, PST3093 as a selective SERCA2a activator can be considered the prototype of a novel pharmacodynamic category for HF treatment.
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Insuficiencia Cardíaca , Corazón , Animales , Humanos , Ratas , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/farmacología , Adenosina Trifosfatasas/uso terapéutico , Etiocolanolona/farmacología , Etiocolanolona/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Miocitos Cardíacos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/uso terapéuticoRESUMEN
TRANSLATIONS: For the Chinese, French, German, and Spanish translations of the abstract see Supplementary Materials section.
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COVID-19 , Cambio Climático , Clima Extremo , Salud Global , Conservación de los Recursos Naturales/tendencias , Política de Salud , Humanos , Cooperación Internacional , Pandemias , SARS-CoV-2Asunto(s)
Cambio Climático , Combustibles Fósiles , Humanos , Salud Global , Política de Salud , Informe de InvestigaciónAsunto(s)
Cambio Climático , Salud Global , Predicción , Salud Global/tendencias , Planificación en Salud , Humanos , Energía RenovableRESUMEN
Cannabinoid receptor type 1 (CB1R) plays an important role in the development of myocardial hypertrophy and fibrosis-2 pathological features of uremic cardiomyopathy. However, it remains unknown whether CB1R is involved in the pathogenesis of uremic cardiomyopathy. Here, we aimed to elucidate the role of CB1R in the development of uremic cardiomyopathy via modulation of Akt signalling. The heart size and myocardial fibrosis were evaluated by echocardiography and immunohistochemical staining, respectively, in 5/6 nephrectomy chronic kidney disease (CKD) mice treated with a CB1R antagonist. CB1R and fibrosis marker expression levels were determined by immunoblotting in H9c2 cells exposed to the uremic toxin indoxyl sulfate (IS), with an organic anion transporter 1 inhibitor or a CB1R antagonist or agonist. Akt phosphorylation was also assessed to examine the signaling pathways downstream of CB1R activation induced by IS in H9c2 cells. CKD mice exhibited marked left ventricular hypertrophy and myocardial fibrosis, which were reversed by treatment with the CB1R antagonist. CB1R, collagen I, transforming growth factor (TGF)-ß, and α-smooth muscle actin (SMA) expression showed time- and dose-dependent upregulation in H9c2 cells treated with IS. The inhibition of CB1R by either CB1R antagonist or small interfering RNA-mediated knockdown attenuated the expression of collagen I, TGF-ß, and α-SMA in IS-treated H9c2 cells, while Akt phosphorylation was enhanced by CB1R agonist and abrogated by CB1R antagonist in these cells. In summary, we conclude that CB1R blockade attenuates LVH and Akt-mediated cardiac fibrosis in a CKD mouse model. Uremic toxin IS stimulates the expression of CB1R and fibrotic markers and CB1R inhibition exerts anti-fibrotic effects via modulation of Akt signaling in H9c2 myofibroblasts. Therefore, the development of drugs targeting CB1R may have therapeutic potential in the treatment of uremic cardiomyopathy.
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Antagonistas de Receptores de Cannabinoides/farmacología , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Cannabinoide CB1/antagonistas & inhibidores , Uremia/tratamiento farmacológico , Animales , Antagonistas de Receptores de Cannabinoides/uso terapéutico , Línea Celular , Colágeno/metabolismo , Evaluación Preclínica de Medicamentos , Fibrosis , Hipertrofia Ventricular Izquierda/etiología , Masculino , Ratones Endogámicos C57BL , Probenecid/farmacología , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismo , Uremia/complicacionesRESUMEN
OBJECTIVE: Hyperphosphatemia-induced endothelial dysfunction has been shown to play a pathogenic role in the development of atherosclerosis in chronic kidney disease (CKD) through unclear mechanisms. Emerging evidence indicates that autophagy is involved in the maintenance of normal cardiovascular function. However, it is unclear whether autophagy participates in the molecular mechanism underlying high phosphate (Pi)-induced endothelial dysfunction. METHODS: The autophagy activity was determined by the immunofluorescence staining of the expression of endothelial microtubule-associated protein 1 light chain 3 (LC3) in the 5/6 nephrectomy rat model of CKD and sham-operated control rats. The LC3-II/LC3-I ratio and the activation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway were determined in cultured human microvascular endothelial cell (HMEC-1) endothelial cells that were exposed to a high concentration of Pi with or without the Pi influx blocker phosphonoformic acid, the autophagy inhibitor 3-methyladenine, and the autophagy inducer rapamycin. The impacts of autophagy on Pi-induced apoptotic damage were assessed by flow cytometry. RESULTS: The in vivo rat model of CKD revealed that hyperphosphatemia is associated with increased endothelial LC3 staining. The exposure of HMEC-1 cells to high Pi induced both dose-dependent and time-dependent increases in the LC3-II/LC3-I expression ratio accompanied by the inhibition of the Akt/mTOR signaling pathway. In HMEC-1 cells, high Pi-induced autophagy and the inhibition of Akt/mTOR signaling were reversed by phosphonoformic acid through the blockage of Pi influx. Apoptosis, characterized by the levels of cleaved caspase 3 and poly(ADP-ribose) polymerase, along with autophagy was induced by high Pi, and the inhibition of autophagy by 3-methyladenine significantly aggravated high Pi-induced apoptosis. The flow cytometry results confirmed that the blockage of autophagy promoted the apoptosis of endothelial cells. CONCLUSIONS: Hyperphosphatemia induces endothelial autophagy, possibly through the inhibition of the Akt/mTOR signaling pathway, which may play a protective role against high Pi-induced apoptosis.
