RESUMEN
BACKGROUND: Cell-type-specific genes exhibit heterogeneity in genomic contexts and may be subject to different epigenetic regulations through different gene transcriptional processes depending on the cell type involved. The gene-body regions (GBRs) of some cardiomyocyte (CM)-specific genes are long and highly hypomethylated in CMs. To explore the cell-type specificities of epigenetic patterns and functions, multiple epigenetic modifications of GBRs were compared among CMs, liver cells and embryonic stem cells (ESCs). RESULTS: We found that most genes show a moderately negative correlation between transcript levels and gene lengths. As CM-specific genes are generally longer than other cell-type-specific genes, we hypothesized that the gene-body epigenetic features of CMs may support the transcriptional regulation of CM-specific genes. We found gene-body DNA hypomethylation in a CM-specific gene subset co-localized with rare gene-body marks, including RNA polymerase II (Pol II) and p300. Interestingly, 5-hydroxymethylcytosine (5hmC) within the gene body marked cell-type-specific genes at neonatal stages and active gene-body histone mark H3K36 trimethylation declined and overlapped with cell-type-specific gene-body DNA hypomethylation and selective Pol II/p300 accumulation in adulthood. Different combinations of gene-body epigenetic modifications were also observed with genome-wide scale cell-type specificity, revealing the occurrence of dynamic epigenetic rearrangements in GBRs across different cell types. CONCLUSIONS: As 5hmC enrichment proceeded to hypomethylated GBRs, we considered that hypomethylation may not represent a static state but rather an equilibrium state of turnover due to the balance between local methylation linked to transcription and Tet oxidative modification causing demethylation. Accordingly, we conclude that demethylation in CMs can be a used to establish such cell-type-specific epigenetic domains in relation to liver cells. The establishment of cell-type-specific epigenetic control may also change genomic contexts of evolution and may contribute to the development of cell-type-specific transcriptional coordination.
Asunto(s)
Metilación de ADN , Desmetilación , Epigénesis Genética , Ligamiento Genético , Miocitos Cardíacos/metabolismo , Transcripción Genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animales , Linaje de la Célula/genética , Células Madre Embrionarias , Femenino , Genes Esenciales , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BLRESUMEN
Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming.
Asunto(s)
Transdiferenciación Celular/fisiología , Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , MicroARNs/metabolismo , Miocitos Cardíacos/metabolismo , Factores de Transcripción/genética , Análisis de Varianza , Animales , Western Blotting , Transdiferenciación Celular/genética , Clonación Molecular , Fibroblastos/citología , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes , Humanos , Inmunohistoquímica , Ratones , Análisis por Micromatrices , Miocitos Cardíacos/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismoRESUMEN
Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca(2+) oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.
Asunto(s)
Fibroblastos/metabolismo , Regulación de la Expresión Génica , Proteínas Musculares/biosíntesis , Miocitos Cardíacos/metabolismo , Factores de Transcripción/biosíntesis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Células Cultivadas , Niño , Preescolar , Femenino , Fibroblastos/citología , Humanos , Lactante , Masculino , Ratones , Persona de Mediana Edad , Proteínas Musculares/genética , Miocitos Cardíacos/citología , Factores de Transcripción/genéticaRESUMEN
Dichloroacetate (DCA) promotes pyruvate entry into the Krebs cycle by inhibiting pyruvate dehydrogenase (PDH) kinase and thereby maintaining PDH in the active dephosphorylated state. DCA has recently gained attention as a potential metabolic-targeting therapy for heart failure but the molecular basis of the therapeutic effect of DCA in the heart remains a mystery. Once-daily oral administration of DCA alleviates pressure overload-induced left ventricular remodeling. We examined changes in the metabolic fate of pyruvate carbon (derived from glucose) entering the Krebs cycle by metabolic interventions of DCA. (13)C6-glucose pathway tracing analysis revealed that instead of being completely oxidized in the mitochondria for ATP production, DCA-mediated PDH dephosphorylation results in an increased acetyl-CoA pool both in control and pressure-overloaded hearts. DCA induces hyperacetylation of histone H3K9 and H4 in a dose-dependent manner in parallel to the dephosphorylation of PDH in cultured cardiomyocytes. DCA administration increases histone H3K9 acetylation in in vivo mouse heart. Interestingly, DCA-dependent histone acetylation was associated with an up-regulation of 2.3% of genes (545 out of 23,474 examined). Gene ontology analysis revealed that these genes are highly enriched in transcription-related categories. This evidence suggests that sustained activation of PDH by DCA results in an overproduction of acetyl-CoA, which exceeds oxidation in the Krebs cycle and results in histone acetylation. We propose that DCA-mediated PDH activation has the potential to induce epigenetic remodeling in the heart, which, at least in part, forms the molecular basis for the therapeutic effect of DCA in the heart.
