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
BACKGROUND: Glucagon like peptide-1 receptor agonists (GLP-1RAs) have shown to reduce mortality and cardiovascular events in patients with type 2 diabetes mellitus (T2DM). Since the impairment in number and function of vasculotrophic circulating CD34+ hematopoietic stem progenitor cells (HSPCs) in T2D has been reported to increase cardiovascular (CV) risk, we hypothesized that one of the mechanisms whereby GLP-1 RAs exert CV protective effects may be related to the ability to improve CD34+ HSPC function. METHODS: In cord blood (CB)-derived CD34+ HSPC, the expression of GLP-1 receptor (GLP-1R) mRNA, receptor protein and intracellular signaling was evaluated by RT-qPCR and Western Blot respectively. CD34+ HSPCs were exposed to high glucose (HG) condition and GLP-1RA liraglutide (LIRA) was added before as well as after functional impairment. Proliferation, CXCR4/SDF-1α axis activity and intracellular ROS production of CD34+ HSPC were evaluated. RESULTS: CD34+ HSPCs express GLP-1R at transcriptional and protein level. LIRA treatment prevented and rescued HSPC proliferation, CXCR4/SDF-1α axis activity and metabolic imbalance from HG-induced impairment. LIRA stimulation promoted intracellular cAMP accumulation as well as ERK1/2 and AKT signaling activation. The selective GLP-1R antagonist exendin (9-39) abrogated LIRA-dependent ERK1/2 and AKT phosphorylation along with the related protective effects. CONCLUSION: We provided the first evidence that CD34+ HSPC express GLP-1R and that LIRA can favorably impact on cell dysfunction due to HG exposure. These findings open new perspectives on the favorable CV effects of GLP-1 RAs in T2DM patients.
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Diabetes Mellitus Tipo 2 , Liraglutida , Quimiocina CXCL12 , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Glucosa/toxicidad , Humanos , Hipoglucemiantes/farmacología , Liraglutida/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Madre/metabolismoRESUMEN
We report the rational design, synthesis, and in vitro preliminary evaluation of a new small library of non-peptide ligands of Gastrin Releasing Peptide Receptor (GRP-R), able to antagonize its natural ligand bombesin (BN) in the nanomolar range of concentration. GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation. Being overexpressed on the surface of different human cancer cell lines, GRP-R is ideal for the selective delivery to tumor cells of both anticancer drug and diagnostic devices. What makes very challenging the design of non-peptide BN analogues is that the 3D structure of the GRP-R is not available, which is the case for many membrane-bound receptors. Thus, the design of GRP-R ligands has to be based on the structure of its natural ligands, BN and GRP. We recently mapped the BN binding epitope by NMR and here we exploited the same spectroscopy, combined with MD, to define BN conformation in proximity of biological membranes, where the interaction with GRP-R takes place. The gained structural information was used to identify a rigid C-galactosidic scaffold able to support pharmacophore groups mimicking the BN key residues' side chains in a suitable manner for binding to GRP-R. Our BN antagonists represent hit compounds for the rational design and synthesis of new ligands and modulators of GRP-R. The further optimization of the pharmacophore groups will allow to increase the biological activity. Due to their favorable chemical properties and stability, they could be employed for the active receptor-mediated targeting of GRP-R positive tumors.
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Antineoplásicos/farmacología , Bombesina/farmacología , Diseño de Fármacos , Receptores de Bombesina/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Bombesina/análogos & derivados , Bombesina/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Receptores de Bombesina/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) has revolutionized the complex scientific field of disease modelling and personalized therapy. Cardiac differentiation of human iPSCs into cardiomyocytes (hiPSC-CMs) has been used in a wide range of healthy and disease models by deriving CMs from different somatic cells. Unfortunately, hiPSC-CMs have to be improved because existing protocols are not completely able to obtain mature CMs recapitulating physiological properties of human adult cardiac cells. Therefore, improvements and advances able to standardize differentiation conditions are needed. Lately, evidences of an epigenetic memory retained by the somatic cells used for deriving hiPSC-CMs has led to evaluation of different somatic sources in order to obtain more mature hiPSC-derived CMs.
