RESUMEN
BACKGROUND: The molecular pathomechanisms underlying atrial thrombogenesis are multifactorial and still require detailed investigations. Transgenic mice with cardiomyocyte-directed expression of the transcriptional repressor CREM-IbΔC-X (CREM-TG) represent an experimental model of atrial fibrillation (AF) that shows a gradual, age-dependent progression from atrial ectopy to persistent AF. Importantly, this model develops biatrial thrombi. The molecular characteristics related to the thrombogenesis in CREM-TG mice have not been studied, yet. METHODS: The inflammatory and prothrombotic state was evaluated at the transcriptional (qRT-PCR) and protein level in the left (LA) and right atria (RA) from CREM-TG mice at the age of 20weeks and compared to wild-type controls. Moreover, histological analyses of atrial thrombi were performed. RESULTS: The endocardial dysfunction was mirrored by diminished levels of eNOS-mRNA in both atria (RA: 0.79±0.04, LA: 0.72±0.06; each P<0.05). Moreover, the PAI-1/t-PA mRNA ratio was significantly increased in both atria (RA: 3.6±0.6; P<0.01, LA: 4.0±1.0; P<0.05) indicating a high risk of thrombus formation. However, the inflammatory phenotype was more pronounced in the RA and was reflected by a significant increase in the mRNA levels encoding adhesion molecules ICAM-1 (2.1±0.2; P<0.01), VCAM-1 (2.3±0.5; P<0.05), and selectin P (3.6±0.5: P<0.05). CONCLUSIONS: CREM-TG mice represent a valuable model for studying atrial thrombogenesis and assessing therapeutic approaches preventing embolic events in the systemic and pulmonary circulation.
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Fibrilación Atrial/genética , Trombosis/genética , Animales , Fibrilación Atrial/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Trombosis/metabolismoRESUMEN
AIMS: In atrial fibrillation, increased function of the Na+/Ca2+-exchanger (NCX) is one among several electrical remodeling mechanisms. METHODS/RESULTS: Using the patch-clamp- and Ca2+ imaging-methods, we investigated atrial myocytes from NCX-homozygous-overexpressor (OE)- and heterozygous-knockout (KO)-mice and their corresponding wildtypes (WTOE; WTKO). NCX mediated Ca2+ extrusion capacity was reduced in KO and increased in OE. There was no evidence for structural or molecular remodeling. During a proarrhythmic pacing-protocol, the number of low amplitude delayed afterdepolarizations (DADs) was unaltered in OE vs. WTOE and KO vs. WTKO. However, DADs triggered full spontaneous action potentials (sAP) significantly more often in OE vs. WTOE (ratio sAP/DAD: OE:0.18±0.05; WTOE:0.02±0.02; p<0.001). Using the same protocol, a DAD triggered an sAP by tendency less often in KO vs. WTKO (p=0.06) and significantly less often under a more aggressive proarrhythmic protocol (ratio sAP/DAD: KO:0.01±0.003; WT KO: 0.12±0.05; p=0.007). The DAD amplitude was increased in OE vs. WTOE and decreased in KO vs. WTKO. There were no differences in SR-Ca2+-load, the number of spontaneous Ca2+-release-events or IKACh/IK1. CONCLUSIONS: Atrial myocytes with increased NCX expression exhibited increased vulnerability towards sAPs while atriomyocytes with reduced NCX expression were protected. The underlying mechanism consists of a modification of the DAD-amplitude by the level of NCX-activity. Thus, although the number of spontaneous Ca2+-releases and therefore DADs is unaltered, the higher DAD-amplitude in OE made a transgression of the voltage-threshold of an sAP more likely. These findings indicate that the level of NCX activity could influence triggered activity in atrial myocytes independent of possible remodeling processes.