Asunto(s)
Ácido Dicloroacético/farmacología , Epigénesis Genética/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismo , Remodelación Ventricular/efectos de los fármacos , Remodelación Ventricular/genética , Ácido 3-Hidroxibutírico/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Corazón/efectos de los fármacos , Histonas/metabolismo , Masculino , Metaboloma , Metabolómica/métodos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Miocardio/metabolismo , Especificidad de Órganos/genética , Fosforilación , Complejo Piruvato Deshidrogenasa/farmacología , Ratas , Transcripción GenéticaRESUMEN
Cardiac muscle differentiation in vivo is guided by sequential growth factor signals, including endoderm-derived diffusible factors, impinging on cardiogenic genes in the developing mesoderm. Previously, by RNA interference in AB2.2 mouse embryonic stem cells (mESCs), we identified the endodermal transcription factor Sox17 as essential for Mesp1 induction in primitive mesoderm and subsequent cardiac muscle differentiation. However, downstream effectors of Sox17 remained to be proven functionally. In this study, we used genome-wide profiling of Sox17-dependent genes in AB2.2 cells, RNA interference, chromatin immunoprecipitation, and luciferase reporter genes to dissect this pathway. Sox17 was required not only for Hhex (a second endodermal transcription factor) but also for Cer1, a growth factor inhibitor from endoderm that, like Hhex, controls mesoderm patterning in Xenopus toward a cardiac fate. Suppressing Hhex or Cer1 blocked cardiac myogenesis, although at a later stage than induction of Mesp1/2. Hhex was required but not sufficient for Cer1 expression. Over-expression of Sox17 induced endogenous Cer1 and sequence-specific transcription of a Cer1 reporter gene. Forced expression of Cer1 was sufficient to rescue cardiac differentiation in Hhex-deficient cells. Thus, Hhex and Cer1 are indispensable components of the Sox17 pathway for cardiopoiesis in mESCs, acting at a stage downstream from Mesp1/2.