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Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citologíaRESUMEN
ß3-Adrenoceptors (AR) stimulate cardiac Na+/K+ pump in healthy hearts. ß3-ARs are upregulated by persistent sympathetic hyperactivity; however, their effect on Na+/K+ ATPase activity and ventricular function in this condition is still unknown. Here, we investigate preventive effects of additional ß3-AR activation (BRL) on Na+/K+ ATPase activity and in vivo hemodynamics in a model of noradrenaline-induced hypertrophy. Rats received NA or NA plus simultaneously administered BRL in vivo infusion for 14 days; their cardiac function was investigated by left ventricular pressure-volume analysis. Moreover, fibrosis and apoptosis were also assessed histologically. NA induced an hypertrophic pattern, as detected by morphological, histological, and biochemical markers. Additional BRL exposure reversed the hypertrophic pattern and restored Na+/K+ ATPase activity. NA treatment increased systolic function and depressed diastolic function (slowed relaxation). Additional BRL treatment reversed most NA-induced hemodynamic changes. NA decreased Na+/K+ pump α2 subunit expression selectively, a change also reversed by additional BRL treatment. Increasing ß3-AR stimulation may prevent the consequences of chronic NA exposure on Na+/K+ pump and in vivo hemodynamics. ß3-AR agonism may thus represent a new therapeutic strategy for pharmacological modulation of hypertrophy under conditions of chronically enhanced sympathetic activity.
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Apoptosis/efectos de los fármacos , Cardiomegalia/metabolismo , Miocardio/metabolismo , Norepinefrina/efectos adversos , Receptores Adrenérgicos beta 3/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Fibrosis , Masculino , Miocardio/patología , Norepinefrina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Aim: Repolarization response to ß-adrenergic (ß-AR) stimulation differs between guinea-pig and canine myocytes and, within the latter, between myocardial layers. Correlative analysis suggests that this may be due to differences in action potential (AP) contour. Here we tested whether AP contour may set the response of current and of repolarization to ß-AR stimulation (10 nM isoproterenol, ISO). Methods and results: The responses of AP and current to ISO were measured under I-clamp and "AP-clamp" in guinea-pig (GP), dog epicardial (DEPI) and dog subendocardial (DENDO) myocytes. Dynamic-clamp (DC) was used to evaluate the impact of AP features on AP response to ISO. ISO prolonged AP duration (APD) in GP myocytes, did not affect it in DENDO and shortened it in DEPI ones. The current induced by ISO (IISO) sharply differed between GP and canine myocytes and, to a lesser extent, between DENDO and DEPI ones. Differences in IISO profile likely important in setting APD response (time-to-peak, time-to-reversal), were minimized when canine myocytes where clamped with GP AP-waveforms and vice versa. Introduction of a "notch" in GP AP (by DC) was alone insufficient to affect the APD response to ISO; nevertheless, when incorporated in a GP AP-waveform, the main "canine" AP features ("notch" and low plateau potential) caused IISO of GP myocytes to acquire canine features. Conclusion: Early repolarization contour and level of plateau potential contribute to species-specificity of IISO profile. Changes in AP contour, also when generated by modulation of ISO-insensitive currents, may be crucial in setting APD response to ß-AR stimulation.
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Potenciales de Acción/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Isoproterenol/farmacología , Miocitos Cardíacos/efectos de los fármacos , Animales , Perros , Endocardio/citología , Cobayas , Técnicas de Placa-Clamp , Pericardio/citología , Especificidad de la EspecieRESUMEN
PURPOSE: Exposure to hypoxia has been suggested to activate multiple adaptive pathways so that muscles are better able to maintain cellular energy homeostasis. However, there is limited research regarding the tissue specificity of this response. The aim of this study was to investigate the influence of tissue specificity on mitochondrial adaptations of rat skeletal and heart muscles after 4 weeks of normobaric hypoxia (FiO2: 0.10). METHODS: Twenty male Wistar rats were randomly assigned to either normobaric hypoxia or normoxia. Mitochondrial respiration was determined in permeabilised muscle fibres from left and right ventricles, soleus and extensorum digitorum longus (EDL). Citrate synthase activity and the relative abundance of proteins associated with mitochondrial biogenesis were also analysed. RESULTS: After hypoxia exposure, only the soleus and left ventricle (both predominantly oxidative) presented a greater maximal mass-specific respiration (+48 and +25%, p < 0.05) and mitochondrial-specific respiration (+75 and +28%, p < 0.05). Citrate synthase activity was higher in the EDL (0.63 ± 0.08 vs 0.41 ± 0.10 µmol min- 1 µg- 1) and lower in the soleus (0.65 ± 0.17 vs 0.87 ± 0.20 µmol min- 1 µg- 1) in hypoxia with respect to normoxia. There was a lower relative protein abundance of PGC-1α (-25%, p < 0.05) in the right ventricle and a higher relative protein abundance of PGC-1ß (+43%, p < 0.05) in the left ventricle of rats exposed to hypoxia, with few differences for protein abundance in the other muscles. CONCLUSION: Our results show a muscle-specific response to 4 weeks of normobaric hypoxia. Depending on fibre type, and the presence of ventricular hypertrophy, muscles respond differently to the same degree of environmental hypoxia.
