RESUMEN
The 2015 European Guidelines on Diagnosis and Treatment of Pulmonary Hypertension (PH) are also valid for Germany. While the guidelines contain detailed recommendations regarding clinical aspects of pulmonary arterial hypertension (PAH) and other forms of PH, they contain only a relatively short paragraph on novel findings on the pathobiology, pathology, and genetics. However, these are of great importance for our understanding of this complex disease both from a clinical and scientific point of view, and they are essential for the development of novel treatment strategies. To this end, a number of current data are relevant, prompting a detailed commentary to the guidelines, and the consideration of new scientific data. In June 2016, a Consensus Conference organized by the PH working groups of the German Society of Cardiology (DGK), the German Society of Respiratory Medicine (DGP) and the German Society of Pediatric Cardiology (DGPK) was held in Cologne, Germany. This conference aimed to solve practical and controversial issues surrounding the implementation of the European Guidelines in Germany. To this end, a number of working groups was initiated, one of which was specifically dedicated to the pathobiology, pathology and genetics of PH. This article summarizes the results and recommendations of this working group.
Asunto(s)
Cardiología/normas , Hipertensión Pulmonar/diagnóstico , Hipertensión Pulmonar/terapia , Guías de Práctica Clínica como Asunto , Neumología/normas , Antihipertensivos/uso terapéutico , Terapia Combinada/normas , Endarterectomía/normas , Alemania , Humanos , Hipertensión Pulmonar/genéticaRESUMEN
Intermittent hypoxia seems to be a major pathomechanism of obstructive sleep apnea-associated progression of atherosclerosis. The goal of the present study was to assess the influence of hypoxia on endothelial function depending on the initial stage of vasculopathy. We used 16 ApoE-/- mice were exposed to a 6-week-intermittent hypoxia either immediately (early preatherosclerosis) or after 5 weeks of high-cholesterol diet (advanced preatherosclerosis). Another 16 ApoE-/- mice under normoxia served as corresponding controls. Endothelial function was measured by an organ bath technique. Blood plasma CD31+/annexin V+ endothelial microparticles as well as sca1/flk1+ endothelial progenitor cells in blood and bone marrow were analyzed by flow cytometry. The findings were that intermittent hypoxia impaired endothelial function (56.6±6.2% of maximal phenylephrine-induced vasoconstriction vs. 35.2±4.1% in control) and integrity (increased percentage of endothelial microparticles: 0.28±0.05% vs. 0.15±0.02% in control) in early preatherosclerosis. Peripheral repair capacity expressed as the number of endothelial progenitor cells in blood was attenuated under hypoxia (2.0±0.5% vs. 5.3±1.9% in control), despite the elevated number of these cells in the bone marrow (2.0±0.4% vs. 1.1±0.2% in control). In contrast, endothelial function, as well as microparticle and endothelial progenitor cell levels were similar under hypoxia vs. control in advanced preatherosclerosis. We conclude that hypoxia aggravates endothelial dysfunction and destruction in early preatherosclerosis.