Asunto(s)
Células Madre Embrionarias/metabolismo , Proteínas HMGB/metabolismo , Proteínas de Homeodominio/metabolismo , Mesodermo/embriología , Miocardio/metabolismo , Proteínas/metabolismo , Factores de Transcripción SOXF/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Animales , Sitios de Unión/genética , Tipificación del Cuerpo/efectos de los fármacos , Diferenciación Celular/genética , Citocinas , Células Madre Embrionarias/citología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genoma , Subunidades beta de Inhibinas/metabolismo , Mesodermo/citología , Ratones , Modelos Biológicos , Desarrollo de Músculos/genética , Miocardio/citología , Proteína Nodal/metabolismo , Unión Proteica/genética , Transducción de Señal/genéticaRESUMEN
RATIONALE: After myocardial infarction (MI), massive cell death in the myocardium initiates fibrosis and scar formation, leading to heart failure. We recently found that a combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), reprograms fibroblasts directly into functional cardiomyocytes in vitro. OBJECTIVE: To investigate whether viral gene transfer of GMT into infarcted hearts induces cardiomyocyte generation. METHODS AND RESULTS: Coronary artery ligation was used to generate MI in the mouse. In vitro transduction of GMT retrovirus converted cardiac fibroblasts from the infarct region into cardiomyocyte-like cells with cardiac-specific gene expression and sarcomeric structures. Injection of the green fluorescent protein (GFP) retrovirus into mouse hearts, immediately after MI, infected only proliferating noncardiomyocytes, mainly fibroblasts, in the infarct region. The GFP expression diminished after 2 weeks in immunocompetent mice but remained stable for 3 months in immunosuppressed mice, in which cardiac induction did not occur. In contrast, injection of GMT retrovirus into α-myosin heavy chain (αMHC)-GFP transgenic mouse hearts induced the expression of αMHC-GFP, a marker of cardiomyocytes, in 3% of virus-infected cells after 1 week. A pooled GMT injection into the immunosuppressed mouse hearts induced cardiac marker expression in retrovirus-infected cells within 2 weeks, although few cells showed striated muscle structures. To transduce GMT efficiently in vivo, we generated a polycistronic retrovirus expressing GMT separated by 2A "self-cleaving" peptides (3F2A). The 3F2A-induced cardiomyocyte-like cells in fibrotic tissue expressed sarcomeric α-actinin and cardiac troponin T and had clear cross striations. Quantitative RT-PCR also demonstrated that FACS-sorted 3F2A-transduced cells expressed cardiac-specific genes. CONCLUSIONS: GMT gene transfer induced cardiomyocyte-like cells in infarcted hearts.
Asunto(s)
Diferenciación Celular/genética , Factor de Transcripción GATA4/genética , Técnicas de Transferencia de Gen , Infarto del Miocardio/patología , Miocitos Cardíacos/patología , Factores Reguladores Miogénicos/genética , Proteínas de Dominio T Box/genética , Animales , Diferenciación Celular/fisiología , Fibroblastos/patología , Factor de Transcripción GATA4/fisiología , Proteínas Fluorescentes Verdes/genética , Factores de Transcripción MEF2 , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Desnudos , Ratones Transgénicos , Modelos Animales , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/fisiología , Factores Reguladores Miogénicos/fisiología , Regeneración/genética , Regeneración/fisiología , Retroviridae/genética , Proteínas de Dominio T Box/fisiologíaRESUMEN
Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.
Asunto(s)
Separación Celular/métodos , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Coloración y Etiquetado/métodos , Animales , Animales Recién Nacidos , Callithrix , Diferenciación Celular , Trasplante de Células , Células Cultivadas , Embrión de Mamíferos/citología , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Colorantes Fluorescentes/análisis , Corazón/embriología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mitocondrias/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/trasplante , Ratas , Rodaminas/análisisRESUMEN
MicroRNAs (miRNAs) play a pivotal role during embryonic development and are required for proper organogenesis, including hematopoiesis. Recent studies suggest that, in the early mesoderm, there is an interaction between the hematopoietic and cardiac lineages. However, whether miRNAs can affect other lineages remains unknown. Therefore, we investigated whether hematopoietic miR-142-3p modulated the mesoderm formation. We report that knockdown (KD) of miR-142-3p, a hematopoietic-specific miRNA, in zebrafish resulted in loss of hematopoiesis during embryonic development. Intriguingly, we observed abnormal cardiac phenotypes and insufficiency of somitegenesis in KD-morphants. In the early developmental stage, a tiny heart, contractile dysfunction in the ventricle, cardiac arrhythmia (e.g. a 2:1 ratio of atrial:ventricular beating), and bradycardia were consistently observed. Histological examination revealed severe hypoplasia of the ventricle and disrupted muscle alignment. To determine the mechanism, we performed DNA microarray analysis. The results revealed that the expression of several mesodermal genes essential for the formation of cardiac and somatic mesoderm, such as no tail, T-box gene 16, mesoderm posterior a, one eye pinhead, and rho-associated, coiled-coil containing protein kinase (Rock2a), were increased in miR-142-3p KD-morphants. The luciferase reporter assay revealed that miR-142-3p repressed luciferase activity on the Rock2a 3'-UTR. The findings of the present study indicate that miR-142-3p plays a critical role in hematopoiesis, cardiogenesis, and somitegenesis in the early stage of mesoderm formation via regulation of Rock2a.