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Adaptación Fisiológica , Hipoxia/metabolismo , Mitocondrias Cardíacas/metabolismo , Músculo Esquelético/metabolismo , Animales , Respiración de la Célula , Citrato (si)-Sintasa/metabolismo , Hipoxia/fisiopatología , Masculino , Miocardio/metabolismo , Especificidad de Órganos , Biogénesis de Organelos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Ratas , Ratas WistarRESUMEN
BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavß2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavß2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavß2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavß2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavß2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavß2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavß2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.
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Materiales Biomiméticos/administración & dosificación , Materiales Biomiméticos/metabolismo , Canales de Calcio Tipo L/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Peptidomiméticos/administración & dosificación , Peptidomiméticos/metabolismo , Secuencia de Aminoácidos , Animales , Materiales Biomiméticos/química , Canales de Calcio Tipo L/genética , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Células Cultivadas , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Peptidomiméticos/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Estudios RetrospectivosRESUMEN
Blockade of the late Na+ current (I NaL) protects from ischemia/reperfusion damage; nevertheless, information on changes in I NaL during acute ischemia and their effect on intracellular milieu is missing. I NaL, cytosolic Na+ and Ca2+ activities (Nacyt, Cacyt) were measured in isolated rat ventricular myocytes during 7 min of simulated ischemia (ISC); in all the conditions tested, effects consistently exerted by ranolazine (RAN) and tetrodotoxin (TTX) were interpreted as due to I NaL blockade. The results indicate that I NaL was enhanced during ISC in spite of changes in action potential (AP) contour; I NaL significantly contributed to Nacyt rise, but only marginally to Cacyt rise. The impact of I NaL on Cacyt was markedly enhanced by blockade of the sarcolemmal(s) Na+/Ca2+ exchanger (NCX) and was due to the presence of (Na+-sensitive) Ca2+ efflux through mitochondrial NCX (mNCX). sNCX blockade increased Cacyt and decreased Nacyt, thus indicating that, throughout ISC, sNCX operated in the forward mode, in spite of the substantial Nacyt increment. Thus, a robust Ca2+ source, other than sNCX and including mitochondria, contributed to Cacyt during ISC. Most, but not all, of RAN effects were shared by TTX. (1) The paradigm that attributes Cacyt accumulation during acute ischemia to decrease/reversal of sNCX transport may not be of general applicability; (2) I NaL is enhanced during ISC, when the effect of Nacyt on mitochondrial Ca2+ transport may substantially contribute to I NaL impact on Cacyt; (3) RAN may act mostly, but not exclusively, through I NaL blockade during ISC.
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Calcio/metabolismo , Isquemia Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Sodio/metabolismo , Potenciales de Acción , Animales , Homeostasis/fisiología , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-DawleyRESUMEN
Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage.
