RESUMEN
Lamotrigine is widely prescribed to treat bipolar neurological disorder and epilepsy. It exerts its antiepileptic action by blocking voltage-gated sodium channels in neurons. Recently, the US Food and Drug Administration issued a warning on the use of Lamotrigine after observations of conduction anomalies and Brugada syndrome patterns on the electrocardiograms of epileptic patients treated with the drug. Brugada syndrome and conduction disturbance are both associated with alterations of the cardiac sodium current (INa) kinetics and amplitude. In this study, we used the patch clamp technique on cardiomyocytes from epileptic rats to test the hypothesis that Lamotrigine also blocks INa in the heart. We found that Lamotrigine inhibited 60% of INa peak amplitude and reduced cardiac excitability in epileptic rats but had little effect in sham animals. Moreover, Lamotrigine inhibited 67% of INaL and, more importantly, prolonged the action potential refractory period in epileptic animals. Our results suggest that enhanced affinity of Lamotrigine for INa may in part explain the clinical phenotypes observed in epileptic patients.
RESUMEN
Voltage-gated ion channels play a key role in the action potential (AP) initiation and its propagation in sensory neurons. Modulation of their activity during chronic inflammation creates a persistent pain state. In this study, we sought to determine how peripheral inflammation caused by complete Freund's adjuvant (CFA) alters the fast sodium (INa ), L-type calcium (ICaL ), and potassium (IK ) currents in primary afferent fibers to increase nociception. In our model, intraplantar administration of CFA induced mechanical allodynia and thermal hyperalgesia at day 14 post-injection. Using whole-cell patch-clamp recording in dissociated small (C), medium (Aδ), and large-sized (Aß) rat dorsal root ganglion (DRG) neurons, we found that CFA prolonged the AP duration and increased the amplitude of the tetrodotoxin-resistant (TTX-r) INa in Aß fibers. In addition, CFA accelerated the recovery of INa from inactivation in C and Aδ nociceptive fibers but enhanced the late sodium current (INaL ) only in Aδ and Aß neurons. Inflammation similarly reduced the amplitude of ICaL in each neuronal cell type. Fourteen days after injection, CFA reduced both components of IK (IKdr and IA ) in Aδ fibers. We also found that IA was significantly larger in C and Aδ neurons in normal conditions and during chronic inflammation. Our data, therefore, suggest that targeting the transient potassium current IA represents an efficient way to shift the balance toward antinociception during inflammation, since its activation will selectively decrease the AP duration in nociceptive fibers. Altogether, our data indicate that complex interactions between IK , INa , and ICaL reduce pain threshold by concomitantly enhancing the activity of nociceptive neurons and reducing the inhibitory action of Aß fibers during chronic inflammation.
Asunto(s)
Potenciales de Acción , Neuronas Aferentes/metabolismo , Dolor Nociceptivo/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Ganglios Espinales/fisiología , Masculino , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Nocicepción , Dolor Nociceptivo/fisiopatología , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
BACKGROUND/AIMS: Apelin and its G protein-coupled receptor APJ (gene symbol Aplnr) are strongly expressed in magnocellular vasopressinergic neurons suggesting that the apelin/APJ system plays a key role at the central level in regulating salt and water balance by counteracting the antiduretic action of vasopressin (AVP). Likewise, recent studies revealed that apelin exerts opposite effects to those of vasopressin induced on water reabsorption via a direct action on the kidney collecting duct. However, the underlying mechanisms of the peripheral action of apelin are not clearly understood. Here, we thus investigated the role of the apelin/APJ system in the regulation of water balance in the kidney, and more specifically its involvement in modulating the function of aquaporin-2 (AQP2) in the collecting duct. METHODS: Mouse cortical collecting duct cells (mpkCCD) were incubated in the presence of dDAVP and treated with or without apelin-13. Changes in AQP2 expression and localization were determined by immunoblotting and confocal immunofluorescence staining. RESULTS: Herein, we showed that the APJ was present in mpkCCD cells. Treatment of mpkCCD with apelin-13 reduced the cAMP production and antagonized the AVP-induced increase in AQP2 mRNA and protein expressions. Immunofluorescent experiments also revealed that the AVP-induced apical cell surface expression of AQP2, and notably its phosphorylated isoform AQP2-pS269, was considerably reduced following apelin-13 application to mpkCCD cells. CONCLUSION: Our data reinforce the aquaretic role of the apelin/APJ system in the fine regulation of body fluid homeostasis at the kidney level and its physiological opposite action to the antiduretic activity of AVP.