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Atrios Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Potenciales de Acción/genética , Animales , Calcio/metabolismo , Señalización del Calcio , Femenino , Expresión Génica , Masculino , Potenciales de la Membrana/genética , Ratones , Ratones Transgénicos , Contracción Miocárdica/genética , Miocardio/metabolismo , Retículo Sarcoplasmático/metabolismo , Intercambiador de Sodio-Calcio/genéticaRESUMEN
Chronic ß-adrenergic stimulation is regarded as a pivotal step in the progression of heart failure which is associated with a high risk for arrhythmia. The cAMP-dependent transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) mediate transcriptional regulation in response to ß-adrenergic stimulation and CREM repressor isoforms are induced after stimulation of the ß-adrenoceptor. Here, we investigate whether CREM repressors contribute to the arrhythmogenic remodeling in the heart by analyzing arrhythmogenic alterations in ventricular cardiomyocytes (VCMs) from mice with transgenic expression of the CREM repressor isoform CREM-IbΔC-X (TG). Patch clamp analyses, calcium imaging, immunoblotting and real-time quantitative RT-PCR were conducted to study proarrhythmic alterations in TG VCMs vs. wild-type controls. The percentage of VCMs displaying spontaneous supra-threshold transient-like Ca(2+) releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca(2+) waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca(2+) transport and NCX1 protein level in TG. An increase in I NCX and decrease in I to and its accessory channel subunit KChIP2 was associated with action potential prolongation and an increased proportion of TG VCMs showing early afterdepolarizations. Finally, ventricular extrasystoles were augmented in TG mice underlining the in vivo relevance of our findings. Transgenic expression of CREM-IbΔC-X in mouse VCMs leads to distinct arrhythmogenic alterations. Since CREM repressors are inducible by chronic ß-adrenergic stimulation our results suggest that the inhibition of CRE-dependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease.
Asunto(s)
Arritmias Cardíacas/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Potenciales de Acción , Animales , Arritmias Cardíacas/fisiopatología , Calcio/metabolismo , Células Cultivadas , Modulador del Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ventrículos Cardíacos/fisiopatología , Isoproterenol , Ratones , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Transcription factors of the cAMP response element-binding protein (Creb)/cAMP response element modulator (Crem) family were linked to the switch from a contractile to a proliferating phenotype in vascular smooth muscle cells (VSMCs). Here, we analyzed the vascular function of Crem in mice with a global inactivation of Crem (Crem(-/-)). CRE-mediated transcriptional activity was enhanced in primary Crem(-/-) VSMCs under nonstimulated conditions and under stimulation with Forskolin and platelet-derived growth factor (Pdgf) whereas stimulation with nitric oxide or cGMP showed no effect. This elevated CRE-mediated transcriptional activity as a result of Crem inactivation did not alter aortic contractility or fractions of proliferating or apoptotic aortic VSMCs in situ, and no impact of Crem inactivation on the development of atherosclerotic plaques was observed. Crem(-/-) mice exhibited an increased neointima formation after carotid ligation associated with an increased proliferation of VSMCs in the carotid media. Pdgf-stimulated proliferation of primary aortic Crem(-/-) VSMCs was increased along with an upregulation of messenger RNA (mRNA) levels of Pdgf receptor, alpha polypeptide (Pdgfra), cyclophilin A (Ppia), the regulator of G-protein signaling 5 (Rgs5), and Rho GTPase-activating protein 12 (Arhgap12). Taken together, our data reveal the inhibition of Pdgf-stimulated proliferation of VSMCs by repressing the Pdgf-stimulated CRE-mediated transcriptional activation as the predominant function of Crem in mouse vasculature suggesting an important role of Crem in vasculoproliferative diseases.
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Proliferación Celular , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Ciclofilina A/genética , Ciclofilina A/metabolismo , Masculino , Ratones , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/fisiología , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Regulación hacia ArribaRESUMEN
Human congestive heart failure is accompanied by structural and electrical alterations leading to the development of an arrhythmogenic substrate. This substrate is associated with the "sudden cardiac death" due to ventricular tachycardia or ventricular fibrillation. Multiple studies link distinct transcription factors to the transcriptional regulation of genes related to the formation of an arrhythmogenic substrate. In addition to cardiac hypertrophy the up- or downregulation of ion channels, calcium-handling proteins, and proteins forming gap junctions play a pivotal role in the progression of heart failure. This review summarizes the transcriptional regulation of selected genes implicated in the formation of an arrhythmogenic substrate. In this context we provide an overview of relevant transcription factors, activating stimuli and pathways, the evidence of binding to respective elements in the promoter of target genes and the associated mRNA regulation in animal models. Finally, possible therapeutic consequences are discussed.
