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1.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-33803193

RESUMEN

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.


Asunto(s)
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-Dawley
2.
J Mol Cell Cardiol ; 114: 10-19, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29024690

RESUMEN

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.


Asunto(s)
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/metabolismo
3.
Heart Rhythm ; 21(3): 331-339, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38008367

RESUMEN

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.


Asunto(s)
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/metabolismo
4.
Stem Cell Res ; 73: 103239, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37922745

RESUMEN

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.


Asunto(s)
Fibrilación Atrial , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Diferenciación Celular , Línea Celular , Plásmidos
5.
Stem Cell Res ; 63: 102847, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35772296

RESUMEN

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.


Asunto(s)
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/metabolismo
6.
Stem Cell Res ; 60: 102717, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35231795

RESUMEN

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 , Masculino
7.
Front Cell Dev Biol ; 8: 528742, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33134290

RESUMEN

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.

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