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Acuaporina 2/metabolismo , Desamino Arginina Vasopresina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Transporte de Proteínas/efectos de los fármacos , Animales , Receptores de Apelina/metabolismo , Acuaporina 2/genética , Línea Celular , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Ratones , Fosforilación/efectos de los fármacosRESUMEN
BACKGROUND: In utero exposure to tobacco smoke is associated with sudden infant death syndrome (SIDS) and cardiac arrhythmias in newborns. The arrhythmogenic mechanisms seem linked to alterations of the cardiac sodium current (INa). We previously reported that in utero exposure to nicotine delays the postnatal development of the heart sinoatrial node in rabbits and altered expression of the sodium channels NaV1.5 and NaV1.1 in the atrium surrounding it. These channels react differently to sympathetic stimulation. OBJECTIVE: The purpose of this study was to test whether nicotine altered the response of INa to stimulation by the ß-adrenoreceptor agonist isoproterenol in atrial myocytes. Our hypothesis is that changes in the sympathetic response of sinoatrial node peripheral cells may create a substrate for arrhythmia. METHODS: Using the patch-clamp technique we measured the effect of nicotine on the response of INa to adrenergic stimulation in isolated cardiomyocytes. RESULTS: Isoproterenol increased INa by 50% in newborn sham rabbits but had no effect in newborn rabbits exposed to nicotine in utero. Our data also show that nicotine increases the late sodium current, an effect that may promote QT prolongation. CONCLUSION: We provide the first evidence linking fetal exposure to nicotine to long-term alterations of INa response to isoproterenol. These changes may impair INa adaptation to sympathetic tone and prevent awakening from sleep apnea, thus leading to arrhythmias that could potentially be involved in SIDS. Our data also raise concerns about the use of nicotine replacement therapies for pregnant women.
Asunto(s)
Potenciales de Acción/fisiología , Atrios Cardíacos/fisiopatología , Isoproterenol/farmacología , Síndrome de QT Prolongado/metabolismo , Miocitos Cardíacos/metabolismo , Preñez , Sodio/metabolismo , Potenciales de Acción/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Femenino , Atrios Cardíacos/metabolismo , Síndrome de QT Prolongado/fisiopatología , Nicotina/farmacología , Técnicas de Placa-Clamp , Embarazo , Conejos , Nodo Sinoatrial/fisiopatologíaRESUMEN
INTRODUCTION: Mutations within SCN5A are found in a significant proportion (15-30%) of Brugada syndrome (BrS) cases and impair sodium transport across excitable cardiac cells that mediate ventricular contractions. Genetic testing offers a means to clinically assess and manage affected individuals and their family members. METHODS AND RESULTS: The proband at age 44â¯years old exhibited a syncopal event during exercise, and presented later with a spontaneous type-I BrS pattern on 12lead resting electrocardiogram (ECG). Mutational analysis performed across all SCN5A exons revealed a unique three base-pair deletion p.M741_T742delinsI (c.2223_2225delGAC), in a heterozygous state in the proband and 2 siblings. This mutation was not seen in a cohort of 105 ethnicity-matched controls or in public genome databases. Patch clamp electrophysiology study conducted in TSA201 cells showed an abolishment of sodium current (INa). The proband, and several relatives, also harboured a known SCN5A variant, p.R1193Q (c.3578G>A). CONCLUSION: Our study has demonstrated the deleterious effect of a novel SCN5A mutation p.M741_T742delinsI (c.2223_2225delGAC). The findings highlight the complex effects of gender and age in phenotype manifestation. It also offers insights into improving the long-term management of BrS, and the utility of cascade genetic screening for risk stratification.