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Antiarrítmicos/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Factores de Transcripción/metabolismo , Animales , Calcio/metabolismo , Regulación de la Expresión Génica , Homeostasis , HumanosRESUMEN
Anti-insect depressant toxins represent a subfamily of scorpion venom-derived ß-toxins that are polypeptides composed of 61-65 amino acid residues stabilized by four disulfide bridges. These toxins affect the activation of voltage-sensitive sodium channels (NaScTx) and exhibit the preferential ability to induce flaccid paralysis in insect larvae. Here we demonstrate the recombinant expression of the novel cardiac inotropic peptide (Bj-IP) that was classified as an anti-insect depressant ßNaScTx isolated from the venom of Hottentotta judaicus. By using "splicing by overlap extension" (SOE)-PCR, allowing for the first time one step de novo synthesis of long-chain scorpion toxin genes, we generated a codon-optimized DNA fragment of Bj-IP for cloning into the Escherichia coli vector pQE30. Moreover, the gene of interest was fused to a 6xHis coding DNA sequence. Subsequent recombinant expression was performed in E. coli KRX. The purification of the polypeptide was achieved by a combination of NiNTA agarose columns and RP (C(18)) high-performance liquid chromatography. The purified fusion protein was digested with factor Xa resulting in the elution of Bj-IP. The yield of recombinant Bj-IP expression was approximately 4.5 mg per liter of culture. Mass spectrometry confirmed the theoretical total mass of Bj-IP (6608 Da). Tag-free Bj-IP was refolded in guanidine chloride buffer with a glutathione redox system which was supplemented with different additives at 16 °C. Supplementation with 10% glycerol produced Bj-IP folding forms that exhibited reproducible biological activity in mouse cardiomyocytes. Cell contractility was increased by almost 3-fold and decay kinetics were hasten by 47% after administration of Bj-IP. Taken together, here we show the recombinant expression of the functionally active cardiac inotropic peptide Bj-IP, a new ßNaScTx from H. judaicus, for promising pharmacological applications. Furthermore, our data suggest that the use of SOE-PCR may help to facilitate in future the high throughput of cloning and/or modification of scorpion toxin genes.
Asunto(s)
Péptidos/genética , Péptidos/toxicidad , Venenos de Escorpión/química , Escorpiones , Animales , Secuencia de Bases , Western Blotting , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Espectrometría de Masas , Mutagénesis , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido NucleicoRESUMEN
The cAMP response element binding protein (CREB) belongs to the CREB/cAMP response element binding modulator/activating transcription factor 1 family of cAMP-dependent transcription factors mediating a regulation of gene transcription in response to cAMP. Chronic stimulation of ß-adrenergic receptors and the cAMP-dependent signal transduction pathway by elevated plasma catecholamines play a central role in the pathogenesis of heart failure. Ion channel remodeling, particularly a decreased transient outward current (I(to)), and subsequent action potential (AP) prolongation are hallmarks of the failing heart. Here, we studied the role of CREB for ion channel regulation in mice with a cardiomyocyte-specific knockout of CREB (CREB KO). APs of CREB KO cardiomyocytes were prolonged with increased AP duration at 50 and 70% repolarization and accompanied by a by 51% reduction of I(to) peak amplitude as detected in voltage-clamp measurements. We observed a 29% reduction of Kcnd2/Kv4.2 mRNA in CREB KO cardiomyocytes mice while the other I(to)-related channel subunits Kv4.3 and KChIP2 were not different between groups. Accordingly, Kv4.2 protein was reduced by 37% in CREB KO. However, we were not able to detect a direct regulation of Kv4.2 by CREB. The I(to)-dependent AP prolongation went along with an increase of I(Na) and a decrease of I(Ca,L) associated with an upregulation of Scn8a/Nav1.6 and downregulation of Cacna1c/Cav1.2 mRNA in CREB KO cardiomyocytes. Our results from mice with cardiomyocyte-specific inactivation of CREB definitively indicate that CREB critically regulates the AP shape and duration in the mouse ventricle, which might have an impact on ion channel remodeling in situations of altered cAMP-dependent signaling like heart failure.