Asunto(s)
Aorta Torácica/fisiopatología , Aterosclerosis/fisiopatología , Células Endoteliales/patología , Hipoxia/fisiopatología , Apnea Obstructiva del Sueño/fisiopatología , Células Madre/patología , Animales , Anexina A5/genética , Anexina A5/metabolismo , Antígenos Ly/genética , Antígenos Ly/metabolismo , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/complicaciones , Aterosclerosis/metabolismo , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/metabolismo , Colesterol/administración & dosificación , Dieta Alta en Grasa , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Femenino , Expresión Génica , Hipoxia/complicaciones , Hipoxia/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Técnicas de Cultivo de Órganos , Fenilefrina/farmacología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Apnea Obstructiva del Sueño/complicaciones , Apnea Obstructiva del Sueño/metabolismo , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Factores de Tiempo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Vasoconstricción/efectos de los fármacosRESUMEN
The aim of this study was to investigate whether the L-type calcium current (I(Ca.L)) may be altered in aged hearts and whether the classical calcium antagonist verapamil may exhibit altered pharmacological profile in aged hearts. We examined male New Zealand rabbits aged either 6 months or 26 months. To examine I(Ca.L) whole-cell patch-clamp technique was performed on isolated cells. Moreover, activation-recovery intervals (ARI) of isolated hearts (Langendorff method) were assessed using an epicardial 256 channel mapping system. We found that the I(Ca.L) density, normalised to the cell volume was significantly reduced (p<0.001). Maximum conductance was also significantly decreased (p=0.01) and steady state inactivation was shifted to more positive potentials in aged hearts (p<0.001). A slightly reduced effect of beta-adrenergic modulation of the I(Ca.L) in aged hearts, and a significantly reduced effect of carbachol on isoprenaline-stimulated I(Ca.L) in aged hearts was observed. L-type alpha 1c subunit, SERCA2-ATPase and the Na(+)/Ca(2+)-exchanger expression were neither significantly different in atrial and ventricular tissues nor between young and old animals. Using the mapping system, isolated hearts were exposed to verapamil (0.005, 0.01, 0.02, 0.05 microM/L). While verapamil did not affect ARI in young hearts, in aged hearts ARI was concentration-dependently reduced and the negative inotropic effect of verapamil was significantly attenuated in aged hearts (p<0.05). From these results we conclude that there are distinct alterations in the electrophysiology of I(Ca.L) (reduced maximum conductance, a shift of the steady state inactivation) in the aged heart which may influence the response to verapamil.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Verapamilo/farmacología , Factores de Edad , Envejecimiento , Animales , Bloqueadores de los Canales de Calcio/administración & dosificación , Canales de Calcio Tipo L/metabolismo , Carbacol/farmacología , Técnicas Electrofisiológicas Cardíacas , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Isoproterenol/farmacología , Masculino , Técnicas de Placa-Clamp , Conejos , Verapamilo/administración & dosificaciónRESUMEN
Collagen XVIII is an important component of the extracellular matrix and is expressed in basement membranes. Its degradation results in the generation of endostatin claimed to possess antiangiogenic activity. To date, only limited knowledge exists with regard to the cellular signaling of this molecule. We show in single-cell measurements using the Ca2+ indicator fura-2 acetoxy methylester (fura-2 AM) and the nitric oxide (NO) indicator 4,5-diaminofluorescein diacetate that application of endostatin (ES) (5 pmol/L, 100 ng/mL) induced Ca2+ spikes and an increase of NO production in human and murine endothelial cells. The NO response was independent of an increase in cytosolic Ca2+ and blocked by the endothelial NO synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester and by incubation with pertussis toxin known to inhibit G(i/o) proteins. The physiological relevance of this novel signaling pathway of ES was assessed with isometric force measurements in large and small arteries of mouse. Physiological concentrations of ES were found to decrease vascular tone in an endothelium-dependent manner. This occurred via an Arg-Gly-Asp (RGD) peptide-independent pathway through activation of G(i/o) proteins, phosphatidylinositol 3-kinase, Akt, and eNOS. We conclude that the proteolytic matrix fragment ES is a prominent vasorelaxing agent. Because ES is constantly released into the blood, it is a novel regulator of blood pressure and, therefore, represents an interesting pharmacological target.
Asunto(s)
Endostatinas/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Vasodilatación , Vasodilatadores/farmacología , Animales , Células Cultivadas , Células Endoteliales/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Ratones , Óxido Nítrico/metabolismo , Proteínas Recombinantes/farmacología , Vasoconstricción/efectos de los fármacosRESUMEN
The aim of this study was to identify and functionally characterize cardiac subtypes during early stages of development. For this purpose, transgenic embryonic stem cells were generated using the alpha-myosin heavy chain promoter driving the expression of the enhanced green fluorescent protein (EGFP). EGFP-positive clusters of cells were first observed as early as 7 days of development, thus, even before the initiation of the contractile activity. Flow cytometry and single-cell fluorescence measurements evidenced large diversities of EGFP intensity. Patch-clamp experiments showed EGFP expression exclusively in pacemaker and atrial but not ventricular cells. The highest fluorescence intensities were detected in pacemaker-like cardiomyocytes. In accordance, multielectrode-array recordings of whole embryoid bodies confirmed that the pacemaker center coincided with strongly EGFP-positive areas. The cardiac subtypes displayed already at this early stage differential characteristics of electrical activity and ion channel expression. Thus, quantitation of the alpha-myosin heavy chain driven reporter gene expression allows identification and functional characterization of early cardiac subtypes.