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Autofagia , Células Endoteliales/enzimología , Hiperfosfatemia/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Autofagia/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Activación Enzimática , Foscarnet/farmacología , Humanos , Hiperfosfatemia/etiología , Hiperfosfatemia/patología , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Ratas Wistar , Insuficiencia Renal Crónica/complicaciones , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Factores de Tiempo , TransfecciónRESUMEN
Gender is known to be associated with longevity and oestrogen administration induced longevity-associated gene expression is one of the potential mechanisms underlying the benefits of oestrogen on lifespan, whereas the role of testosterone in the regulation of longevity-associated gene expressions remains largely unclear. The klotho gene, predominantly expressed in the kidney, has recently been discovered to be an aging suppressor gene. In the present study, we investigated the regulatory effects of testosterone on renal klotho gene expression in vivo and in vitro. In testosterone-administered mouse kidney and NRK-52E cells, increased klotho expression was accompanied by the up-regulation of the nuclear androgen receptor (AR). Overexpression of AR enhanced the expression of klotho mRNA and protein. Conversely, testosterone-induced klotho expression was attenuated in the presence of flutamide, an AR antagonist. A reporter assay and a chromatin immunoprecipitation (ChIP) assay demonstrated that AR directly binds to the klotho promoter via androgen response elements (AREs) which reconfirmed its importance for AR binding via the element mutation. In summary, our study demonstrates that testosterone up-regulates anti-aging klotho together with AR expression in the kidney in vivo and in vitro by recruiting AR on to the AREs of the klotho promoter.
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Envejecimiento/genética , Glucuronidasa/biosíntesis , Longevidad/genética , Receptores Androgénicos/biosíntesis , Envejecimiento/efectos de los fármacos , Animales , Estrógenos/metabolismo , Flutamida/farmacología , Regulación de la Expresión Génica , Riñón/citología , Riñón/metabolismo , Proteínas Klotho , Longevidad/efectos de los fármacos , Masculino , Ratones , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Elementos de Respuesta/genética , Transducción de Señal/genética , Testosterona/administración & dosificación , Testosterona/metabolismoRESUMEN
BACKGROUND: Chronic renal failure (CRF) is associated with increased cardiovascular mortality, and medial vascular smooth muscle cell (VSMC) hypertrophy, proliferation, and calcification play a pivotal role in uremic vasculopathy. Glucose transporter-1 (GLUT1) facilitates the transport of glucose into VSMCs, and GLUT1 overexpression associated with high glucose influx leads to a stimulation of VSMC proliferation. However, the role of GLUT1 in uremic vasculopathy remains unclear. This study aimed to identify changes in the expression of GLUT1 in VSMCs in the setting of experimental uremia and investigate whether Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) signaling, which plays a crucial role in VSMC proliferation and glucose metabolism, is involved in the regulation of GLUT1 expression. METHODS: In vivo experimental CRF was induced in Wistar rats by 5/6 nephrectomy, and the GLUT1 expression in aortic tissue was determined by the reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemical staining. Indoxyl sulfate (IS) is a uremic retention solute proven with pro-proliferative effect on rat VSMCs, and we further studied the expression of GLUT1 in rat A7r5 rat embryonic aortic cells stimulated by IS in the presence or absence of phloretin, a GLUT1 inhibitor, to explore the pathogenic role of GLUT1 in uremic vasculopathy. The contribution of Akt/TSC2/mTOR/S6K signaling in modifying the GLUT1 expression was also assessed. RESULTS: Eight weeks after 5/6 nephrectomy, aortic tissue obtained from CRF rats exhibited increased wall thickness and VSMC hypertrophy, hyperplasia, and degeneration. Compared with the sham-operated control group, the messenger (m)RNA and protein abundance of GLUT1 were both markedly increased in CRF rats. In vitro, IS induced a significant increase in expression of GLUT1 protein as well as pro-proliferative cyclin D1 and p21 mRNA and a modest increase in expression of antiapoptotic p53 mRNA in A7r5 cells, whereas inhibition of GLUT1 mediated glucose influx reduced the pro-proliferative and antiapoptotic effects of IS. In addition to increased GLUT1 expression, IS significantly suppressed Akt and TSC2 phosphorylation after 6-hour and 12-hour treatment, but increased S6K phosphorylation after 3-hour treatment. Inactivation of mTOR downstream signaling by rapamycin treatment inhibited S6K phosphorylation and abolished the stimulatory effect of IS on GLUT1 expression. CONCLUSIONS: In vivo and in vitro experimental CRF displayed prominent GLUT1 upregulation in VSMCs. The uremic toxin IS stimulated proliferation of VSMCs possibly through induction of GLUT1 expression. The Akt/TSC/mTOR/S6K signaling pathway may be one of the mechanisms underlying the upregulation of GLUT1 expression in uremic VSMCs.
