RESUMEN
Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by fibrofatty replacement of the myocardium. Deleterious variants in desmosomal genes are the main cause of ACM and lead to common and gene-specific molecular alterations, which are not yet fully understood. This article presents the first systematic in vitro study describing gene and protein expression alterations in desmosomes, electrical conduction-related genes, and genes involved in fibrosis and adipogenesis. Moreover, molecular and functional alterations in calcium handling were also characterized. This study was performed d with HL1 cells with homozygous knockouts of three of the most frequently mutated desmosomal genes in ACM: PKP2, DSG2, and DSC2 (generated by CRISPR/Cas9). Moreover, knockout and N-truncated clones of DSP were also included. Our results showed functional alterations in calcium handling, a slower calcium re-uptake was observed in the absence of PKP2, DSG2, and DSC2, and the DSP knockout clone showed a more rapid re-uptake. We propose that the described functional alterations of the calcium handling genes may be explained by mRNA expression levels of ANK2, CASQ2, ATP2A2, RYR2, and PLN. In conclusion, the loss of desmosomal genes provokes alterations in calcium handling, potentially contributing to the development of arrhythmogenic events in ACM.
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Displasia Ventricular Derecha Arritmogénica , Calcio , Humanos , Displasia Ventricular Derecha Arritmogénica/genética , Displasia Ventricular Derecha Arritmogénica/metabolismo , Desmosomas/genética , Desmosomas/metabolismo , Miocardio/metabolismo , CorazónRESUMEN
The SCN5A gene encodes the α-subunit of the voltage-gated cardiac sodium channel (NaV1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human SCN5A have been largely associated with inherited cardiac arrhythmias. Increasing evidence also suggests that aberrant expression of the SCN5A gene could increase susceptibility to arrhythmogenic diseases, but the mechanisms governing SCN5A expression are not yet well understood. To gain insights into the molecular basis of SCN5A gene regulation, we used rat gastrocnemius muscle four days following denervation, a process well known to stimulate Scn5a expression. Our results show that denervation of rat skeletal muscle induces the expression of the adult cardiac Scn5a isoform. RNA-seq experiments reveal that denervation leads to significant changes in the transcriptome, with Scn5a amongst the fifty top upregulated genes. Consistent with this increase in expression, ChIP-qPCR assays show enrichment of H3K27ac and H3K4me3 and binding of the transcription factor Gata4 near the Scn5a promoter region. Also, Gata4 mRNA levels are significantly induced upon denervation. Genome-wide analysis of H3K27ac by ChIP-seq suggest that a super enhancer recently described to regulate Scn5a in cardiac tissue is activated in response to denervation. Altogether, our experiments reveal that similar mechanisms regulate the expression of Scn5a in denervated muscle and cardiac tissue, suggesting a conserved pathway for SCN5A expression among striated muscles.
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Epigénesis Genética , Desnervación Muscular , Músculo Esquelético/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/biosíntesis , Elementos de Respuesta , Transcriptoma , Animales , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Masculino , Músculo Esquelético/inervación , Músculo Esquelético/patología , RNA-Seq , Ratas , Ratas Sprague-DawleyRESUMEN
Brugada syndrome is an inherited, rare cardiac arrhythmogenic disease, associated with sudden cardiac death. It accounts for up to 20% of sudden deaths in patients without structural cardiac abnormalities. The majority of mutations involve the cardiac sodium channel gene SCN5A and give rise to classical abnormal electrocardiogram with ST segment elevation in the right precordial leads V1 to V3 and a predisposition to ventricular fibrillation. The pathophysiological mechanisms of Brugada syndrome have been investigated using model systems including transgenic mice, canine heart preparations, and expression systems to study different SCN5A mutations. These models have a number of limitations. The recent development of pluripotent stem cell technology creates an opportunity to study cardiomyocytes derived from patients and healthy individuals. To date, only a few studies have been done using Brugada syndrome patient-specific iPS-CM, which have provided novel insights into the mechanisms and pathophysiology of Brugada syndrome. This review provides an evaluation of the strengths and limitations of each of these model systems and summarizes the key mechanisms that have been identified to date.