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Potenciales de Acción/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Ventrículos Cardíacos/citología , Canales Iónicos/fisiología , Miocitos Cardíacos/fisiología , Función Ventricular/fisiología , Animales , Canales de Calcio Tipo L/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación hacia Abajo/fisiología , Ratones , Ratones Noqueados , Modelos Animales , Miocitos Cardíacos/citología , Canal de Sodio Activado por Voltaje NAV1.6 , Proteínas del Tejido Nervioso/fisiología , Técnicas de Placa-Clamp , Transducción de Señal/fisiología , Canales de Sodio/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Classically, stimulation of muscarinic cholinoceptors exerts negative inotropic and chronotropic effects in the atrium of mammalian hearts. These effects are crucial to the vagal regulation of the heart beat. This effect is assumed to be mediated via GTP binding (G) proteins, because they can be abolished by Pertussis toxin. However, it is unknown which G proteins are involved. EXPERIMENTAL APPROACH: We studied contractility in isolated left or right atrium from genetically manipulated mice with deletion of one of two G proteins, either of the alpha subunit of G(i2) protein (G(i2)alpha) or of the alpha subunit of G(o) protein (G(o)alpha). Preparations were stimulated with carbachol alone or after pretreatment with the beta-adrenoceptor agonist isoprenaline. For comparison, the effects of carbachol on L-type Ca(2+)-channels in isolated ventricular cardiomyocytes were studied. KEY RESULTS: The negative inotropic and chronotropic effects of carbachol alone or in the presence of isoprenaline were identical in atria from knockout or wild-type mice. However, the effect of carbachol on isoprenaline-activated L-type Ca(2+)-channel in isolated ventricular cardiomyocytes was greatly attenuated in both types of knockout mice studied. CONCLUSIONS AND IMPLICATIONS: These data imply that there is either redundancy of G proteins for signal transduction or that Pertussis toxin-sensitive proteins other than G(i2)alpha and G(o)alpha mediate the vagal stimulation in the atrium. Moreover, different G proteins mediate the effect of carbachol in ventricle compared with atrium.
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Carbacol/farmacología , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Isoproterenol/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Agonistas Colinérgicos/farmacología , Femenino , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Masculino , Ratones , Ratones Noqueados , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The loss of NO synthase (NOS) from the sarcolemma was assumed to be associated with development of Duchenne muscular dystrophy (DMD). We have, however, recently reported that, in contrast to the commonly accepted view, NOS expression in DMD myofibres is up-regulated. This poses the question of the fibre type-specific NOS expression in DMD muscles and how the NOS expression is related to the regeneration or degeneration status. To address this issue, we examined localization of NOS isoforms I, II and III in skeletal muscles of DMD patients employing immunohistochemical labelling with tyramide signal amplification complemented with enzyme histochemistry. We found that NOS immunolabelling as well as metabolic enzyme activity in DMD muscles were heterogeneously distributed along the fibre length of DMD muscle fibres revealing regenerating and degenerate (hypercontracted) fibres as well as normal segments. Like in normal muscles, positive NOS immunoreactivity was found to be associated with fast-oxidative glycolytic (FOG) phenotype. The regeneration status of NOS-positive segments was deduced from the presence of neonatal and developmental myosin heavy chains. High NOS expression in regenerating DMD muscle fibres can be well reconciled with reports about the protective role of endogenous NO in inflammatory diseases and in muscle repair.
Asunto(s)
Fibras Musculares Esqueléticas/enzimología , Músculo Esquelético/enzimología , Distrofia Muscular de Duchenne/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Preescolar , Humanos , Inmunohistoquímica , Isoenzimas/metabolismo , Masculino , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Cadenas Pesadas de Miosina/metabolismo , Regeneración/fisiologíaRESUMEN
Idiopathic-dilated cardiomyopathy (IDC) is a common primary myocardial disease of unknown etiology associated with apoptosis, cardiac dilatation, progressive heart failure and increased mortality. An elevation of the transcription factor activator protein 2alpha (AP-2alpha) is involved in vertebrate embryonic development and oncogenesis. Here, we show that AP-2alpha protein is expressed in the human heart and increased in human failing myocardium with IDC. Adenovirus-mediated overexpression of human AP-2alpha triggered apoptosis and increased mRNA levels of Bcl-2 family members Bax and Bcl-x in rat cardiomyocytes. Immunohistological analysis of human myocardium revealed an increased percentage of AP-2alpha-positive nuclei in IDC and, interestingly, a colocalization of AP-2alpha-positive but not -negative cells with a caspase-cleaved fragment of poly(ADP-ribose)polymerase. We suggest AP-2alpha as a novel cardiac regulator implicated in the activation of apoptosis in IDC.