Asunto(s)
Síndrome de Brugada/genética , Canal de Sodio Activado por Voltaje NAV1.5/genética , Eliminación de Secuencia , Adulto , Síndrome de Brugada/complicaciones , Femenino , Humanos , Masculino , Linaje , Fenotipo , Síncope/etiologíaRESUMEN
The apelinergic system is an important player in the regulation of both vascular tone and cardiovascular function, making this physiological system an attractive target for drug development for hypertension, heart failure and ischemic heart disease. Indeed, apelin exerts a positive inotropic effect in humans whilst reducing peripheral vascular resistance. In this study, we investigated the signaling pathways through which apelin exerts its hypotensive action. We synthesized a series of apelin-13 analogs whereby the C-terminal Phe13 residue was replaced by natural or unnatural amino acids. In HEK293 cells expressing APJ, we evaluated the relative efficacy of these compounds to activate Gαi1 and GαoA G-proteins, recruit ß-arrestins 1 and 2 (ßarrs), and inhibit cAMP production. Calculating the transduction ratio for each pathway allowed us to identify several analogs with distinct signaling profiles. Furthermore, we found that these analogs delivered i.v. to Sprague-Dawley rats exerted a wide range of hypotensive responses. Indeed, two compounds lost their ability to lower blood pressure, while other analogs significantly reduced blood pressure as apelin-13. Interestingly, analogs that did not lower blood pressure were less effective at recruiting ßarrs. Finally, using Spearman correlations, we established that the hypotensive response was significantly correlated with ßarr recruitment but not with G protein-dependent signaling. In conclusion, our results demonstrated that the ßarr recruitment potency is involved in the hypotensive efficacy of activated APJ.
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Antihipertensivos/farmacología , Receptores de Apelina/metabolismo , Presión Sanguínea/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/farmacología , beta-Arrestinas/metabolismo , Animales , Antihipertensivos/química , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Hipotensión/tratamiento farmacológico , Hipotensión/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Masculino , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The apelin receptor generates increasing interest as a potential target across several cardiovascular indications. However, the short half-life of its cognate ligands, the apelin peptides, is a limiting factor for pharmacological use. In this study, we systematically explored each position of apelin-13 to find the best position to cyclize the peptide, with the goal to improve its stability while optimizing its binding affinity and signaling profile. Macrocyclic analogues showed a remarkably higher stability in rat plasma (half-life >3 h versus 24 min for Pyr-apelin-13), accompanied by improved affinity (analogue 15, Ki 0.15 nM and t1/2 6.8 h). Several compounds displayed higher inotropic effects ex vivo in the Langendorff isolated heart model in rats (analogues 13 and 15, maximum response at 0.003 nM versus 0.03 nM of apelin-13). In conclusion, this study provides stable and active compounds to better characterize the pharmacology of the apelinergic system.
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Azetidinas/síntesis química , Péptidos y Proteínas de Señalización Intercelular/síntesis química , Monoacilglicerol Lipasas/química , Tomografía de Emisión de Positrones/métodos , Radiofármacos/síntesis química , Animales , Azetidinas/farmacocinética , Encéfalo/diagnóstico por imagen , Encéfalo/enzimología , Encéfalo/metabolismo , Radioisótopos de Carbono , Radioisótopos de Flúor , Péptidos y Proteínas de Señalización Intercelular/farmacocinética , Macaca mulatta , Masculino , Ratones , Trazadores Radiactivos , Radiofármacos/farmacocinética , Ratas Sprague-Dawley , Especificidad por Sustrato , Distribución TisularRESUMEN
OBJECTIVES: Apelin-13 was recently proposed as an alternative to the recommended ß-adrenergic drugs for supporting endotoxin-induced myocardial dysfunction. Since Apelin-13 signals through its receptor (Apelin peptide jejunum) to exert singular inotropic/vasotropic actions and to optimize body fluid balance, this candidate pathway might benefit septic shock management. Whether the newly discovered ELABELA (ELA), a second endogenous ligand of the Apelin peptide jejunum receptor highly expressed in the kidney, further improves cardio-renal impairment remains unknown. DESIGN, SETTING, AND SUBJECTS: Interventional study in a rat model of septic shock (128 adult males) to assess the effects of ELA and Apelin-13 on vascular and cardio-renal function. Experiments were performed in a tertiary care University-based research institute. INTERVENTIONS: Polymicrobial sepsis-induced cardiac dysfunction was produced by cecal ligation puncture to assess hemodynamic efficacy, cardioprotection, and biomechanics under acute or continuous infusions of the apelinergic agonists ELA or Apelin-13 (39 and 15 µg/kg/hr, respectively) versus normal saline. MEASUREMENTS AND MAIN RESULTS: Apelinergic agonists improved 72-hour survival after sepsis induction, with ELA providing the best clinical outcome after 24 hours. Apelinergic agonist infusion counteracted cecal ligation puncture-induced myocardial dysfunction by improving left ventricular pressure-volume relationship. ELA-treated cecal ligation puncture rats were the only group to 1) display a significant improvement in left ventricular filling as shown by increased E-wave velocity and left ventricular end-diastolic volume, 2) exhibit a higher plasma volume, and 3) limit kidney injury and free-water clearance. These beneficial renal effects were superior to Apelin-13, likely because full-length ELA enabled a distinctive regulation of pituitary vasopressin release. CONCLUSIONS: Activation of the apelinergic system by exogenous ELA or Apelin-13 infusion improves cardiovascular function and survival after cecal ligation puncture-induced sepsis. However, ELA proved better than Apelin-13 by improving fluid homeostasis, cardiovascular hemodynamics recovery, and limiting kidney dysfunction in a vasopressinergic-dependent manner.