Asunto(s)
Embrión de Mamíferos/citología , Atrios Cardíacos/citología , Sistema de Conducción Cardíaco/citología , Miocitos Cardíacos/citología , Células Madre/citología , Animales , Carbacol/farmacología , Diferenciación Celular , Separación Celular , Células Clonales , Electrofisiología , Citometría de Flujo , Fluorescencia , Expresión Génica , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Atrios Cardíacos/embriología , Sistema de Conducción Cardíaco/embriología , Ratones , Ratones Transgénicos , Microscopía Confocal , Agonistas Muscarínicos/farmacología , Miocitos Cardíacos/clasificación , Miocitos Cardíacos/metabolismo , Cadenas Pesadas de Miosina/genética , Regiones Promotoras Genéticas/genética , Células Madre/clasificación , Células Madre/metabolismo , Transfección , Miosinas Ventriculares/genéticaRESUMEN
Nitric oxide (NO) is thought to play an important role as a signaling molecule in embryonic and adult cardiomyocytes; however, its involvement in muscarinic signaling is still unclear. The aim of the present work was to analyze the muscarinic modulation of the L-type Ca2+ current (ICa) in early- and late-stage embryonic ventricular cardiomyocytes. Muscarinic stimulation depressed basal ICa by 30.1 +/- 3.2% (n=27) in early-stage cardiomyocytes. Pharmacological evidence suggested that the muscarinic modulation was mediated through generation of NO, activation of cGMP-dependent phosphodiesterase (PDE) 2, and ensuing lowering of cyclic AMP/protein kinase A (cAMP/PKA) levels. Conversely, in late-stage cardiomyocytes, muscarinic regulation of ICa occurred in a NO-independent manner via inhibition of prestimulated adenylyl cyclase (AC). To unequivocally prove the involvement of NO and to identify the nitric oxide synthase (NOS) isoform(s), we analyzed muscarinic signaling in embryonic ventricular cardiomyocytes of NOS2 (-/-) and NOS3 (-/-) mice. The early-stage NOS3 (-/-) cardiomyocytes lacked muscarinic modulation, whereas it was preserved in NOS2 (-/-) cells. Moreover, at the late embryonic stage, muscarinic modulation of ICa was intact in both strains. Thus, NO is the key regulator of muscarinic signaling in the early embryonic ventricle, whereas at later stages, signaling occurs through a NO-independent pathway.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Corazón Fetal/fisiología , Óxido Nítrico/fisiología , Transducción de Señal/fisiología , Animales , Animales no Consanguíneos , Carbacol/farmacología , Colinérgicos/farmacología , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Femenino , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/farmacología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/embriología , Transporte Iónico/efectos de los fármacos , Ratones , Ratones Noqueados , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Toxina del Pertussis/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/fisiología , Transducción de Señal/efectos de los fármacos , omega-N-Metilarginina/farmacologíaRESUMEN
Replacement of damaged myocardium with electrically functional, contracting syncytium with a balanced blood supply remains a key goal for the treatment of hearts damaged by coronary heart disease or other disorders. Stem cell therapy offers a potential solution. This paper describes the value of in vitro stem cell research to unravel the roles of key regulatory molecules in embryogenesis of myocardium and blood vessels. Studies have shown that functioning myocytes can be derived from stem cells in vitro and engrafted into infarcted areas of heart where they develop into functional adult like cardiomyocytes with action potentials and capacity for beta adrenergic and muscarinic regulation. Further studies have identified specific roles for platelet endothelial cell adhesion molecule (PECAM), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) in the sequential differentiation of blood vessels and capillaries.