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Síndrome de Brugada/etiología , Síndrome de Brugada/fisiopatología , Modelos Animales de Enfermedad , Animales , Animales Modificados Genéticamente , Biomarcadores , Síndrome de Brugada/diagnóstico , Síndrome de Brugada/terapia , Diferenciación Celular , Susceptibilidad a Enfermedades , Perros , Predisposición Genética a la Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Mutación , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genéticaRESUMEN
Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G>A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1-45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters. Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems.
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Síndrome de Brugada/metabolismo , Síndrome de Brugada/patología , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Secuencia de Bases , Biomarcadores/metabolismo , Biotinilación , Membrana Celular/metabolismo , Forma de la Célula , Células HEK293 , Humanos , Activación del Canal Iónico , Proteínas Mutantes/metabolismoRESUMEN
INTRODUCTION: The California antitobacco culture may have influenced home smoking bans in Mexico. Based on the Behavioral Ecological Model, exposure to socially reinforcing contingencies or criticism may explain adoption of home smoking bans in Tijuana, Mexico, approximating rates relative to San Diego, California, and higher than those in Guadalajara, Mexico. METHODS: A representative cross-sectional population survey of Latinos (N = 1,901) was conducted in San Diego, Tijuana, and Guadalajara between June 2003 and September 2004. Cities were selected to represent high-, medium-, and low-level exposure to antitobacco social contingencies of reinforcement in a quasiexperimental analysis of possible cultural influences across borders. RESULTS: Complete home smoking ban prevalence was 91% in San Diego, 66% in Tijuana, and 38% in Guadalajara (p < .001). Sample cluster-adjusted logistic regression showed significantly lower odds of complete home smoking bans in Guadalajara (odds ratio [OR] = .048) and in Tijuana (OR = .138) compared to San Diego after control for demographics. Odds of complete home smoking bans in both Guadalajara and Tijuana in comparison with San Diego were weakened when mediators for bans were controlled in predictive models. Direction of association was consistent with theory. When theoretical mediators were explored as possible moderators, weak and nonsignificant associations were obtained for all interaction terms. Bootstrap analyses demonstrated that our multivariable logistic regression results were reliable. CONCLUSIONS: Results suggest that California antismoking social contingencies mediate complete home smoking bans in all 3 cities and may account for the greater effects in Tijuana contrasted with Guadalajara.
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Política para Fumadores , Fumar/epidemiología , Control Social Formal , Contaminación por Humo de Tabaco/legislación & jurisprudencia , Adulto , California/epidemiología , Análisis por Conglomerados , Estudios Transversales , Difusión de Innovaciones , Exposición a Riesgos Ambientales , Femenino , Humanos , Modelos Logísticos , Masculino , México/epidemiología , Modelos Teóricos , Fumar/psicología , Control Social Formal/métodos , Factores Socioeconómicos , Contaminación por Humo de Tabaco/prevención & controlRESUMEN
BACKGROUND: Brugada syndrome (BrS) is an inherited cardiac arrhythmogenic disease that predisposes patients to sudden cardiac death. It is associated with mutations in SCN5A, which encodes the cardiac sodium channel alpha subunit (NaV1.5). BrS-related mutations have incomplete penetrance and variable expressivity within families. OBJECTIVE: The purpose of this study was to determine the role of patient-specific genetic background on the cellular and clinical phenotype among carriers of NaV1.5_p.V1525M. METHODS: We studied sodium currents from patient-specific human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and heterologously transfected human embryonic kidney (HEK) tsA201 cells using the whole-cell patch-clamp technique. We determined gene and protein expression by quantitative polymerase chain reaction, RNA sequencing, and western blot and performed a genetic panel for arrhythmogenic diseases. RESULTS: Our results showed a large reduction in INa density in hiPSC-CM derived from 2 V1525M single nucleotide variant (SNV) carriers compared with hiPSC-CM derived from a noncarrier, suggesting a dominant-negative effect of the NaV1.5_p.V1525M channel. INa was not affected in hiPSC-CMs derived from a V1525M SNV carrier who also carries the NaV1.5_p.H558R polymorphism. Heterozygous expression of V1525M in HEK-293T cells produced a loss of INa function, not observed when this variant was expressed together with H558R. In addition, the antiarrhythmic drug mexiletine rescued INa function in hiPSC-CM. SCN5A expression was increased in the V1525M carrier who also expresses NaV1.5_p.H558R. CONCLUSION: Our results in patient-specific hiPSC-CM point to a dominant-negative effect of NaV1.5_p.V1525M, which can be reverted by the presence of NaV1.5_p.H558R. Overall, our data points to a role of patient-specific genetic background as a determinant for incomplete penetrance in BrS.