Asunto(s)
Corazón/embriología , Hematopoyesis , MicroARNs/metabolismo , Organogénesis , Pez Cebra/embriología , Animales , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Reporteros , Corazón/fisiología , Luciferasas/biosíntesis , Luciferasas/genética , MicroARNs/genética , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
RATIONALE: The transcriptional networks guiding heart development remain poorly understood, despite the identification of several essential cardiac transcription factors. OBJECTIVE: To isolate novel cardiac transcription factors, we performed gene chip analysis and found that Zac1, a zinc finger-type transcription factor, was strongly expressed in the developing heart. This study was designed to investigate the molecular and functional role of Zac1 as a cardiac transcription factor. METHODS AND RESULTS: Zac1 was strongly expressed in the heart from cardiac crescent stages and in the looping heart showed a chamber-restricted pattern. Zac1 stimulated luciferase reporter constructs driven by ANF, BNP, or alphaMHC promoters. Strong functional synergy was seen between Zac1 and Nkx2-5 on the ANF promoter, which carries adjacent Zac1 and Nkx2-5 DNA-binding sites. Zac1 directly associated with the ANF promoter in vitro and in vivo, and Zac1 and Nkx2-5 physically associated through zinc fingers 5 and 6 in Zac1, and the homeodomain in Nkx2-5. Zac1 is a maternally imprinted gene and is the first such gene found to be involved in heart development. Homozygous and paternally derived heterozygous mice carrying an interruption in the Zac1 locus showed decreased levels of chamber and myofilament genes, increased apoptotic cells, partially penetrant lethality and morphological defects including atrial and ventricular septal defects, and thin ventricular walls. CONCLUSIONS: Zac1 plays an essential role in the cardiac gene regulatory network. Our data provide a potential mechanistic link between Zac1 in cardiogenesis and congenital heart disease manifestations associated with genetic or epigenetic defects in an imprinted gene network.
Asunto(s)
Proteínas de Ciclo Celular/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Cardiopatías Congénitas/genética , Corazón/embriología , Factores de Transcripción/genética , Animales , Apoptosis/genética , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Sitios de Unión , Células COS , Proteínas de Ciclo Celular/metabolismo , Chlorocebus aethiops , Perfilación de la Expresión Génica/métodos , Genes Supresores de Tumor , Impresión Genómica , Edad Gestacional , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/patología , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Mutantes , Morfogénesis/genética , Mutación , Péptido Natriurético Tipo-C/genética , Péptido Natriurético Tipo-C/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Ratas , Factores de Transcripción/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
OBJECTIVE: We recently demonstrated that primitive neural crest-derived (NC) cells migrate from the cardiac neural crest during embryonic development and remain in the heart as dormant stem cells, with the capacity to differentiate into various cell types, including cardiomyocytes. Here, we examined the migration and differentiation potential of these cells on myocardial infarction (MI). METHODS AND RESULTS: We obtained double-transgenic mice by crossing protein-0 promoter-Cre mice with Floxed-enhanced green fluorescent protein mice, in which the NC cells express enhanced green fluorescent protein. In the neonatal heart, NC stem cells (NCSCs) were localized predominantly in the outflow tract, but they were also distributed in a gradient from base to apex throughout the ventricular myocardium. Time-lapse video analysis revealed that the NCSCs were migratory. Some NCSCs persisted in the adult heart. On MI, NCSCs accumulated at the ischemic border zone area (BZA), which expresses monocyte chemoattractant protein-1 (MCP-1). Ex vivo cell migration assays demonstrated that MCP-1 induced NCSC migration and that this chemotactic effect was significantly depressed by an anti-MCP-1 antibody. Small NC cardiomyocytes first appeared in the BZA 2 weeks post-MI and gradually increased in number thereafter. CONCLUSIONS: These results suggested that NCSCs migrate into the BZA via MCP-1/CCR2 signaling and contribute to the provision of cardiomyocytes for cardiac regeneration after MI.