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Diferenciación Celular/fisiología , MicroARNs/biosíntesis , Miocitos Cardíacos/metabolismo , Animales , Línea Celular , Células Cultivadas , Humanos , Ratones , MicroARNs/administración & dosificación , Miocitos Cardíacos/efectos de los fármacos , Organogénesis/efectos de los fármacos , Organogénesis/fisiologíaRESUMEN
Recent evidence of beneficial effects of ranolazine (RAN) in type II diabetes motivates interest in the role of the late sodium current (INaL) in glucose-stimulated insulin secretion. In the present work, we characterize INaL and its function in rat INS-1E cells and human islets cells. INaL was identified as steady-state current blocked by 10 µM RAN (IRAN) or 0.5 µM tetrodotoxin (TTX) (ITTX). Veratridine (VERA, 40 µM) was used as INaL enhancer. Baseline INaL was similar between INS-1E and human islet cells. In INS-1E cells, activated by glucose or tolbutamide, TTX or RAN hyperpolarized membrane potential (V m). VERA-induced depolarization was countered by TTX or RAN. ITTX and IRAN reversal potentials were negative to Na(+) equilibrium one, but they approached it after Na(+) substitution with Li(+) or when K(+) channels were blocked. This revealed INaL coupling with Na(+)-activated K(+) current (IKNa); expression of IKNa channels (Slick/Slack) was confirmed by transcript analysis and Western blot. RAN or TTX blunted cytosolic Ca(2+) response to depolarization. Long-term incubation in high (33 mM) glucose (CHG) constitutively enhanced INaL. VERA immediately increased glucose-stimulated insulin secretion. CHG increased glucose-independent secretion instead and abolished the secretory response to glucose. RAN or TTX countered VERA- and CHG-induced changes in insulin secretion. Our study demonstrated that (1) INaL was expressed in insulin-secreting cells and coupled to IKNa; INaL affected cytosolic Ca(2+) but, unless enhanced, barely contributed to glucose-stimulated insulin secretion (GSIS); and (2) sustained hyperglycemic stress enhanced INaL, which contributed to the attending increase of glucose-independent insulin "leak" and GSIS impairment.
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Células Secretoras de Insulina/metabolismo , Sodio/metabolismo , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Línea Celular , Glucosa/farmacología , Humanos , Hipoglucemiantes/farmacología , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Potenciales de la Membrana , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de potasio activados por Sodio , ARN Mensajero/metabolismo , Ratas , Factores de Tiempo , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Canales de Sodio Activados por Voltaje/efectos de los fármacos , Canales de Sodio Activados por Voltaje/genéticaRESUMEN
Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor kappaB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca(2+)/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cgamma2 activation, influx of extracellular Ca(2+) and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.
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Células Dendríticas/citología , Células Dendríticas/inmunología , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/inmunología , Factores de Transcripción NFATC/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular , Supervivencia Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Fosfolipasa C gamma/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Mutations underlying genetic cardiomyopathies might affect differentiation commitment of resident progenitor cells. Cardiac mesoangioblasts (cMabs) are multipotent progenitor cells resident in the myocardium. A switch from cardiac to skeletal muscle differentiation has been recently described in cMabs from ß-sarcoglycan-null mice (ßSG(-/-)), a murine model of genetic myopathy with early myocardial involvement. Although complementation with ßSG gene was inconsequential, knock-in of miRNA669a (missing in ßSG(-/-) cMabs) partially rescued the mutation-induced molecular phenotype. Here, we undertook a detailed evaluation of functional differentiation of ßSG(-/-) cMabs and tested the effects of miRNA669a-induced rescue in vitro. To this end, cMabs were compared with neonatal cardiomyocytes (CMs) and skeletal muscle C2C12 cells, representative of cardiac and skeletal muscle respectively. Consistent with previous data on molecular patterns, electrophysiological and Ca(2+)-handling properties of ßSG(-/-) cMabs were closer to C2C12 cells than to CM ones. Nevertheless, subtler aspects, including action potential contour, Ca(2+)-spark properties and RyR isoform expression, distinguished ßSG(-/-) cMabs from C2C12 cells. Contrary to previous reports, wild-type cMabs failed to show functional differentiation towards either cell type. Knock-in of miRNA669a in ßSG(-/-) cMabs rescued the wild-type functional phenotype, i.e. it completely prevented development of skeletal muscle functional responses. We conclude that miRNA669a expression, ablated by ßSG deletion, may prevent functional differentiation of cMabs towards the skeletal muscle phenotype.
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Corazón/fisiopatología , MicroARNs/genética , Músculo Esquelético/citología , Enfermedades Musculares/patología , Miocitos Cardíacos/citología , Sarcoglicanos/fisiología , Células Madre/citología , Potenciales de Acción , Animales , Calcio/metabolismo , Células Cultivadas , Electrofisiología , Ratones , Ratones Noqueados , Contracción Muscular , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Fenotipo , Células Madre/metabolismoRESUMEN
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific IK1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach improves the translational relevance of the hiPSC-CM model for studying mechanisms underlying inherited or acquired atrial arrhythmias in human CM, and for screening anti-arrhythmic agents.