Asunto(s)
Infarto del Miocardio/terapia , Miocitos Cardíacos/trasplante , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular/fisiología , Clonación Molecular , Humanos , RatonesRESUMEN
The capacity of pluripotent embryonic stem cells (ESC) to differentiate in vitro into various tissues provides the opportunity to develop an in vitro assay for investigating mechanisms of developmental toxicity. ESC clones carrying tissue specific reporter gene constructs are currently being developed. The clones should allow the quantification of the effects of chemicals on the development of germ layers and main target tissues. We report the establishment of the alpha-fetoprotein_GFP/D3 reporter gene clone: alpha-fetoprotein (AFP) enhancers and the homologous promoter regulate green fluorescent protein (GFP) expression in cells of the D3-ESC clone. AFP was used as a marker for endodermal cells. Differentiation of this clone via embryoid bodies (EBs, spheroids of cells) leads to green fluorescence on the surfaces of EBs. AFP- related GFP expression was confirmed. An easy and quick image analysis-based endpoint measurement was developed for quantifying low amounts of cells expressing GFP. As demonstrated with the embryotoxic chemical diphenylhydantoin, image analysis can be used to distinguish between a general effect on EB growth and a specific effect on the development of GFP-positive endodermal cells. Endoderm development was inhibited at a different dose than cardiomyocyte development.
Asunto(s)
Alternativas a las Pruebas en Animales , Endodermo/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Proteínas Luminiscentes/metabolismo , Células Madre Pluripotentes/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Clonales , Relación Dosis-Respuesta a Droga , Endodermo/efectos de los fármacos , Genes Reporteros/genética , Proteínas Fluorescentes Verdes , Indicadores y Reactivos , Proteínas Luminiscentes/genética , Células Madre Pluripotentes/efectos de los fármacos , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacos , Xenobióticos/toxicidad , alfa-Fetoproteínas/genéticaRESUMEN
Nitric oxide (NO) is an important signalling molecule that plays a relevant role in different cell systems, among them the adult heart. The effects of NO are primarily mediated through modulation of Ca(2+) homeostasis, myofibrillar contractility, and metabolic regulation in cardiomyocytes. Recent evidence also suggests an important role of NO for cardiomyogenesis by modulating proliferation and differentiation and regulating cardiac function. In the embryonic, but also the healthy and diseased, adult mammalian heart, the inducible (iNOS) and the endothelial (eNOS) nitric oxide synthases (NOS) are detected. However, the expression pattern of NO and its function differ during development. Furthermore, under pathophysiological conditions NOS expression can also change and cause impairment of cardiac performance and cytotoxic effects. The present review focuses on the role and function of NO during cardiomyogenesis, the mechanisms responsible for eNOS availability, and the paracrine effects of NO generated by cardiomyocytes.
Asunto(s)
Corazón/embriología , Miocardio/metabolismo , Óxido Nítrico Sintasa/metabolismo , Animales , Calcio/metabolismo , Humanos , Miocardio/enzimología , Transducción de SeñalRESUMEN
Expression of the enhanced green fluorescent protein (EGFP) under control of a thymidine kinase promoter/nestin second intron was specifically detected in nestin immunoreactive neural precursor cells after selection of murine embryonic stem (ES) cells in chemically defined medium. Allowing differentiation in vitro, the capacity of these cells to give rise to astroglia, oligodendroglia, and neurones was investigated. After intracerebral transplantation, long-lasting integration of precursor cells into the host tissue was observed, serving as a pool for successive neuronal and glial differentiation. EGFP expression by ES cell-derived neural precursor cells may be a valuable tool to optimize protocols for maintenance and expansion of these cells in vitro as well as in vivo after intracerebral transplantation. In addition, preparative fluorescence-activated cell sorting of EGFP-labeled neural precursor cells should be useful for standardization of a donor cell population for cell replacement therapies.
Asunto(s)
Embrión de Mamíferos/citología , Trasplante de Células Madre Hematopoyéticas , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas del Tejido Nervioso , Animales , Diferenciación Celular , Línea Celular , Separación Celular , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Nestina , Regiones Promotoras Genéticas , Unión Proteica , Ratas , Ratas Wistar , Factores de Tiempo , TransgenesRESUMEN
beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.