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Movimiento Celular , Infarto del Miocardio/patología , Miocitos Cardíacos/patología , Cresta Neural/embriología , Células Madre/patología , Animales , Proliferación Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Integrasas/genética , Ratones , Ratones Transgénicos , Proteína P0 de la Mielina/genética , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Activadores Plasminogénicos/genética , Regiones Promotoras Genéticas , Interferencia de ARN , Receptores CCR2/genética , Receptores CCR2/metabolismo , Regeneración , Transducción de Señal , Células Madre/metabolismo , Factores de Tiempo , Grabación en Video , Proteína Wnt1/genéticaRESUMEN
Gene expression programs and concomitant chromatin regulation change dramatically during the maturation of postmitotic neurons. Subnuclear positioning of gene loci is relevant to transcriptional regulation. However, little is known about subnuclear genome positioning in neuronal maturation. Using cultured murine hippocampal neurons, we found genomic locus 14qD2 to be enriched with genes that are upregulated during neuronal maturation. Reportedly, the locus is homologous to human 8p21.3, which has been extensively studied in neuropsychiatry and neurodegenerative diseases. Mapping of the 14qD2 locus in the nucleus revealed that it was relocated from the nuclear periphery to the interior. Moreover, we found a concomitant decrease in lamin B1 expression. Overexpression of lamin B1 in neurons using a lentiviral vector prevented the relocation of the 14qD2 locus and repressed the transcription of the Egr3 gene on this locus. Taken together, our results suggest that reduced lamin B1 expression during the maturation of neurons is important for appropriate subnuclear positioning of the genome and transcriptional programs.
Asunto(s)
Lamina Tipo B , Neuronas , Animales , Núcleo Celular , Humanos , Lamina Tipo B/genética , Ratones , NeurogénesisRESUMEN
Reactive oxygen species (ROS) attack polyunsaturated fatty acids of the membrane and trigger lipid peroxidation, which results in the generation of alpha,beta-unsaturated aldehydes, such as 4-hydroxy-2-nonenal (4-HNE). There is compelling evidence that high concentrations of aldehydes are responsible for much of the damage elicited by cardiac ischemia-reperfusion injury, while sublethal concentrations of aldehydes stimulate stress resistance pathways, to achieve cardioprotection. We investigated the mechanism of cardioprotection mediated by 4-HNE. For cultured cardiomyocytes, 4-HNE was cytotoxic at higher concentrations (>or=20 microM) but had no appreciable cytotoxicity at lower concentrations. Notably, a sublethal concentration (5muM) of 4-HNE primed cardiomyocytes to become resistant to cytotoxic concentrations of 4-HNE. 4-HNE induced nuclear translocation of transcription factor NF-E2-related factor 2 (Nrf2), and enhanced the expression of gamma-glutamylcysteine ligase (GCL) and the core subunit of the Xc(-) high-affinity cystine transporter (xCT), thereby increasing 1.45-fold the intracellular GSH levels. Cardiomyocytes treated with either Nrf2-specific siRNA or the GCL inhibitor l-buthionine sulfoximine (BSO) were less tolerant to 4-HNE. Moreover, the cardioprotective effect of 4-HNE pretreatment against subsequent glucose-free anoxia followed by reoxygenation was completely abolished in these cells. Intravenous administration of 4-HNE (4 mg/kg) activated Nrf2 in the heart and increased the intramyocardial GSH content, and consequently improved the functional recovery of the left ventricle following ischemia-reperfusion in Langendorff-perfused hearts. This cardioprotective effect of 4-HNE was not observed for Nrf2-knockout mice. In summary, 4-HNE activates Nrf2-mediated gene expression and stimulates GSH biosynthesis, thereby conferring on cardiomyocytes protection against ischemia-reperfusion injury.