Asunto(s)
Fibrilación Atrial , Células Madre Pluripotentes Inducidas , Humanos , Miocitos Cardíacos , Constricción , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
Insuficiencia Cardíaca , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Animales , Arritmias Cardíacas , Calcio/metabolismo , Cobayas , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , RatasRESUMEN
Rationale: Aging in the heart is a gradual process, involving continuous changes in cardiovascular cells, including cardiomyocytes (CMs), namely cellular senescence. These changes finally lead to adverse organ remodeling and resulting in heart failure. This study exploits CMs from human induced pluripotent stem cells (iCMs) as a tool to model and characterize mechanisms involved in aging. Methods and Results: Human somatic cells were reprogrammed into human induced pluripotent stem cells and subsequently differentiated in iCMs. A senescent-like phenotype (SenCMs) was induced by short exposure (3 hours) to doxorubicin (Dox) at the sub-lethal concentration of 0.2 µM. Dox treatment induced expression of cyclin-dependent kinase inhibitors p21 and p16, and increased positivity to senescence-associated beta-galactosidase when compared to untreated iCMs. SenCMs showed increased oxidative stress, alteration in mitochondrial morphology and depolarized mitochondrial membrane potential, which resulted in decreased ATP production. Functionally, when compared to iCMs, SenCMs showed, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence, two well-known arrhythmogenic indexes. These effects were largely ascribable to augmented late sodium current (INaL) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca2+ content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca2+ leak. Electrical and intracellular Ca2+ alterations were mostly justified by increased CaMKII activity in SenCMs. Finally, SenCMs phenotype was furtherly confirmed by analyzing physiological aging in CMs isolated from old mice in comparison to young ones. Conclusions: Overall, we showed that SenCMs recapitulate the phenotype of aged primary CMs in terms of senescence markers, electrical and Ca2+ handling properties and metabolic features. Thus, Dox-induced SenCMs can be considered a novel in vitro platform to study aging mechanisms and to envision cardiac specific anti-aging approach in humans.
Asunto(s)
Células Madre Pluripotentes Inducidas , Potenciales de Acción , Anciano , Animales , Calcio/metabolismo , Senescencia Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Pro-arrhythmic risk strongly limits the therapeutic value of current inotropic interventions. Istaroxime (previously PST2744) is a novel inotropic agent, significantly less pro-arrhythmic than digoxin that, in addition to block Na(+)/K(+) pump, stimulates sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2). Here we compare istaroxime and digoxin effects to further address the role of SR modulation in reducing the toxicity associated with Na(+)/K(+) pump blockade. In murine ventricular myocytes both compounds increased cell twitch (inotropy) in a concentration-dependent fashion. At high concentrations digoxin, but not istaroxime, induced unstimulated contractions, a sign of pro-arrhythmic toxicity. To evaluate the mechanism of this difference, we compared the two drugs at concentrations exerting equal inotropy but different toxicity. At these concentrations: (1) the two drugs equally inhibited the Na(+)/K(+) pump; (2) digoxin induced larger increases in resting Ca(2+) and in diastolic Ca(2+) during pacing; (3) neither drug affected the relationship between RyR-mediated SR Ca(2+) leak and Ca(2+) content; (4) istaroxime, but not digoxin, enhanced SR Ca(2+) reuptake rate. In conclusion, digoxin toxicity was associated to larger accumulation of cytosolic Ca(2+), which did not result from RyR facilitation, but which might ultimately induce it to promote unstimulated Ca(2+) release. The lower toxicity of Na(+)/K(+) pump blockade by istaroxime may thus reflect improved Ca(2+) confinement within the SR, likely to result from concomitant SERCA2 stimulation.
Asunto(s)
Calcio/metabolismo , Cardiotónicos/farmacología , Digoxina/farmacología , Etiocolanolona/análogos & derivados , Animales , Células Cultivadas , Electrofisiología , Etiocolanolona/farmacología , Femenino , Ratones , Ratones Endogámicos C57BL , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
In this study we describe the generation and characterization of an human induced pluripotent stem cell (hiPSC) line from a long QT syndrome type 1 (LQT1) patient carrying the KCNQ1 c.940 G > A (p.Gly314Ser) mutation. This patient-specific iPSC line has been obtained by using non-integrational Sendai reprogramming method, expresses pluripotency markers and has the capacity to differentiate into the three germ layers and into spontaneously beating cardiomyocytes (iPSC-CMs).