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Transportador de Glucosa de Tipo 1/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Insuficiencia Renal/enzimología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Aorta/enzimología , Apoptosis , Western Blotting , Línea Celular , Proliferación Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/antagonistas & inhibidores , Transportador de Glucosa de Tipo 1/genética , Hiperplasia , Hipertrofia , Inmunohistoquímica , Indicán/metabolismo , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Nefrectomía , Floretina/farmacología , Fosforilación , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Insuficiencia Renal/genética , Insuficiencia Renal/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Factores de Tiempo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Uremia/enzimologíaRESUMEN
BACKGROUND: Polluting fuels and inefficient stove technologies are still a leading cause of premature deaths worldwide, particularly in low-income and middle-income countries. Previous studies of global household air pollution (HAP) have neither considered the estimation of PM2·5 at national level nor the corresponding attributable mortality burden. Additionally, the effects of climate and ambient air pollution on the global estimation of HAP-PM2·5 exposure for different urban and rural settings remain largely unknown. In this study, we include climatic effects to estimate the HAP-PM2·5 exposure from different fuel types and stove technologies in rural and urban settings separately and the related attributable global mortality burden. METHODS: Bayesian hierarchical models were developed to estimate an annual average HAP-PM2·5 personal exposure and HAP-PM2·5 indoor concentration (including both outdoor and indoor sources). Model variables were selected from sample data in 282 peer-reviewed studies drawn and updated from the WHO Global HAP dataset. The PM2·5 exposure coefficients from the developed model were applied to the external datasets to predict the HAP-PM2·5 exposure globally (personal exposure in 62 countries and indoor concentration in 69 countries). Attributable mortality rate was estimated using a comparative risk assessment approach. Using weighted averages, the national level 24 h average HAP-PM2·5 exposure due to polluting and clean fuels and related death rate per 100â000 population were estimated. FINDINGS: In 2020, household use of polluting solid fuels for cooking and heating led to a national-level average personal exposure of 151 µg/m3 (95% CI 133-169), with rural households having an average of 171 µg/m3 (153-189) and urban households an average of 92 µg/m3 (77-106). Use of clean fuels gave rise to a national-level average personal exposure of 69 µg/m3 (62-76), with a rural average of 76 µg/m3 (69-83) and an urban average of 49 µg/m3 (46-53). Personal exposure-attributable premature mortality (per 100â000 population) from the use of polluting solid fuels at national level was on average 78 (95% CI 69-87), with a rural average of 82 (73-90) and an urban average of 66 (57-75). The average attributable premature mortality (per 100â000 population) from the use of clean fuels at the national level is 62 (54-70), with a rural average of 66 (58-74) and an urban average of 52 (47-57). The estimated HAP-PM2·5 indoor concentration shows that the use of polluting solid fuels resulted in a national-level average of 412 µg/m3 (95% CI 353-471), with a rural average of 514 µg/m3 (446-582) and an urban average of 149 µg/m3 (126-173). The use of clean fuels (gas and electricity) led to an average PM2·5 indoor concentration of 135 µg/m3 (117-153), with a rural average of 174 µg/m3 (154-195) and an urban average of 71 µg/m3 (63-80). Using time-weighted HAP-PM2·5 indoor concentrations, the attributable premature death rate (per 100â000 population) from the use of polluting solid fuels at the national level is on average 78 (95% CI 72-84), the rural average being 84 (78-91) and the urban average 60 (54-66). From the use of clean fuels, the average attributable premature death rate (per 100â000 population) at the national level is 59 (53-64), the rural average being 68 (62-74) and the urban average 45 (41-50). INTERPRETATION: A shift from polluting to clean fuels can reduce the average PM2·5 personal exposure by 53% and thereby lower the death rate. For all fuel types, the estimated average HAP-PM2·5 personal exposure and indoor concentrations exceed the WHO's Interim Target-1 average annual threshold. Policy interventions are urgently needed to greatly increase the use of clean fuels and stove technologies by 2030 to achieve the goal of affordable clean energy access, as set by the UN in 2015, and address health inequities in urban-rural settings. FUNDING: Wellcome Trust, The Lancet Countdown, the Engineering and Physical Sciences Research Council, and the Natural Environment Research Council.