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Péptidos y Proteínas de Señalización Intercelular/farmacología , Hormonas Peptídicas/farmacología , Choque Séptico/tratamiento farmacológico , Animales , Biomarcadores , Citocinas/inmunología , Modelos Animales de Enfermedad , Ecocardiografía , Hemodinámica/efectos de los fármacos , Masculino , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
In-utero exposure to tobacco smoke remains the highest risk factor for sudden infant death syndrome (SIDS). To alleviate the risks, nicotine replacement therapies are often prescribed to women who wish to quit smoking during their pregnancy. Cardiac arrhythmias is considered the final outcome leading to sudden death. Our goal in this study was to determine if exposing rabbit fetus to nicotine altered the cardiac conduction system of newborn kittens in a manner susceptible to cause SIDS. Using neuronal markers and a series of immunohistological and electrophysiological techniques we found that nicotine delayed the development of the cardiac pacemaker center (sinoatrial node) and decreased its innervation. At the molecular level, nicotine favored the expression of cardiac sodium channels with biophysical properties that will tend to slow heart rate and diminish electrical conduction. Our results show that alterations of the cardiac sodium current may contribute to the bradycardia, conduction disturbances and other cardiac arrhythmias often associated to SIDS and raise awareness on the use of replacement therapy during pregnancy.
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Nicotina/toxicidad , Nodo Sinoatrial/fisiología , Muerte Súbita del Lactante/etiología , Animales , Animales Recién Nacidos , Cotinina/sangre , Femenino , Frecuencia Cardíaca/fisiología , Humanos , Lactante , Miocitos Cardíacos/fisiología , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Técnicas de Placa-Clamp , Embarazo , Efectos Tardíos de la Exposición Prenatal , Conejos , Nodo Sinoatrial/fisiopatologíaRESUMEN
OBJECTIVE: Dobutamine is the currently recommended ß-adrenergic inotropic drug for supporting sepsis-induced myocardial dysfunction when cardiac output index remains low after preload correction. Better and safer therapies are nonetheless mandatory because responsiveness to dobutamine is limited with numerous side effects. Apelin-13 is a powerful inotropic candidate that could be considered as an alternative noncatecholaminergic support in the setting of inflammatory cardiovascular dysfunction. DESIGN: Interventional controlled experimental animal study. SETTING: Tertiary care university-based research institute. SUBJECTS: One hundred ninety-eight adult male rats. INTERVENTIONS: Using a rat model of "systemic inflammation-induced cardiac dysfunction" induced by intraperitoneal lipopolysaccharide injection (10 mg/kg), hemodynamic efficacy, cardioprotection, and biomechanics were assessed under IV osmotic pump infusions of apelin-13 (0.25 µg/kg/min) or dobutamine (7.5 µg/kg/min). MEASUREMENTS AND MAIN RESULTS: In this model and in both in vivo and ex vivo studies, apelin-13 compared with dobutamine provoked distinctive effects on cardiac function: 1) optimized cardiac energy-dependent workload with improved cardiac index and lower vascular resistance, 2) upgraded hearts' apelinergic responsiveness, and 3) consecutive downstream advantages, including increased urine output, enhanced plasma volume, reduced weight loss, and substantially improved overall outcomes. In vitro studies confirmed that these apelin-13-driven processes encompassed a significant and rapid reduction in systemic cytokine release with dampening of myocardial inflammation, injury, and apoptosis and resolution of associated molecular pathways. CONCLUSIONS: In this inflammatory cardiovascular dysfunction, apelin-13 infusion delivers distinct and optimized hemodynamic support (including positive fluid balance), along with cardioprotective effects, modulation of circulatory inflammation and extended survival.