Asunto(s)
Apoptosis/fisiología , Cardiomiopatía Dilatada/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Miocitos Cardíacos/metabolismo , Factores de Transcripción/metabolismo , Adenoviridae/genética , Animales , Caspasas/metabolismo , Células Cultivadas , Clonación Molecular , Proteínas de Unión al ADN/genética , Genes bcl-2/fisiología , Humanos , Miocardio , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , Factor de Transcripción AP-2 , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: A variety of pathologic stimuli lead to apoptosis of cardiomyocytes. Survival factors like insulin-like growth factor-I (IGF-I) exert anti-apoptotic effects in the heart. Yet the underlying signaling pathways are poorly understood. METHODS AND RESULTS: In a model of hypoxia-induced apoptosis of cultured neonatal cardiomyocytes, IGF-I prevented cell death in a dose-dependent manner. Antiapoptotic signals induced by IGF-I are mediated by more than one signaling pathway, because pharmacological inhibition of the phosphatidylinositol-3-OH-kinase (PI3K) or the mitogen-activated protein kinase kinase (MEK1) signaling pathway both antagonize the protective effect of IGF-I in an additive manner. IGF-I-stimulation was followed by a PI3K-dependent phosphorylation of AKT and BAD and an MEK1-dependent phosphorylation of extracellular signal-regulated kinase (ERK) 1 and ERK2. IGF-I also induced phosphorylation of cAMP response element-binding protein (CREB) in a PI3K- and MEK1-dependent manner. Ectopic overexpression of a dominant-negative mutant of CREB abolished the antiapoptotic effect of IGF-I. Protein levels of the antiapoptotic factor bcl-2 increased after longer periods of IGF-I-stimulation, which could be reversed by pharmacological inhibition of PI3K as well as MEK1 and also by overexpression of dominant-negative CREB. CONCLUSIONS: In summary, our data demonstrate that in cardiomyocytes, the antiapoptotic effect of IGF-I requires both PI3K- and MEK1-dependent pathways leading to the activation of the transcription factor CREB, which then induces the expression of the antiapoptotic factor bcl-2.
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Hipoxia de la Célula/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Proteínas Portadoras/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , MAP Quinasa Quinasa 1 , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Miocardio/citología , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transcripción Genética/efectos de los fármacos , Transfección , Proteína Letal Asociada a bclRESUMEN
The aim of this study was to investigate the functional properties of the promoter of the protein phosphatase 1alpha catalytic subunit. Luciferase plasmids with different fragments of the rat catalytic subunit of the protein phosphatase 1alpha promoter ranging from -3.7 kbp to -59 bp were transiently transfected into cells by the calcium-phosphate precipitation method. The promoter activity was determined in the absence and presence of inotropic agents which influencing the cAMP-depending pathway. The basal transcriptional activity decreased at fragment -124 bp and shorter fragments. To identify regions of regulatory importance we investigated the cAMP-dependent influence on the transcriptional activity. Stimulation of the complete promoter region with forskolin (1-100 microM) for 6 h led to a concentration-dependent decrease of transcriptional activity. Moreover, regions shorter than 3.7 kbp were inhibited by forskolin (10 microM). Short time stimulation (10 min) with forskolin (10 microM) increased the transcriptional activity of only the 3.7 kbp fragment. The effects were antagonized by Rp-cAMPS, a specific antagonist of protein kinase A, indicating cAMP-dependent effects. The results provide evidence for cAMP-dependent regulation of the protein phosphatase 1alpha promotor.
