RESUMEN
LMNA mutations in patients are responsible for a dilated cardiomyopathy. Molecular mechanisms underlying the origin and development of the pathology are unknown. Herein, using mouse pluripotent embryonic stem cells (ESCs) and a mouse model both harboring the p.H222P Lmna mutation, we found early defects in cardiac differentiation of mutated ESCs and dilatation of mutated embryonic hearts at E13.5, pointing to a developmental origin of the disease. Using mouse ESCs, we demonstrated that cardiac differentiation of LmnaH222P/+ was impaired at the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene involved in epithelial-to-mesenchymal transition of epiblast cells, as well as Snai1 and Twist expression, was decreased in LmnaH222P/+ cells compared with WT cells in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. ChIP assay of H3K4me1 in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of Mesp1 and Twist in LmnaH222P/+ cells. Downregulation or inhibition of LSD1 that specifically demethylated H3K4me1 rescued the epigenetic landscape of mesodermal LmnaH222P/+ cells and in turn contraction of cardiomyocytes. Inhibition of LSD1 in pregnant mice or neonatal mice prevented cardiomyopathy in E13.5 LmnaH222P/H222P offspring and adults, respectively. Thus, LSD1 appeared to be a therapeutic target to prevent or cure dilated cardiomyopathy associated with a laminopathy.
Asunto(s)
Cardiomiopatías/enzimología , Cardiomiopatías/prevención & control , Histona Demetilasas/metabolismo , Laminopatías/complicaciones , Laminopatías/enzimología , Miocitos Cardíacos/enzimología , Sustitución de Aminoácidos , Animales , Cardiomiopatías/genética , Diferenciación Celular , Modelos Animales de Enfermedad , Histona Demetilasas/genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Laminopatías/genética , Ratones , Ratones Mutantes , Células Madre Embrionarias de Ratones/enzimología , Células Madre Embrionarias de Ratones/patología , Mutación Missense , Miocitos Cardíacos/patologíaRESUMEN
Pluripotent stem cells feature the capacity to differentiate into any somatic cell types including cardiomyocytes. We report a cost-effective and simple protocol for the differentiation of specific ventricular cardiomyocytes. These cells are elongated, do not spontaneously beat, and do not feature any Ca2+-transient, an index of their stage of maturation toward adult cardiac cells. They represent a suitable model to screen both the efficiency and toxicology of drugs.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Animales , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ratones , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismoRESUMEN
Hyper-activation of extracellular signal-regulated kinase (ERK) 1/2 contributes to heart dysfunction in cardiomyopathy caused by mutations in the lamin A/C gene (LMNA cardiomyopathy). The mechanism of how this affects cardiac function is unknown. We show that active phosphorylated ERK1/2 directly binds to and catalyzes the phosphorylation of the actin depolymerizing factor cofilin-1 on Thr25. Cofilin-1 becomes active and disassembles actin filaments in a large array of cellular and animal models of LMNA cardiomyopathy. In vivo expression of cofilin-1, phosphorylated on Thr25 by endogenous ERK1/2 signaling, leads to alterations in left ventricular function and cardiac actin. These results demonstrate a novel role for cofilin-1 on actin dynamics in cardiac muscle and provide a rationale on how increased ERK1/2 signaling leads to LMNA cardiomyopathy.
Asunto(s)
Actinas/metabolismo , Cardiomiopatía Dilatada/patología , Cofilina 1/metabolismo , Lamina Tipo A/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación , Actinas/genética , Adolescente , Adulto , Animales , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Estudios de Casos y Controles , Cofilina 1/genética , Femenino , Corazón/fisiología , Humanos , Lamina Tipo A/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosforilación , Transducción de Señal , Adulto JovenRESUMEN
Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.