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Cardiomiopatías/fisiopatología , Cardiotónicos/farmacología , Dobutamina/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Animales , Peso Corporal/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Cardiomiopatías/inducido químicamente , Cardiomiopatías/prevención & control , Cardiotónicos/uso terapéutico , Citocinas/sangre , Modelos Animales de Enfermedad , Dobutamina/uso terapéutico , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Lipopolisacáridos , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/enzimología , Óxido Nítrico Sintasa de Tipo II/sangre , Peroxidasa/metabolismo , Fosforilación/efectos de los fármacos , Volumen Plasmático/efectos de los fármacos , Ratas , Tasa de Supervivencia , Resistencia Vascular/efectos de los fármacos , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
Apelin is the endogenous ligand for the G protein-coupled receptor APJ and exerts a key role in regulating cardiovascular functions. We report herein a novel series of macrocyclic analogues of apelin-13 in which the N- and C-terminal residues as well as the macrocycle composition were chemically modified to modulate structure-activity relationships on the APJ receptor. To this end, the binding affinity and the ability to engage G protein-dependent and G protein-independent signalling pathways of the resulting analogues were assessed. In this series, the position and the nature of the C-terminal aromatic residue is a determinant for APJ interaction and ß-arrestin recruitment, as previously demonstrated for linear apelin-13 derivatives. We finally discovered compounds 1, 4, 11 and 15, four potent G protein-biased apelin receptor agonists exhibiting affinity in the nanomolar range for APJ. These macrocyclic compounds represent very useful pharmacological tools to explore the therapeutic potential of the apelinergic system.
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Receptores de Apelina/agonistas , Péptidos y Proteínas de Señalización Intercelular/farmacología , Compuestos Macrocíclicos/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Péptidos y Proteínas de Señalización Intercelular/síntesis química , Péptidos y Proteínas de Señalización Intercelular/química , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Masculino , Conformación Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
ELABELA (ELA) was recently discovered as a novel endogenous ligand of the apelin receptor (APJ), a G protein-coupled receptor. ELA signaling was demonstrated to be crucial for normal heart and vasculature development during embryogenesis. We delineate here ELA's structure-activity relationships and report the identification of analogue 3 (ELA(19-32)), a fragment of ELA that binds to APJ, activates the Gαi1 and ß-arrestin-2 signaling pathways, and induces receptor internalization similarly to its parent endogenous peptide. An alanine scan performed on 3 revealed that the C-terminal residues are critical for binding to APJ and signaling. Finally, using isolated-perfused hearts and in vivo hemodynamic and echocardiographic measurements, we demonstrate that ELA and 3 both reduce arterial pressure and exert positive inotropic effects on the heart. Altogether, these results present ELA and 3 as potential therapeutic options in managing cardiovascular diseases.
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Fármacos Cardiovasculares/farmacología , Fragmentos de Péptidos/farmacología , Hormonas Peptídicas/química , Relación Estructura-Actividad , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Receptores de Apelina , Presión Sanguínea/efectos de los fármacos , Fármacos Cardiovasculares/química , Electrocardiografía , Corazón/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/fisiopatología , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Masculino , Datos de Secuencia Molecular , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Hormonas Peptídicas/metabolismo , Hormonas Peptídicas/farmacología , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
AIMS: Brugada syndrome (BrS) is a rare heritable ventricular arrhythmia. Genetic defects in SCN5A, a gene that encodes the α-subunit of the sodium ion channel Nav1.5, are present in 15-30% of BrS cases. SCN5A remains by far, the highest yielding gene for BrS. We studied a young male who presented with syncope at age 11. This proband was screened for possible disease causing SCN5A mutations. The inheritance pattern was also examined amongst his first-degree family members. METHODS AND RESULTS: The proband had a baseline electrocardiogram that showed Type 2 BrS changes, which escalated to a characteristic Type I BrS pattern during a treadmill test before polymorphic ventricular tachycardia onset at a cycle length of 250 ms. Mutational analysis across all 29 exons in SCN5A of the proband and first-degree relatives of the family revealed that the proband inherited a compound heterozygote mutation in SCN5A, specifically p.A226V and p.R1629X from each parent. To further elucidate the functional changes arising through these mutations, patch-clamp electrophysiology was performed in TSA201 cells expressing the mutated SCN5A channels. The p.A226V mutation significantly reduced peak sodium current (INa) to 24% of wild type (WT) whereas the p.R1629X mutation abolished the current. To mimic the functional state in our proband, functional expression of the compound variants A226V + R1629X resulted in overall peak INa of only 13% of WT (P < 0.01). CONCLUSION: Our study is the first to report a SCN5A compound heterozygote in a Singaporean Chinese family. Only the proband carrying both mutations displayed the BrS phenotype, thus providing insights into the expression and penetrance of BrS in an Asian setting.