Asunto(s)
Citoesqueleto/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Integrina beta1/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Muscarínicos/metabolismo , Acetilcolina/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Factor Natriurético Atrial/farmacología , Canales de Calcio Tipo L/metabolismo , Compartimento Celular , Células Cultivadas , Citocalasina D/farmacología , Adhesiones Focales , Integrina beta1/genética , Isoproterenol/farmacología , Ratones , Antagonistas Muscarínicos/farmacología , Miocardio/citología , Óxido Nítrico/farmacología , Canales de Potasio/metabolismo , Transducción de SeñalRESUMEN
1. The regulation of L-type Ca(2+) current (I(Ca)) by the two nitric oxide (NO) donors sodium nitroprusside (SNP, 1 microM to 1 mM) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 3 or 10 microM) was investigated in frog ventricular myocytes using double voltage clamp and double-barrelled microperfusion techniques. 2. SNP and SNAP depressed the isoprenaline (ISO, 10-100 nM)- or forskolin (FSK, 1 microM)-mediated stimulation of I(Ca) via cGMP activation of the cGMP-stimulated phosphodiesterase (PDE2). Complete inhibition of the ISO (100 nM) response was observed at 1 mM SNP. 3. When SNP was applied locally, i.e. to one-half of the cell, and ISO to the whole cell, the response of I(Ca) to ISO was strongly antagonized in the cell half exposed to SNP (up to 100 % inhibition at 1 mM SNP) but a relatively small depression was observed in the other half of the cell (only 20 % inhibition at 1 mM SNP). 4. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO, 1 mM) reversed the local effect of SNAP (3 microM) on FSK-stimulated I(Ca) when applied to the same side as the NO donor, but had no effect when applied to the other side of the cell. 5. A local application of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 microM), a selective inhibitor of PDE2, fully reversed the local effect of SNP (100 microM) or SNAP (10 microM) on I(Ca) but had no effect on the distant response. 6. When EHNA was applied on the distant side, with SNP (1 mM) and ISO (100 nM) applied locally, the distant effect of SNP was fully reversed. 7. Our results demonstrate that in frog ventricular myocytes stimulation of guanylyl cyclase by NO leads to a strong local depletion of cAMP near the L-type Ca(2+) channels due to activation of PDE2, but only to a modest reduction of cAMP in the rest of the cell. This may be explained by the existence of a tight microdomain between L-type Ca(2+) channels and PDE2.
Asunto(s)
Adenina/análogos & derivados , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/fisiología , Óxido Nítrico/farmacología , Función Ventricular/efectos de los fármacos , Adenina/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Benzoatos/farmacología , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2 , Conductividad Eléctrica , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Membranas Intracelulares/metabolismo , Isoproterenol/farmacología , Miocardio/citología , Óxido Nítrico/antagonistas & inhibidores , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Técnicas de Placa-Clamp , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/metabolismo , Rana esculenta , Distribución TisularRESUMEN
Annexin A7 has been proposed to function in the fusion of vesicles, acting as a Ca(2+) channel and as Ca(2+)-activated GTPase, thus inducing Ca(2+)/GTP-dependent secretory events. To understand the function of annexin A7, we have performed targeted disruption of the Anxa7 gene in mice. Matings between heterozygous mice produced offspring showing a normal Mendelian pattern of inheritance, indicating that the loss of annexin A7 did not interfere with viability in utero. Mice lacking annexin A7 showed no obvious phenotype and were fertile. To assay for exocytosis, insulin secretion from isolated islets of Langerhans was examined. Ca(2+)-induced and cyclic AMP-mediated potentiation of insulin secretion was unchanged in the absence of annexin A7, suggesting that it is not directly implicated in vesicle fusion. Ca(2+) regulation studied in isolated cardiomyocytes, showed that while cells from early embryos displayed intact Ca(2+) homeostasis and expressed all of the components required for excitation-contraction coupling, cardiomyocytes from adult Anxa7(-/-) mice exhibited an altered cell shortening-frequency relationship when stimulated with high frequencies. This suggests a function for annexin A7 in electromechanical coupling, probably through Ca(2+) homoeostasis.