Asunto(s)
Aldehídos/uso terapéutico , Factor 2 Relacionado con NF-E2/metabolismo , Daño por Reperfusión/prevención & control , Aldehídos/farmacología , Animales , Western Blotting , Muerte Celular/efectos de los fármacos , Células Cultivadas , Glutatión/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Factor 2 Relacionado con NF-E2/genética , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Epigenetic alterations are implicated in the development of cardiac hypertrophy and heart failure, but little is known of which epigenetic changes in which regions of the genome play such a role. We now show that trimethylation of histone H3 on lysine-4 (K4TM) or lysine-9 (K9TM) is markedly affected in cardiomyocytes in association with the development of heart failure in a rat disease model. High-throughput pyrosequencing performed with ChIP products for K4TM or K9TM prepared from human left ventricular tissue with retained or damaged function also revealed that protein-coding genes located in the vicinity of K4TM marks differ between functional and disabled myocytes, yet both sets of genes encode proteins that function in the same signal transduction pathways for cardiac function, indicative of differential K4TM marking during the development of heart failure. However, K9TM mark-profile was less dependent on the disease status compared to that of K4TM. Our data collectively reveal global epigenetic changes in cardiac myocytes associated with heart failure.
Asunto(s)
Genoma/genética , Insuficiencia Cardíaca/genética , Histonas/metabolismo , Anciano , Animales , Proteínas de Unión al Calcio , Inmunoprecipitación de Cromatina , Mapeo Cromosómico , Análisis por Conglomerados , Epigénesis Genética , Femenino , Regulación de la Expresión Génica , Humanos , Lisina/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metilación , Persona de Mediana Edad , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Miocardio/patología , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas DahlRESUMEN
Circulating levels of fibroblast growth factor-21 (FGF21) start increasing in patients with chronic kidney disease (CKD) since early stages during the cause of disease progression. FGF21 is a liver-derived hormone that induces responses to stress through acting on hypothalamus to activate the sympathetic nervous system and the hypothalamus-pituitary-adrenal endocrine axis. However, roles that FGF21 plays in pathophysiology of CKD remains elusive. Here we show in mice that FGF21 is required to survive CKD but responsible for blood pressure dysregulation. When introduced with CKD, Fgf21-/- mice died earlier than wild-type mice. Paradoxically, these Fgf21-/- CKD mice escaped several complications observed in wild-type mice, including augmentation of blood pressure elevating response and activation of the sympathetic nervous system during physical activity and increase in serum noradrenalin and corticosterone levels. Supplementation of FGF21 by administration of an FGF21-expressing adeno-associated virus vector recapitulated these complications in wild-type mice and restored the survival period in Fgf21-/- CKD mice. In CKD patients, high serum FGF21 levels are independently associated with decreased baroreceptor sensitivity. Thus, increased FGF21 in CKD can be viewed as a survival response at the sacrifice of blood pressure homeostasis.
