RESUMEN
The human ether-a-go-go-related gene KCNH2 encodes the voltage-gated potassium channel underlying IKr, a current critical for the repolarization phase of the cardiac action potential. Mutations in KCNH2 that cause a reduction of the repolarizing current can result in cardiac arrhythmias associated with long-QT syndrome. Here, we investigate the regulation of KCNH2 and identify multiple active enhancers. A transcribed enhancer â¼85 kbp downstream of Kcnh2 physically contacts the promoters of two Kcnh2 isoforms in a cardiac-specific manner in vivo. Knockdown of its ncRNA transcript results in reduced expression of Kcnh2b and two neighboring mRNAs, Nos3 and Abcb8, in vitro. Genomic deletion of the enhancer, including the ncRNA transcription start site, from the mouse genome causes a modest downregulation of both Kcnh2a and Kcnh2b in the ventricles. These findings establish that the regulation of Kcnh2a and Kcnh2b is governed by a complex regulatory landscape that involves multiple partially redundantly acting enhancers.
Asunto(s)
Canal de Potasio ERG1/genética , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Miocardio/metabolismo , Transcripción Genética , Animales , Sistemas CRISPR-Cas/genética , Línea Celular , Canal de Potasio ERG1/metabolismo , Femenino , Sitios Genéticos , Ventrículos Cardíacos/metabolismo , Humanos , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Eliminación de Secuencia , Pez CebraRESUMEN
RATIONALE: Alternative cleavage and polyadenylation (APA) of mRNA represents a layer of gene regulation that to date has remained unexplored in the heart. This phenomenon may be relevant, as the positioning of the poly(A) tail in mRNAs influences the length of the 3'-untranslated region (UTR), a critical determinant of gene expression. OBJECTIVE: To investigate whether the 3'UTR length is regulated by APA in the human heart and whether this changes in the failing heart. METHODS AND RESULTS: We used 3'end RNA sequencing (e3'-Seq) to directly measure global patterns of APA in healthy and failing human heart specimens. By monitoring polyadenylation profiles in these hearts, we identified disease-specific APA signatures in numerous genes. Interestingly, many of the genes with shortened 3'UTRs in heart failure were enriched for functional groups such as RNA binding, whereas genes with longer 3'UTRs were enriched for cytoskeletal organization and actin binding. RNA sequencing in a larger series of human hearts revealed that these APA candidates are often differentially expressed in failing hearts, with an inverse correlation between 3'UTR length and the level of gene expression. Protein levels of the APA regulator, poly(A)-binding protein nuclear-1 were substantially downregulated in failing hearts. CONCLUSIONS: We provide genome-wide, high-resolution polyadenylation maps of the human heart and show that the 3'end formation of mRNA is dynamic in heart failure, suggesting that APA-mediated 3'UTR length modulation represents an additional layer of gene regulation in failing hearts.
Asunto(s)
Regiones no Traducidas 3' , Insuficiencia Cardíaca/genética , Poliadenilación , ARN Mensajero/genética , Adulto , Anciano , Secuencia de Bases , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteína I de Unión a Poli(A)/metabolismo , ARN Mensajero/metabolismoRESUMEN
Perturbations in sarcomeric function may in part underlie systolic and diastolic dysfunction of the failing heart. Sarcomeric dysfunction has been ascribed to changes in phosphorylation status of sarcomeric proteins caused by an altered balance between intracellular kinases and phosphatases during the development of cardiac disease. In the present review we discuss changes in phosphorylation of the thick filament protein myosin binding protein C (cMyBP-C) reported in failing myocardium, with emphasis on phosphorylation changes observed in familial hypertrophic cardiomyopathy caused by mutations in MYBPC3. Moreover, we will discuss assays which allow to distinguish between functional consequences of mutant sarcomeric proteins and (mal)adaptive changes in sarcomeric protein phosphorylation.