Asunto(s)
Anexina A7/metabolismo , Calcio/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Contracción Muscular/fisiología , Miocardio/metabolismo , Animales , Anexina A7/genética , Cafeína/farmacología , Cardiotónicos/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Colforsina/farmacología , Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Femenino , Marcación de Gen , Glucosa/farmacología , Homeostasis , Hipoglucemiantes/farmacología , Immunoblotting , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/fisiología , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Contracción Muscular/efectos de los fármacos , Miocardio/citología , Técnicas de Placa-Clamp , Tolbutamida/farmacologíaRESUMEN
This study is based on the unique potential of pluripotent embryonic stem (ES) cells to differentiate in vitro into embryoid bodies containing cell lineages representative of most cell types found in the mammalian fetus. However, the use of wild type ES cells as an in vitro assay for embryotoxicological studies is complicated by the simultaneous development of various cellular phenotypes. This prevents a quantitative assessment of drug effects on one specific cell type. Here we report the effects of 15 chemicals on cardiac differentiation as determined by various specific toxicological endpoints such as morphological inspection (contractile activity), quantitative mRNA analysis and cardiac-specific expression of green fluorescent protein (GFP), used as a quantitative reporter. The data from the different endpoints have been subjected to a statistical analysis, and a preliminary prediction model is proposed. The results demonstrate that genetically-engineered ES cells could provide a valuable tool for estimating the developmental cardiotoxic potential of compounds in vitro and form the basis for automated analysis in a high-throughput system.
Asunto(s)
Embrión de Mamíferos/citología , Genes Reporteros , Corazón/efectos de los fármacos , Células Madre/efectos de los fármacos , Pruebas de Toxicidad/métodos , Animales , Animales Modificados Genéticamente , Diferenciación Celular , Línea Celular , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes , Corazón/embriología , Ratones , Modelos Biológicos , Miocardio/metabolismo , Valor Predictivo de las Pruebas , Regiones Promotoras Genéticas/genética , Sensibilidad y Especificidad , Células Madre/metabolismo , TransfecciónRESUMEN
The aim of the present study was to investigate the expression and functional role of outwards currents during the early stages of cardiomyogenesis. The predominant repolarizing current in early-stage, embryonic stem (ES) cell-derived cardiomyocytes was a 4-aminopyridine (4-AP) sensitive [concentration for half-maximal inhibition (IC50) 1.7 mM], transient outward current (Ito) with a current density of 10.3+/-2.1 pA/pF (n=72). We observed two additional, rapidly activating, outwardly rectifying current components, I(K),sus and Ires, in early- and late-stage cardiomyocytes. These currents were characterized by slow and no inactivation, respectively, during the depolarizing voltage step. I(K),sus was detected in about 25% of cells investigated and displayed 4-AP hypersensitivity (IC50 29 microM), whereas Ires was found in all cells of both differentiation stages and was 4-AP insensitive. In contrast to early-stage cells, Ires formed the larger portion of the aggregate, whole-cell current in late-stage, ES cell-derived cardiomyocytes. The current densities of all three current components increased during development, however, the most prominent increase was observed for I(res) from 3.6+/-0.8 pA/pF (n=72) to 8+/-1.1 pA/pF (n=35). In current-clamp recordings in early-stage, spontaneously contracting cardiomyocytes, 4-AP depolarized the cells, lengthened the action potential duration (APD) and increased the action potential frequency. In late-stage cells 4-AP had no effect on action potential frequency. We conclude that in early-stage cardiomyocytes I(to) plays an important role in controlling electrical activity.