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Síndrome de Brugada , Humanos , Sodio/metabolismo , Arritmias Cardíacas/metabolismo , Trastorno del Sistema de Conducción Cardíaco/metabolismo , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismoRESUMEN
Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%-25% of BrS patients carry genetic defects that cause loss-of-function of the voltage-gated cardiac sodium channel. Thus, 70%-75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium ß2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa ) from cells coexpressing Nav 1.5 and wild-type (ß2WT) or mutant (ß2D211G) ß2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav 1.5 was coexpressed with the ß2D211G. Single channel analysis showed that the mutation did not affect the Nav 1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that ß2D211G decreases Nav 1.5 cell surface expression. The effect of the mutant ß2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.
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Síndrome de Brugada/genética , Predisposición Genética a la Enfermedad , Mutación Missense , Subunidad beta-2 de Canal de Sodio Activado por Voltaje/genética , Muerte Súbita Cardíaca/etiología , Femenino , Humanos , Persona de Mediana Edad , Canales de Sodio/genética , Canales de Sodio/metabolismo , Subunidad beta-2 de Canal de Sodio Activado por Voltaje/metabolismoRESUMEN
We studied principal neurons from canine intracardiac (IC) ganglia to determine whether large-conductance calcium-activated potassium (BK) channels play a role in their excitability. We performed whole cell recordings in voltage- and current-clamp modes to measure ion currents and changes in membrane potential from isolated canine IC neurons. Whole cell currents from these neurons showed fast- and slow-activated outward components. Both current components decreased in the absence of calcium and following 1-2 mM tetraethylammonium (TEA) or paxilline. These results suggest that BK channels underlie these current components. Single-channel analysis showed that BK channels from IC neurons do not inactivate in a time-dependent manner, suggesting that the dynamic of the decay of the fast current component is akin to that of intracellular calcium. Immunohistochemical studies showed that BK channels and type 2 ryanodine receptors are coexpressed in IC principal neurons. We tested whether BK current activation in these neurons occurred via a calcium-induced calcium release mechanism. We found that the outward currents of these neurons were not affected by the calcium depletion of intracellular stores with 10 mM caffeine and 10 µM cyclopiazonic acid. Thus, in canine intracardiac neurons, BK currents are directly activated by calcium influx. Membrane potential changes elicited by long (400 ms) current injections showed a tonic firing response that was decreased by TEA or paxilline. These data strongly suggest that the BK current present in canine intracardiac neurons regulates action potential activity and could increase these neurons excitability.
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Corazón/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Perros , Ganglios/efectos de los fármacos , Ganglios/metabolismo , Ganglios/fisiología , Corazón/efectos de los fármacos , Indoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Tetraetilamonio/farmacologíaRESUMEN
The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)] has been reported as a novel large-conductance Ca²âº-activated K⺠(BK) channel activator with selectivity for its ß1- or ß4-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory ß1-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the ß1-subunit, we explored the effects of DiBAC4(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC4(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic ß1-knockout mice in excised patches. BK channels from brain arteries, with or without the ß1-subunit, were similarly activated by DiBAC4(3). In addition, we found that saturating concentrations of DiBAC4(3) (~30 µM) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as -300 mV. This "sweet spot" for persistent activation is independent of Ca²âº and/or the ß1â4-subunits and is fully achieved when DiBAC4(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC4(3) varies across species and/or vascular beds. DiBAC4(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators.