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Contaminación del Aire Interior , Contaminación del Aire , Humanos , Contaminación del Aire Interior/efectos adversos , Exposición a Riesgos Ambientales/efectos adversos , Exposición a Riesgos Ambientales/análisis , Material Particulado/efectos adversos , Teorema de Bayes , Contaminación del Aire/efectos adversosRESUMEN
The stimulation of sarcoplasmic reticulum calcium ATPase SERCA2a emerged as a novel therapeutic strategy to efficiently improve overall cardiac function in heart failure (HF) with reduced arrhythmogenic risk. Istaroxime is a clinical-phase IIb compound with a double mechanism of action, Na+/K+ ATPase inhibition and SERCA2a stimulation. Starting from the observation that istaroxime metabolite PST3093 does not inhibit Na+/K+ ATPase while stimulates SERCA2a, we synthesized a series of bioisosteric PST3093 analogues devoid of Na+/K+ ATPase inhibitory activity. Most of them retained SERCA2a stimulatory action with nanomolar potency in cardiac preparations from healthy guinea pigs and streptozotocin (STZ)-treated rats. One compound was further characterized in isolated cardiomyocytes, confirming SERCA2a stimulation and in vivo showing a safety profile and improvement of cardiac performance following acute infusion in STZ rats. We identified a new class of selective SERCA2a activators as first-in-class drug candidates for HF treatment.
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Insuficiencia Cardíaca , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Animales , Arritmias Cardíacas , Calcio/metabolismo , Cobayas , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , RatasRESUMEN
AIMS: Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes. METHODS AND RESULTS: Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed (i) marked DD not associated to cardiac fibrosis, (ii) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, (iii) reduced LV SERCA2 protein level and activity and (iv) slower SR Ca2+ uptake rate, (v) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, (vi) increased diastolic Ca2+, and (vii) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. CONCLUSIONS: SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment.
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Diabetes Mellitus Experimental , Cardiomiopatías Diabéticas , Animales , Calcio/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Cardiomiopatías Diabéticas/tratamiento farmacológico , Cardiomiopatías Diabéticas/etiología , Cardiomiopatías Diabéticas/prevención & control , Etiocolanolona/análogos & derivados , Etiocolanolona/metabolismo , Etiocolanolona/farmacología , Etiocolanolona/uso terapéutico , Ratas , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
BACKGROUND: Lipotoxicity causes endoplasmic reticulum (ER) stress, leading to cell apoptosis. Sirtuin 1 (Sirt1) regulates gene transcription and cellular metabolism. In this study, we investigated the role of Sirt1 in palmitate-induced ER stress. METHODS: Both H9c2 myoblasts and heart-specific Sirt1 knockout mice fed a palmitate-enriched high-fat diet were used. RESULTS: The high-fat diet induced C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) expression in both Sirt1 knockout mice and controls. The Sirt1 knockout mice showed higher CHOP and ATF4 expression compared to those in the control. Palmitic acid (PA) induced ATF4 and CHOP expression in H9c2 cells. PA-treated H9c2 cells showed decreased cytosolic NAD+/NADH alongside reduced Sirt1's activity. The H9c2 cells showed increased ATF4 and CHOP expression when transfected with plasmid encoding dominant negative mutant Sirt1. Sirt1 activator SRT1720 did not affect CHOP and ATF4 expression. Although SRT1720 enhanced the nuclear translocation of ATF4, the extent of the binding of ATF4 to the CHOP promoter did not increase in PA treated-H9c2 cells. CONCLUSION: PA-induced ER stress is mediated through the upregulation of ATF4 and CHOP. Cytosolic NAD+ concentration is diminished by PA-induced ER stress, leading to decreased Sirt1 activity. The Sirt1 activator SRT1720 promotes the nuclear translocation of ATF4 in PA-treated H9c2 cells.