Asunto(s)
Corazón/fisiología , Miocardio/citología , Células Madre/citología , 4-Aminopiridina/farmacología , Potenciales de Acción/efectos de los fármacos , Diferenciación Celular , Línea Celular , Conductividad Eléctrica , Electrofisiología , Embrión de Mamíferos/fisiología , Embrión no Mamífero , Corazón/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Tiempo de Reacción/efectos de los fármacosRESUMEN
1. The biophysical and pharmacological characteristics of the hyperpolarization activated non- selective cation current (If) were recorded using whole-cell voltage clamp in embryonic stem (ES) cell-derived cardiomyocytes at different stages of development. 2. The cation current was detected in a large percentage (65 %) of early stage (EDS, differentiated for 7 + 3-4 days) cells at a current density of 11.4 +/- 0.6 pA pF-1 (n = 47). In late stage (LDS, differentiated for 7 + 9-12 days) cells the percentage of cells expressing If decreased (45 %), but If densities (15.5 +/- 0.9 pA pF-1, n = 20) were increased. 3. The muscarinic agonist carbachol (CCh, 1 microM) depressed basal If in EDS cells by 45.7 +/- 6.5 %, n = 5) and was without effect in LDS cardiomyocytes (n = 4). The beta-adrenoceptor agonist isoprenaline (ISO, 1 microM) stimulated If in LDS cells by 33 +/- 5.2 % (n = 6) but not in EDS cells (n = 5). 4. Cell infusion with the catalytic subunit of the cAMP-dependent protein kinase (PKA, 7 microM) stimulated If in EDS cells by 37.0 +/- 2.9 %, (n = 4), but subsequent superfusion of 8-bromo-cAMP (200 microM) was without effect. Intracellular perfusion of LDS cardiomyocytes with the highly selective peptide inhibitor of PKA (PKI, 20 microM) completely inhibited the stimulation of the L-type Ca2+ current (ICa,L) as well as of If by ISO (1 microM). 5. Extracellular superfusion with phosphodiesterase (PDE) inhibitors - IBMX, a non-selective antagonist, Erythro-9-(2-hydoxy-3-nonyl)adenine (EHNA), a PDE2 antagonist and rolipram, a PDE4 antagonist - resulted in stimulation of ICa,L and If in EDS cells. By contrast, milrinone and cilostamide, two PDE3 antagonists, stimulated ICa,L, but not If. 6. The present work demonstrates that If is functionally expressed during early cardiomyogenesis. Similar to ICa,L, If is regulated during embryonic development by phosphorylation via PKA. In contrast to ICa,L, If is not regulated by PDE3 suggesting different localization of these ion channels with respect to PDE3.
Asunto(s)
Cationes/metabolismo , Corazón/embriología , Canales Iónicos/biosíntesis , Miocardio/metabolismo , Proteínas del Tejido Nervioso , Células Madre/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales Iónicos/agonistas , Canales Iónicos/antagonistas & inhibidores , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Agonistas Muscarínicos/farmacología , Miocardio/citología , Técnicas de Placa-Clamp , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación/efectos de los fármacos , Canales de Potasio , Células Madre/citologíaRESUMEN
Ischemic disorders of the heart can cause an irreversible loss of cardiomyocytes resulting in a substantial decrease of cardiac output. The therapy of choice is heart transplantation, a technique that is hampered by the low number of donor organs. In the present study, we describe the specific labeling, rapid but gentle purification and characterization of cardiomyocytes derived from mouse pluripotent embryonic stem (ES) cells. To isolate the subpopulation of ventricular-like cardiomyocytes, ES cells were stable transfected with the enhanced green fluorescent protein (EGFP) under transcriptional control of the ventricular-specific 2.1 kb myosin light chain-2v (MLC-2v) promoter and the 0.5 kb enhancer element of the cytomegalovirus (CMV(enh).). First fluorescent cells were detected at day 6 + 8 of differentiation within EBs. Four weeks after initiation of differentiation 25% of the cardiomyocyte population displayed fluorescence. Immunohistochemistry revealed the exclusive cardiomyogenic nature of EGFP-positive cells. This was further corroborated by electrophysiological studies where preferentially ventricular phenotypes, but no pacemaker-like cardiomyocytes, were detected among the EGFP-positive population. The enzymatic digestion of EBs, followed by Percoll gradient centrifugation and fluorescence-activated cell sorting, resulted in a 97% pure population of cardiomyocytes. Based on this study, ventricular-like cardiomyocytes can be generated in vitro from EBs and labeled using CMV(enh)./MLC-2v-driven marker genes facilitating an efficient purification. This method may become an important tool for future cell replacement therapy of ischemic cardiomyopathy especially after the proof of somatic differentiation of human ES cells in vitro.