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Barbitúricos/farmacología , Isoxazoles/farmacología , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/efectos de los fármacos , Canales de Potasio de Gran Conductancia Activados por el Calcio/efectos de los fármacos , Animales , Arterias/efectos de los fármacos , Arterias/metabolismo , Barbitúricos/metabolismo , Interpretación Estadística de Datos , Perros , Isoxazoles/metabolismo , Cinética , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Técnicas de Placa-Clamp , Reacción en Cadena en Tiempo Real de la Polimerasa , Canales de Sodio/efectos de los fármacosRESUMEN
Tissue-specific cells differentiated from patient-derived human induced pluripotent stem cells (hiPSC) are a relevant cellular model to study several diseases. We obtained a hiPSC line from skin fibroblasts of a patient affected by familial atrial fibrillation by nucleofection of non-integrating episomal vectors. The resulting hiPSC line displays a normal karyotype, expresses pluripotency surface markers and pluripotency genes, and differentiates into cells of the 3 germ layers. Therefore, it represents a reliable model to study the disease in a physiologically relevant cellular environment.
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Fibrilación Atrial , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Diferenciación Celular , Línea Celular , PlásmidosRESUMEN
BACKGROUND: it has been demonstrated that children obesity is a multifactorial disease and probably, the alteration of the family dynamic is another potential risk factor. The objective was to identify the association between obesity and family dysfunction in school children who attend to a family medicine unit. METHODS: case and control study at Mexican Social Security Institute in Guadalajara, Jalisco, Mexico. Sociodemographic factors and family dynamic of obese and non-obese subjects (n = 452) of six to nine years old from nuclear families were achieved. RESULTS: the association between family dysfunction and obesity was [OR = 1.63 (1.08-2.46), p = 0.01]. Area II, Identity formation, and area VI, Discipline and methods, showed a lower score in cases of children with obesity (p < 0.001 and p = 0.005, respectively). In a logistic regression model family dysfunction [RM 1.79 (1.19, 2.71), p = 0.005] and low literacy of mothers [RM 1.61 (1.06, 2.45), p = 0.02)] were risk factors for obesity in school children. CONCLUSIONS: the results showed an association between family dysfunction and obesity in school children. We suggest to consider it in the prevention of obesity in Mexican school children.
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Relaciones Familiares , Obesidad/epidemiología , Estudios de Casos y Controles , Niño , Femenino , Humanos , Masculino , México/epidemiología , Factores de RiesgoRESUMEN
The effects of genetic mutations on protein function can be studied in a physiologically relevant environment using tissue-specific cells differentiated from patient-derived induced pluripotent stem cells (iPSC). However, it is crucial to use iPSC derived from healthy individuals as control. We generated an iPS cell line from skin fibroblasts of a healthy Caucasian male by nucleofection of non-integrating episomal vectors. This cell line has normal karyotype, expresses pluripotency surface markers and pluripotency genes, and successfully differentiates into cells of the 3 germ layers. Therefore, it can be used as control for any disease of interest that is modelled using iPSC.
Asunto(s)
Células Madre Pluripotentes Inducidas , Diferenciación Celular , Línea Celular , Fibroblastos , Estratos Germinativos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , MasculinoRESUMEN
Patient-derived induced pluripotent stem cells (iPSC) are a valuable approach to model cardiovascular diseases. We nucleofected non-integrating episomal vectors in skin fibroblasts of three family members carrying a single nucleotide variant (SNV) in SCN5A, which encodes the cardiac-type sodium channel, and of a related healthy control. The SNV SCN5A_c.4573G > A had been previously identified in a Brugada Syndrome patient. The resulting iPS cell lines differentiate into cells of the 3 germ layers, display normal karyotypes and express pluripotency surface markers and genes. Thus, they are a reliable source to study the effect of the identified mutation in a physiologically relevant environment.