Asunto(s)
Presión Sanguínea , Factores de Crecimiento de Fibroblastos/metabolismo , Presorreceptores/metabolismo , Insuficiencia Renal Crónica/metabolismo , Animales , Modelos Animales de Enfermedad , Factores de Crecimiento de Fibroblastos/genética , Humanos , Ratones , Ratones Noqueados , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/fisiopatologíaRESUMEN
Hypertrophic cardiomyopathy (HCM) is a hereditary disease characterized by cardiac hypertrophy with diastolic dysfunction. Gene mutations causing HCM have been found in about half of HCM patients, while the genetic etiology and pathogenesis remain unknown for many cases of HCM. To identify novel mechanisms underlying HCM pathogenesis, we generated a cardiovascular-mutant medaka fish, non-spring heart (nsh), which showed diastolic dysfunction and hypertrophic myocardium. The nsh homozygotes had fewer myofibrils, disrupted sarcomeres and expressed pathologically stiffer titin isoforms. In addition, the nsh heterozygotes showed M-line disassembly that is similar to the pathological changes found in HCM. Positional cloning revealed a missense mutation in an immunoglobulin (Ig) domain located in the M-line-A-band transition zone of titin. Screening of mutations in 96 unrelated patients with familial HCM, who had no previously implicated mutations in known sarcomeric gene candidates, identified two mutations in Ig domains close to the M-line region of titin. In vitro studies revealed that the mutations found both in medaka fish and in familial HCM increased binding of titin to muscle-specific ring finger protein 1 (MURF1) and enhanced titin degradation by ubiquitination. These findings implicate an impaired interaction between titin and MURF1 as a novel mechanism underlying the pathogenesis of HCM.
Asunto(s)
Cardiomiopatía Hipertrófica/etiología , Conectina/genética , Modelos Animales de Enfermedad , Proteínas Musculares/fisiología , Mutación , Proteínas de Motivos Tripartitos/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Conectina/fisiología , Humanos , Proteínas Musculares/genética , Oryzias , Transducción de Señal/fisiología , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The anti-Müllerian hormone gene (Amh) is responsible for regression in males of the Müllerian ducts. The molecular mechanism of regulation of chicken Amh expression is poorly understood. To investigate the regulation of chicken Amh expression, we have cloned Amh cDNAs from quail and duck as well as the promoter regions of the gene from chicken, quail, and duck. The expression patterns of Amh during embryonic development in these three species were found to be similar, suggesting that the regulatory mechanisms of Amh expression are conserved. The sequence of the proximal promoter of Amh contains a putative binding site for steroidogenic factor 1 (SF1), the protein product of which can up-regulate Amh in mammals. We showed here that SF1 is able to activate the chicken Amh promoter and binds to its putative SF1 binding site. These results suggest that SF1 plays a role in regulation of Amh expression in avian species.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/metabolismo , Proteínas de Homeodominio/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Hormonas Testiculares/metabolismo , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Hormona Antimülleriana , Secuencia de Bases , Embrión de Pollo , Pollos , Patos , Glicoproteínas/genética , Proteínas de Homeodominio/metabolismo , Masculino , Datos de Secuencia Molecular , Conductos Paramesonéfricos/metabolismo , Codorniz , Receptores Citoplasmáticos y Nucleares/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factor Esteroidogénico 1 , Hormonas Testiculares/genética , Factores de Transcripción/metabolismoRESUMEN
To identify transforming genes in acute myeloid leukemia (AML) we here constructed a retroviral cDNA expression library from an AML patient, and then used this library to infect a mouse cell line 32Dcl3-mCAT. cDNA inserts of the cell clones which proliferated in the presence of granulocyte colony-stimulating factor were derived from JAK3 encoding a JAK3 mutant with a valine-to-alanine substitution at codon 674 and two additional amino acid substitutions. The transforming activity of JAK3(V674A) was confirmed by its introduction into 32Dcl3-mCAT. Sequencing of the original JAK3 cDNA derived from the patient, however, failed to detect the V674A mutation.