RESUMEN
Although gender differences in the renal handling of calcium have been reported, the overall contribution of androgens to these differences remains uncertain. We determined here whether testosterone affects active renal calcium reabsorption by regulating calcium transport proteins. Male mice had higher urinary calcium excretion than female mice and their renal calcium transporters were expressed at a lower level. We also found that orchidectomized mice excreted less calcium in their urine than sham-operated control mice and that the hypocalciuria was normalized after testosterone replacement. Androgen deficiency increased the abundance of the renal mRNA and protein of both the luminal transient receptor potential vanilloid-subtype 5 (TRPV5) and intracellular calbindin-D(28K) transporters, which in turn were suppressed by testosterone treatment. There were no significant differences in serum estrogen, parathyroid hormone, or 1,25-dihydroxyvitamin D3 levels between control and orchidectomized mice with or without testosterone. Moreover, incubation of primary rabbit connecting tubule and cortical collecting duct cells with a nonaromatizable androgen, dihydrotestosterone, reduced transcellular calcium transport. Thus, our study shows that gender differences in renal calcium handling are, in part, mediated by the inhibitory actions of androgens on TRPV5-mediated active renal calcium transport.
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Andrógenos/metabolismo , Calcio/orina , Riñón/metabolismo , Caracteres Sexuales , Testosterona/metabolismo , Animales , Calbindinas , Canales de Calcio/metabolismo , Células Cultivadas , Dihidrotestosterona , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Orquiectomía , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Conejos , Receptores Androgénicos/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Canales Catiónicos TRPV/metabolismoAsunto(s)
Insuficiencia Cardíaca , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/enzimología , Humanos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Etiocolanolona/análogos & derivados , Etiocolanolona/farmacología , Etiocolanolona/uso terapéutico , Enfermedad Crónica , Activadores de Enzimas/farmacología , Activadores de Enzimas/uso terapéuticoRESUMEN
BACKGROUND: The risk of cardiovascular disease is notably increased in patients with chronic kidney disease (CKD) and cannabinoid receptor type 1 (CB1R) plays an important role in the development of uremic cardiomyopathy. However, the molecular mechanism underlying the uremic toxin-induced upregulation of CB1R remains elusive. METHODS: The expression of the ATF3/c-Jun complex and CB1R in both in vivo and in vitro models of CKD were measured. We also determined the impact of the ATF3/c-Jun complex on CB1R expression by transfecting H9c2 cells with dominant negative mutants of ATF3 or c-Jun. Inhibitors of organic anion transport, specific MAPK pathways and oxidative DNA damage were also used to assess the pathways mediating the effects of indoxyl sulfate (IS). RESULTS: CB1R upregulation was associated with increased ATF3 expression and c-Jun phosphorylation in CKD both in vivo and in vitro. Expression of dominant-negative ATF3 or c-Jun mutants in IS-treated cells significantly reduced CB1R mRNA levels. Moreover, Co-IP revealed that the ATF3/c-Jun complex is formed and ChIP confirmed its binding to the CB1R promoter, suggesting that this complex directly stimulates CB1R transcription in CKD. Blocking the cellular entry of IS using an organic anion transport inhibitor, as well as inhibiting the ERK1/2 and/or JNK pathways, abrogated the effects of IS on CB1R, ATF3, and c-Jun expression. The IS-induced reactive oxygen species (ROS) was observed in the mitochondria. CONCLUSIONS: We demonstrate that uremic toxins induce ATF3/c-Jun complex-mediated CB1R expression both in vivo and in vitro, possibly by modulating the ERK1/2 and JNK signaling pathways and ROS.