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Células Madre Pluripotentes Inducidas , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Nucleótidos/metabolismoRESUMEN
Long QT syndrome (LQTS) is an inherited disorder characterized by prolonged QT intervals and potentially life-threatening arrhythmias. Mutations in 12 different genes have been associated with LQTS. Here we describe a patient with LQTS who has a mutation in KCNQ1 as well as a polymorphism in KCNH2. The proband (MMRL0362), a 32-year-old female, exhibited multiple ventricular extrasystoles and one syncope. Her ECG (QT interval corrected for heart rate (QTc) = 518ms) showed an LQT2 morphology in leads V4-V6 and LQT1 morphology in leads V1-V2. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 7 LQTS susceptibility genes were amplified and sequenced. Variations were detected predicting a novel missense mutation (V110I) in KCNQ1, as well as a common polymorphism in KCNH2 (K897T). We expressed wild-type (WT) or V110I Kv7.1 channels in CHO-K1 cells cotransfected with KCNE1 and performed patch-clamp analysis. In addition, WT or K897T Kv11.1 were also studied by patch clamp. Current-voltage (I-V) relations for V110I showed a significant reduction in both developing and tail current densities compared with WT at potentials >+20 mV (p < 0.05; n = 8 cells, each group), suggesting a reduction in IKs currents. K897T- Kv11.1 channels displayed a significantly reduced tail current density compared with WT-Kv11.1 at potentials >+10 mV. Interestingly, channel availability assessed using a triple-pulse protocol was slightly greater for K897T compared with WT (V0.5 = -53.1 ± 1.13 mV and -60.7 ± 1.15 mV for K897T and WT, respectively; p < 0.05). Comparison of the fully activated I-V revealed no difference in the rectification properties between WT and K897T channels. We report a patient with a loss-of-function mutation in KCNQ1 and a loss-of-function polymorphism in KCNH2. Our results suggest that a reduction of both IKr and IKs underlies the combined LQT1 and LQT2 phenotype observed in this patient.
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Canales de Potasio Éter-A-Go-Go/genética , Canal de Potasio KCNQ1/genética , Síndrome de QT Prolongado/genética , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Canal de Potasio ERG1 , Electrocardiografía , Femenino , Variación Genética , Humanos , Datos de Secuencia Molecular , Mutación , Fenotipo , Polimorfismo GenéticoRESUMEN
Voltage-gated sodium (NaV) channels are transmembrane proteins that initiate and propagate neuronal and cardiac action potentials. NaV channel ß subunits have been widely studied due to their modulatory role. Mice null for Scn1b, which encodes NaV ß1 and ß1b subunits, have defects in neuronal development and excitability, spontaneous generalized seizures, cardiac arrhythmias, and early mortality. A mutation in exon 3 of SCN1B, c.308A>T leading to ß1_p.D103V and ß1b_p.D103V, was previously found in a patient with a history of proarrhythmic conditions with progressive atrial standstill as well as cognitive and motor deficits accompanying structural brain abnormalities. We investigated whether ß1 or ß1b subunits carrying this mutation affect NaV1.5 and/or NaV1.1 currents using a whole cell patch-clamp technique in tsA201 cells. We observed a decrease in sodium current density in cells co-expressing NaV1.5 or NaV1.1 and ß1D103V compared to ß1WT. Interestingly, ß1bD103V did not affect NaV1.1 sodium current density but induced a positive shift in the voltage dependence of inactivation and a faster recovery from inactivation compared to ß1bWT. The ß1bD103V isoform did not affect NaV1.5 current properties. Although the SCN1B_c.308A>T mutation may not be the sole cause of the patient's symptoms, we observed a clear loss of function in both cardiac and brain sodium channels. Our results suggest that the mutant ß1 and ß1b subunits play a fundamental role in the observed electrical dysfunction.