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Síndrome de Brugada/genética , Heterocigoto , Canal de Sodio Activado por Voltaje NAV1.5/genética , Taquicardia Ventricular/genética , Adolescente , Adulto , Pueblo Asiatico , Línea Celular , Análisis Mutacional de ADN , Electrocardiografía , Exones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , Linaje , Fenotipo , Singapur , Adulto JovenRESUMEN
BACKGROUND: Arrhythmias associated with QT prolongation on the ECG often lead to sudden unexpected death in epilepsy. The mechanism causing a prolongation of the QT interval during epilepsy remains unknown. Based on observations showing an upregulation of neuronal sodium channels in the brain during epilepsy, we tested the hypothesis that a similar phenomenon occurs in the heart and contributes to QT prolongation by altering cardiac sodium current properties (INa). METHODS AND RESULTS: We used the patch clamp technique to assess the effects of epilepsy on the cardiac action potential and INa in rat ventricular myocytes. Consistent with QT prolongation, epileptic rats had longer ventricular action potential durations attributable to a sustained component of INa (INaL). The increase in INaL was because of a larger contribution of neuronal Na channels characterized by their high sensitivity to tetrodotoxin. As in the brain, epilepsy was associated with an enhanced expression of the neuronal isoform NaV1.1 in cardiomyocyte. Epilepsy was also associated with a lower INa activation threshold resulting in increased cell excitability. CONCLUSIONS: This is the first study correlating increased expression of neuronal sodium channels within the heart to epilepsy-related cardiac arrhythmias. This represents a new paradigm in our understanding of cardiac complications related to epilepsy.
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Potenciales de Acción/fisiología , Arritmias Cardíacas/genética , ADN/genética , Muerte Súbita/etiología , Epilepsia/metabolismo , Regulación de la Expresión Génica , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Animales , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/metabolismo , Western Blotting , Epilepsia/complicaciones , Epilepsia/mortalidad , Masculino , Canal de Sodio Activado por Voltaje NAV1.5/biosíntesis , Técnicas de Placa-Clamp , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Apelin is the endogenous ligand of the APJ receptor, a member of the G protein-coupled receptor family. This system plays an important role in the regulation of blood pressure and cardiovascular functions. To better understand the role of its C-terminal Phe(13) residue on ligand binding, receptor signaling, and hypotension, we report a series of modified analogues in which Phe(13) was substituted by unnatural amino acids. These modifications delivered new compounds exhibiting higher affinity and potency to inhibit cAMP accumulation compared to apelin-13. In particular, analogues Bpa(13) or (α-Me)Phe(13) were 30-fold more potent to inhibit cAMP accumulation than apelin-13. Tyr(OBn)(13) substitution led to a 60-fold improvement in binding affinity and induced stronger and more sustained drop in blood pressure compared to apelin-13. Our study identified new potent analogues of apelin-13, which represent valuable probes to better understand its structure-function relationship.
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Presión Sanguínea/efectos de los fármacos , AMP Cíclico/metabolismo , Hipotensión/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Sustitución de Aminoácidos , Animales , Apelina , Ligandos , Masculino , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
BACKGROUND: Functional alterations in the properties of Aß afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Na(v)1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Na(v)1.8 in controlling Aß-fiber excitability following persistent inflammation. METHODS: Distribution and expression of Na(v)1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund's adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Na(v)1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Na(v)1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats. RESULTS: Our findings revealed that Na(v)1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Na(v)1.8 peak current densities are enhanced in inflamed large myelinated Aß-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in Aß-fiber neuron excitability by shifting the voltage-dependent activation of Na(v)1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Na(v)1.8 currents in Aß-fiber neurons observed following intraplantar CFA injection and concomitantly blocks CFA-induced mechanical allodynia, suggesting that Na(v)1.8 regulation in Aß-fibers contributes to inflammatory pain. CONCLUSIONS: Collectively, these findings support a key role for Na(v)1.8 in controlling the excitability of Aß-fibers and its potential contribution to the development of mechanical allodynia under persistent inflammation.