Asunto(s)
Factor de Transcripción Activador 3/biosíntesis , Cardiomiopatías/metabolismo , Indicán/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/biosíntesis , Receptor Cannabinoide CB1/biosíntesis , Uremia/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
BACKGROUND: The longevity regulator Sirtuin 1 is an NAD+-dependent histone deacetylase that regulates endoplasmic reticulum stress and influences cardiomyocyte apoptosis during cardiac contractile dysfunction induced by aging. The mechanism underlying Sirtuin 1 function in cardiac contractile dysfunction related to aging has not been completely elucidated. METHODS: We evaluated cardiac contractile function, endoplasmic reticulum stress, apoptosis, and oxidative stress in 6- and 12month-old cardiac-specific Sirtuin 1 knockout (Sirt1-/-) and control (Sirt1f/f) mice using western blotting and immunohistochemistry. Mice were injected with a protein disulphide isomerase inhibitor. For in vitro analysis, cultured H9c2 cardiomyocytes were exposed to either a Sirtuin 1 inhibitor or activator, with or without a mitochondrial inhibitor, to evaluate the effects of Sirtuin 1 on endoplasmic reticulum stress, nitric oxide synthase expression, and apoptosis. The effects of protein disulphide isomerase inhibition on oxidative stress and ER stress-related apoptosis were also investigated. RESULTS: Compared with 6-month-old Sirt1f/f mice, marked impaired contractility was observed in 12-month-old Sirt1-/- mice. These findings were consistent with increased endoplasmic reticulum stress and apoptosis in the myocardium. Measures of oxidative stress and nitric oxide synthase expression were significantly higher in Sirt1-/- mice compared with those in Sirt1f/f mice at 6months. In vitro experiments revealed increased endoplasmic reticulum stress-mediated apoptosis in H9c2 cardiomyocytes treated with a Sirtuin 1 inhibitor; the effects were ameliorated by a Sirtuin 1 activator. Moreover, consistent with the in vitro findings, impaired cardiac contractility was demonstrated in Sirt1-/- mice injected with a protein disulphide isomerase inhibitor. CONCLUSION: The present study demonstrates that the aging heart is characterized by contractile dysfunction associated with increased oxidative stress and endoplasmic reticulum stress and Sirtuin 1 might have the ability to protect the aging hearts from the inhibition of endoplasmic reticulum-mediated apoptosis.
Asunto(s)
Envejecimiento , Apoptosis/efectos de los fármacos , Cardiomiopatías/prevención & control , Estrés del Retículo Endoplásmico/efectos de los fármacos , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Sirtuina 1/farmacología , Animales , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Células Cultivadas , ADN/genética , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Contracción Miocárdica/efectos de los fármacos , Miocardio/patología , Sirtuina 1/biosíntesis , Sirtuina 1/genéticaAsunto(s)
Contaminantes Atmosféricos , Contaminación del Aire Interior , Contaminantes Atmosféricos/análisis , Contaminación del Aire Interior/efectos adversos , Conservación de los Recursos Energéticos , Monitoreo del Ambiente , Vivienda , Humanos , Material Particulado/análisis , Reino Unido , VentilaciónRESUMEN
Chronic inflammation plays a crucial role in the long-term complications in patients with chronic kidney disease (CKD). This study aimed to assess the role of NLR pyrin domain-containing protein (NLRP3) inflammasome in cardiac contractile dysfunctions in CKD. The cardiac contractile function was evaluated and the expression of NLRP3 inflammasome and related cytokines in the heart was assessed in a murine sham-operated and 5/6 nephrectomy CKD model in vivo. In vitro, H9c2 cells were treated with uremic toxin indoxyl sulfate (IS), with or without NLRP3 inflammasome inhibition, which was achieved by using small interfering RNA (siRNA)-mediated knockdown of the NLRP3 gene. Moreover, the activation of nuclear factor κB (NF-κB) signaling and apoptosis marker levels were assessed in the IS-treated H9c2 cells. The results demonstrated that CKD can lead to the development of cardiac contractile dysfunction in vivo associated with the upregulation of NLRP3 inflammasome, IL-1ß, IL-18, and contribute to the myocardial apoptosis. In vitro experiments showed the upregulation of inflammasome, IL-1ß, and IL-18 levels, and cell apoptosis in the IS-treated H9c2 cells through the activation of NF-κB signaling pathway. The transfection of cells with si-NLRP3 was shown to alleviate IL-1ß, IL-18, and cell apoptosis. Moreover, decreased cell viability induced by IS was shown to be attenuated by IL-1ß or IL-18-neutralizing antibody. In summary, CKD can result in the development of cardiac contractile dysfunction associated with the upregulation of NLRP3 inflammasome/IL-1ß/IL-18 axis induced by the uremic toxins.