RESUMEN
OBJECTIVE: This study examines the association between home smoking ban status and home secondhand smoke exposure (SHSE) among Mexican-descent adults and children in three cities representing different levels of exposure to the California Tobacco Control Program. METHOD: From 2003-2004, a cross-sectional tobacco survey was conducted with a population-based sample of Mexican-descent adults in San Diego, California (N=1103) and Mexican adults in Tijuana (N=398) and Guadalajara (N=400), Mexico. RESULTS: After adjustment for demographic, behavioral, cultural, and contextual covariates, a complete home smoking ban was associated with Mexican American and Mexican adults and children being less likely to experience SHSE in the home compared to their counterparts with partial or no smoking restrictions. The association was significant across the three survey sites, but stronger in San Diego than in any of the two Mexican cities. CONCLUSION: Home smoking bans reduce the risk of home SHSE among Mexican Americans and Mexicans, regardless of the smoking status of the household residents and other individual and environmental variables. Even if household residents continue smoking and communitywide tobacco control efforts are suboptimal, the promotion of home smoking bans can protect adults and children from home SHSE.
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Exposición a Riesgos Ambientales/estadística & datos numéricos , Prevención del Hábito de Fumar , Fumar/legislación & jurisprudencia , Contaminación por Humo de Tabaco/legislación & jurisprudencia , Contaminación por Humo de Tabaco/estadística & datos numéricos , Adulto , California/epidemiología , Estudios Transversales , Características Culturales , Emigrantes e Inmigrantes/estadística & datos numéricos , Exposición a Riesgos Ambientales/legislación & jurisprudencia , Exposición a Riesgos Ambientales/prevención & control , Composición Familiar , Femenino , Vivienda , Humanos , Masculino , Americanos Mexicanos , México/epidemiología , Medio Social , Contaminación por Humo de Tabaco/prevención & controlRESUMEN
AIMS: To assess the functional impact of two combined KCNH2 variants involved in atrial fibrillation, syncope and sudden infant death syndrome. METHODS AND RESULTS: Genetic testing of a 4-month old SIDS victim identified a rare missense heterozygous in KCNH2 variant (V483I) and a missense homozygous polymorphism (K897T) which is often described as a genetic modifier. Electrophysiological characterisation of heterologous HERG channels representing two different KCNH2 genotypes within the family, showed significant differences in both voltage and time dependence of activation and inactivation with a global gain-of-function effect of mutant versus wild type channels and, also, differences between both types of recombinant channels. CONCLUSIONS: The rare variant V483I in combination with K897T produces a gain-of-function effect that represents a pathological substrate for atrial fibrillation, syncope and sudden infant death syndrome events in this family. Ascertaining the genotype-phenotype correlation of genetic variants is imperative for the correct assessment of genetic testing and counselling. TRANSLATIONAL PERSPECTIVE: According to the current guidelines for clinical interpretation of sequence variants, functional studies are an essential tool for the ascertainment of variant pathogenicity. They are especially relevant in the context of sudden infant death syndrome and sudden cardiac death, where individuals cannot be clinically evaluated. The patch-clamp technique is a gold-standard for analysis of the biophysical mechanisms of ion channels.
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Fibrilación Atrial/genética , Canal de Potasio ERG1/genética , Mutación Missense , Linaje , Muerte Súbita del Lactante/genética , Heterocigoto , Homocigoto , Humanos , Lactante , Masculino , Persona de Mediana Edad , Fenotipo , Polimorfismo GenéticoRESUMEN
The lysine-specific histone demethylase 1A (LSD1) also known as lysine (K)-specific demethylase 1A (KDM1A) is a central epigenetic regulator of metabolic reprogramming in obesity-associated diseases, neurological disorders, and cancer. Here, we evaluated the ability of oleacein, a biophenol secoiridoid naturally present in extra virgin olive oil (EVOO), to target LSD1. Molecular docking and dynamic simulation approaches revealed that oleacein could target the binding site of the LSD1 cofactor flavin adenosine dinucleotide with high affinity and at low concentrations. At higher concentrations, oleacein was predicted to target the interaction of LSD1 with histone H3 and the LSD1 co-repressor (RCOR1/CoREST), likely disturbing the anchorage of LSD1 to chromatin. AlphaScreen-based in vitro assays confirmed the ability of oleacein to act as a direct inhibitor of recombinant LSD1, with an IC50 as low as 2.5 µmol/L. Further, oleacein fully suppressed the expression of the transcription factor SOX2 (SEX determining Region Y-box 2) in cancer stem-like and induced pluripotent stem (iPS) cells, which specifically occurs under the control of an LSD1-targeted distal enhancer. Conversely, oleacein failed to modify ectopic SOX2 overexpression driven by a constitutive promoter. Overall, our findings provide the first evidence that EVOO contains a naturally occurring phenolic inhibitor of LSD1, and support the use of oleacein as a template to design new secoiridoid-based LSD1 inhibitors.