Asunto(s)
Pruebas Genéticas/métodos , Janus Quinasa 3/genética , Leucemia Mieloide/genética , Mutación , Retroviridae/genética , Enfermedad Aguda , Sustitución de Aminoácidos , Animales , Línea Celular , Transformación Celular Viral , Clonación Molecular , Expresión Génica , Biblioteca de Genes , Humanos , Ratones , Retroviridae/metabolismoRESUMEN
The acetylation status of core histones in cardiomyocytes has been linked to the development of cardiac hypertrophy and heart failure. Little is known, however, of the genes affected by abnormal histone acetylation in such pathological conditions. We recently developed a genome-wide screening method, differential chromatin scanning (DCS), to isolate genomic fragments associated with histones subject to differential acetylation. We have now applied DCS to H9C2 rat embryonic cardiomyocytes incubated with or without trichostatin A (TSA), a specific inhibitor of histone deacetylase (HDAC) activity. About 200 genomic fragments were readily isolated by DCS on the basis of the preferential acetylation of associated histones in TSA-treated cells. Quantitation of the amount of DNA in chromatin immunoprecipitates prepared with antibodies to acetylated histone H3 revealed that 37 of 38 randomly chosen DCS clones were preferentially precipitated from the TSA-treated cells, thus verifying the high fidelity of DCS. Epigenetic regulation of DCS clones was further confirmed in cells treated with sodium butyrate, another HDAC inhibitor, as well as in cardiac myocytes isolated from neonatal rats. The mRNA level of 9 (39%) of 23 genes corresponding to DCS clones changed in parallel with the level of histone acetylation in H9C2 cells. Furthermore, a physiological hypertrophic stimulus, cardiotrophin-1, affected the acetylation level of histones associated with genomic regions corresponding to certain DCS clones. Our data thus establish a genome-wide profile of HDAC targets in cardiomyocytes, which should provide a basis for further investigations into the role of epigenetic modification in cardiac disorders.
Asunto(s)
Epigénesis Genética , Genoma , Histona Desacetilasas/genética , Miocitos Cardíacos/enzimología , Acetilación , Animales , Animales Recién Nacidos , Factor Natriurético Atrial/genética , Butiratos/farmacología , Línea Celular , Cromatina , Corazón/embriología , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Ácidos Hidroxámicos/farmacología , RatasRESUMEN
Histone acetylation has been linked to cardiac hypertrophy and heart failure. However, the pathological implications of changes in histone methylation and the effects of interventions with histone methyltransferase inhibitors for heart failure have not been fully clarified. Here, we focused on H3K9me3 status in the heart and investigated the effects of the histone H3K9 methyltransferase inhibitor chaetocin on prognoses in Dahl salt-sensitive rats, an animal model of chronic heart failure. Chaetocin prolonged survival and restored mitochondrial dysfunction. ChIP-seq analysis demonstrated that chronic stress to the heart induced H3K9me3 elevation in thousands of repetitive elements, including intronic regions of mitochondria-related genes, such as the gene encoding peroxisome proliferator-activated receptor-gamma coactivator 1 alpha. Furthermore, chaetocin reversed this effect on these repetitive loci. These data suggested that excessive heterochromatinization of repetitive elements of mitochondrial genes in the failing heart may lead to the silencing of genes and impair heart function. Thus, chaetocin may be a potential therapeutic agent for chronic heart failure.
Asunto(s)
Cardiomegalia/diagnóstico , Insuficiencia Cardíaca/diagnóstico , Acetilación , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/tratamiento farmacológico , Enfermedad Crónica , Dietoterapia , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/tratamiento farmacológico , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperazinas/uso terapéutico , Pronóstico , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Cloruro de Sodio/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Embryonic stem cells (ESCs) are a hallmark of ideal pluripotent stem cells. Epigenetic reprogramming of induced pluripotent stem cells (iPSCs) has not been fully accomplished. iPSC generation is similar to somatic cell nuclear transfer (SCNT) in oocytes, and this procedure can be used to generate ESCs (SCNT-ESCs), which suggests the contribution of oocyte-specific constituents. Here, we show that the mammalian oocyte-specific linker histone H1foo has beneficial effects on iPSC generation. Induction of H1foo with Oct4, Sox2, and Klf4 significantly enhanced the efficiency of iPSC generation. H1foo promoted in vitro differentiation characteristics with low heterogeneity in iPSCs. H1foo enhanced the generation of germline-competent chimeric mice from iPSCs in a manner similar to that for ESCs. These findings indicate that H1foo contributes to the generation of higher-quality iPSCs.