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Ganglios Espinales/citología , Regulación de la Expresión Génica/fisiología , Inflamación/patología , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Neuronas/metabolismo , Nervio Ciático/metabolismo , Ambroxol/uso terapéutico , Animales , Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad , Adyuvante de Freund , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/inducido químicamente , Inflamación/complicaciones , Masculino , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
STIM1 plays a crucial role in Ca(2+) homeostasis, particularly in replenishing the intracellular Ca(2+) store following its depletion. In cardiomyocytes, the Ca(2+) content of the sarcoplasmic reticulum must be tightly controlled to sustain contractile activity. The presence of STIM1 in cardiomyocytes suggests that it may play a role in regulating the contraction of cardiomyocytes. The aim of the present study was to determine how STIM1 participates in the regulation of cardiac contractility. Atomic force microscopy revealed that knocking down STIM1 disrupts the contractility of cardiomyocyte-derived HL-1 cells. Ca(2+) imaging also revealed that knocking down STIM1 causes irregular spontaneous Ca(2+) oscillations in HL-1 cells. Action potential recordings further showed that knocking down STIM1 induces early and delayed afterdepolarizations. Knocking down STIM1 increased the peak amplitude and current density of T-type voltage-dependent Ca(2+) channels (T-VDCC) and shifted the activation curve toward more negative membrane potentials in HL-1 cells. Biotinylation assays revealed that knocking down STIM1 increased T-VDCC surface expression and co-immunoprecipitation assays suggested that STIM1 directly regulates T-VDCC activity. Thus, STIM1 is a negative regulator of T-VDCC activity and maintains a constant cardiac rhythm by preventing a Ca(2+) overload that elicits arrhythmogenic events.
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Canales de Calcio Tipo T/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Contracción Muscular , Miocitos Cardíacos/metabolismo , Taquicardia , Animales , Western Blotting , Canales de Calcio , Células Cultivadas , Electrofisiología , Inmunoprecipitación , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Ratones , Miocitos Cardíacos/citología , Molécula de Interacción Estromal 1RESUMEN
Voltage gated sodium channels (Na(V)s) are essential to propagate neuronal and cardiac electrical impulses. While the cardiac Na(+) current (I(Na)) is often all attributed to the cardiac isoform, Na(V)1.5, some evidence suggests that other Na(+) channel isoforms are also expressed in the heart ventricle. One way to distinguish Na(+) channels is by their sensitivity to tetrodotoxin (TTX); various "non-cardiac-type" Na(+) channels are relatively sensitive to TTX (denoted tNa(V) channels) compared to Na(V)1.5 channels. tNa(V) channels have been detected in hearts with various pathological conditions such as hypertrophy, infarction and ischemia, where they might enhance the late Na(+) current (I(NaL)) thereby prolonging the action potential under such conditions (resulting in a prolonged QT interval on the EKG). The principal aim of this article is to evaluate the extent to which non-cardiac isotypes contribute to I(NaL) under normal physiological conditions. I(NaL) was measured in acutely dissociated dog cardiomyocytes using the patch-clamp technique. Our results indicate that 44% on average of the late I(Na) current is due to non-cardiac Na(V)s. Previous studies indicated that the overexpression of non-cardiac Na(V) channels is responsible for the prolonged duration of the cardiac action potential (and, thereby, a prolonged QT interval) under pathophysiological conditions associated with various heart diseases. Our finding indicates that non-cardiac Na(V) channels are strong contributors to I(NaL) under physiological conditions thereby suggesting that these channels are also major determinants of the duration of the cardiac action potential even in healthy hearts. Interestingly, these results may explain the observations of cardiac arrhythmias associated with prolonged QT intervals in people with inherited neuronal and musculoskeletal diseases involving mutations that enhance the current from non-cardiac-type Na(V)s, a connection which apparently was never made before.