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Aldehídos/farmacología , Histona Demetilasas/antagonistas & inhibidores , Aceite de Oliva/química , Fenoles/farmacología , Aldehídos/análisis , Sitios de Unión/efectos de los fármacos , Neoplasias de la Mama , Línea Celular Tumoral , Proteínas Co-Represoras/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Histona Demetilasas/química , Histona Demetilasas/metabolismo , Histonas/metabolismo , Humanos , Células MCF-7 , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Células Madre Neoplásicas/metabolismo , Fenoles/análisis , Proteínas Recombinantes/efectos de los fármacos , Factores de Transcripción SOXB1/antagonistas & inhibidores , Factores de Transcripción SOXB1/genéticaRESUMEN
Inward rectifier potassium Kir2.x channels mediate cardiac inward rectifier potassium currents (I(K1)). As a subunit of Kir2.x, the physiological role of Kir2.3 in native cardiomyocytes has not been reported. This study shows that Kir2.3 knock-down remarkably down-regulates Kir2.3 expression (Kir2.3 protein was reduced to 19.91+/-3.24% on the 2nd or 3rd day) and I(K1) current densities (at -120 mV, control vs. knock-down: -5.03+/-0.24 pA/pF, n=5 vs. -1.16+/-0.19 pA/pF, n=7, P<0.001) in neonatal rat cardiomyocytes. The data suggest that Kir2.3 plays a potentially important role in I(K1) currents in neonatal rat cardiomyocytes.
Asunto(s)
Potenciales de la Membrana , Miocitos Cardíacos/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Animales , Células Cultivadas , Expresión Génica , Potenciales de la Membrana/genética , Microscopía Confocal , Miocitos Cardíacos/citología , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/genética , Interferencia de ARN , Ratas , Ratas Sprague-DawleyRESUMEN
Isolation of the pulmonary vein antrum can terminate atrial fibrillation, but the rationale has not been elucidated. In the present study, we show that sheep atrial effective refractory period (ERP) was heterogeneously shortened by acetylcholine administration. After perfusion with 15 muM acetylcholine, the shortest ERP occurred in the pulmonary vein antrum, which was recorded with the standard intracellular microelectrode technique (the ERP results in the pulmonary vein antrum, left atrial posterior wall, roof, free wall and appendage, and right atrial free wall were 52.0 +/- 1.6, 75.1 +/- 2.0, 77.2 +/- 1.7, 85.6 +/- 1.7, 64.3 +/- 2.1, and 90.5 +/- 1.3 ms, respectively; P < 0.05). Immunofluorescent staining revealed that muscarinic type 2 receptors (M(2)R) were also distributed heterogeneously in the atrial myocardium, with the highest density in the antrum (the relative fluorescent intensity results of the M(2)R in the pulmonary vein antrum, left atrial posterior wall, roof, free wall and appendage, and right atrial free wall were 62.64 +/- 2.56, 53.12 +/- 2.76, 51.83 +/- 2.45, 47.90 +/- 2.33, 55.27 +/- 2.08, and 45.53 +/- 2.02, respectively; P < 0.05), which was in accordance with the heterogeneity of ERP distribution. Thus the pulmonary vein antrum is a unique electrophysiological region with high sensitivity to acetylcholine, and its intensive response to acetylcholine is most likely associated with the dense M(2)R distribution of this region. Such an acetylcholine-induced ERP heterogeneity is possibly a substrate for atrial fibrillation and hence one of the potential electrophysiological bases for the isolation therapy.