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Ventrículos Cardíacos/citología , Miocitos Cardíacos/fisiología , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Potenciales de Acción , Animales , Línea Celular , Perros , Metanosulfonato de Etilo/análogos & derivados , Metanosulfonato de Etilo/farmacología , Potenciales de la Membrana , Mesilatos/farmacología , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Tetrodotoxina , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
INTRODUCTION: Neuropeptides arginine-vasopressin (AVP), apelin (APL), and stromal-derived factor-1α (SDF-1α) are involved in the dysfunction of the corticotropic axis observed in septic ICU patients. Study aims were: (i) to portray a distinctive stress-related neuro-corticotropic systemic profile of early sepsis, (ii) to propose a combination data score, for aiding ICU physicians in diagnosing sepsis on admission. METHODS: This prospective one-center observational study was carried out in a medical intensive care unit (MICU), tertiary teaching hospital. Seventy-four out of 112 critically ill patients exhibiting systemic inflammatory response syndrome (SIRS) were divided into two groups: proven sepsis and non sepsis, based on post hoc analysis of microbiological criteria and final diagnosis, and compared to healthy volunteers (n = 14). A single blood sampling was performed on admission for measurements of AVP, copeptin, APL, SDF-1α, adrenocorticotropic hormone (ACTH), cortisol baseline and post-stimulation, and procalcitonin (PCT). RESULTS: Blood baseline ACTH/cortisol ratio was lower and copeptin higher in septic vs. nonseptic patients. SDF-1α was further increased in septic patients vs. normal patients. Cortisol baseline, ACTH, PCT, APACHE II and sepsis scores, and shock on admission, were independent predictors of sepsis diagnosis upon admission. Using the three first aforementioned categorical bio-parameters, a probability score for predicting sepsis yielded an area under the Receiver Operating Curve (ROC) curves better than sepsis score or PCT alone (0.903 vs 0.727 and 0.726: P = 0.005 and P < 0.04, respectively). CONCLUSIONS: The stress response of early admitted ICU patients is different in septic vs. non-septic conditions. A proposed combination of variable score analyses will tentatively help in refining bedside diagnostic tools to efficiently diagnose sepsis after further validation.
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Unidades de Cuidados Intensivos/estadística & datos numéricos , Sepsis/fisiopatología , Estrés Fisiológico/fisiología , Hormona Adrenocorticotrópica/sangre , Apelina , Arginina Vasopresina/sangre , Calcitonina/sangre , Péptido Relacionado con Gen de Calcitonina , Estudios de Casos y Controles , Quimiocina CXCL12/sangre , Femenino , Glicopéptidos/sangre , Humanos , Hidrocortisona/sangre , Péptidos y Proteínas de Señalización Intercelular/sangre , Modelos Logísticos , Masculino , Persona de Mediana Edad , Admisión del Paciente/estadística & datos numéricos , Estudios Prospectivos , Precursores de Proteínas/sangre , Curva ROC , Medición de Riesgo , Sepsis/sangre , Sepsis/diagnóstico , Estadísticas no ParamétricasRESUMEN
Apelin, a ligand of the G protein-coupled putative angiotensin II-like receptor (APJ-R), exerts strong vasodilating, cardiac inotropic and chronotropic actions. Its expression is highly up-regulated during heart failure. Apelin also increases cardiac conduction speed and excitability. While our knowledge of apelin cardiovascular actions is growing, our understanding of the physiological mechanisms behind the cardiac effects remains limited. We tested the effects of apelin on the cardiac sodium current (I(Na)) using patch clamp technique on cardiac myocytes acutely dissociated from dog ventricle. We found that apelin-13 and apelin-17 increased peak I(Na) by 39% and 61% and shifted its mid-activation potential by -6.8+/-0.6 mV and -17+/-1 mV respectively thus increasing channel opening at negative voltage. Apelin also slowed I(Na) recovery from inactivation. The effects of apelin on I(Na) amplitude were linked to activation of protein kinase C. Apelin also increased I(Na) "window" current by up to 600% suggesting that changes in intracellular sodium may contribute to the apelin inotropic effects. Our results reveal for the first time the effects of apelin on I(Na). These effects are likely to modulate cardiac conduction and excitability and may have beneficial antiarrhythmic action in sodium chanelopathies such as Brugada Syndrome where I(Na) amplitude is reduced.