Asunto(s)
AMP Cíclico/análogos & derivados , Fosfoproteínas Fosfatasas/genética , Regiones Promotoras Genéticas , Animales , Animales Recién Nacidos , Secuencia de Bases , Bucladesina/farmacología , Dominio Catalítico , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Miocardio/citología , Fosfoproteínas Fosfatasas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Tionucleótidos/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
OBJECTIVE: Chronic beta-adrenergic stimulation of the cAMP-dependent signalling pathway is implicated in functionally relevant expressional changes in congestive heart failure. We studied activation and inactivation of the cardiac gene transcription mediated by the cAMP-response element (CRE) and the CRE-binding protein (CREB) as an important mechanism of a cAMP-dependent gene regulation. METHODS: We investigated the transcriptional activation by forskolin, an activator of the adenylyl cyclase, in chick embryonic cardiomyocytes transfected with a CRE-controlled luciferase construct in comparison to the phosphorylation and expression of CREB determined on immunoblots. RESULTS: Forskolin (10 micromol/l; 8 h) increased CRE-mediated transcription and phosphorylation of CREB 13- and 1.5-fold, respectively. The phosphorylation was further elevated in combination with cantharidin, an inhibitor of type 1+2A protein phosphatases. The transcriptional response to forskolin was desensitized by pretreatment with forskolin (1 micromol/l; 24 h) while CREB phosphorylation was increased. In forskolin-pretreated cells, total CREB protein levels were decreased. Cantharidin did not restore the attenuated transcriptional response. CONCLUSIONS: In cardiomyocytes, there is an activation of the CRE-mediated gene transcription by forskolin that is attenuated after prolonged stimulation, and this attenuation is not dependent from a dephosphorylation of CREB. We suggest that attenuation of the CRE-mediated transcription through chronic stimulation of the cAMP-pathway, e.g. by elevated catecholamines, contributes to the altered expressional regulation in congestive heart failure.
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Adenilil Ciclasas/metabolismo , Proteínas de Ciclo Celular , Colforsina/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , Propano/análogos & derivados , Transcripción Genética/efectos de los fármacos , 1-Metil-3-Isobutilxantina/farmacología , Animales , Cantaridina/farmacología , Cardiotónicos/farmacología , Células Cultivadas , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Etanolaminas , Expresión Génica/efectos de los fármacos , Isoproterenol/farmacología , Luciferasas/genética , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Proteínas de Plantas , Estimulación Química , Factores de TiempoRESUMEN
We studied the effects of the protein phosphatase (PP) inhibitor cantharidin (Cant) on time parameters and force of contraction (FOC) in isometrically contracting electrically driven guinea pig papillary muscles. We correlated the mechanical parameters of contractility with phosphorylation of the inhibitory subunit of troponin (TnI-P) and with the site-specific phosphorylation of phospholamban (PLB) at serine-16 (PLB-Ser-16) and threonine-17 (PLB-Thr-17). Cant (after 30 min) started to increase FOC (112 +/- 4% of control, n = 10) and TnI-P and PLB-Thr-17 (120 +/- 5 and 128 +/- 7% of control) without any alteration of relaxation time. Cant (10 microM) started to increase PLB-Ser-16, but the relaxation was shortened at only 100 microM (from 140 +/- 9 to 116 +/- 12 ms, n = 9). Moreover, 100 microM Cant, 3 min after application, started to increase PLB-Thr-17, TnI-P, and FOC. Cant (100 microM) began to increase PLB-Ser-16 after 20 min. This was accompanied by shortening of relaxation time. Differences in protein kinase activation or different substrate specificities of PP may explain the difference in Cant-induced site-specific phosphorylation of PLB in isometrically contracting papillary muscles. Moreover, PLB-Thr-17 may be important for inotropy, whereas PLB-Ser-16 could be a major determinant of relaxation time.
Asunto(s)
Contracción Miocárdica/fisiología , Miocardio/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Animales , Anticuerpos/farmacología , Calcio/análisis , Proteínas de Unión al Calcio/metabolismo , Cantaridina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Cobayas , Técnicas In Vitro , Masculino , Fibras Musculares Esqueléticas/enzimología , Miocardio/citología , Músculos Papilares/citología , Músculos Papilares/enzimología , Perfusión , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/inmunología , Fosforilación , Ratas , Troponina/metabolismoRESUMEN
In mammalian species, including man, the duration of myocardial contraction is shorter in atria than ventricles. Total contraction time depends at least in part on phosphorylation and dephosphorylation of cardiac regulatory proteins. Dephosphorylation reactions are mediated by protein phosphatases. In the mammalian heart more than 90% of the protein phosphatase (PP) activity consists of PP1 and PP2A. Therefore, the aim of this study was to investigate which isoforms of PP1 and PP2A are present in human myocardium and whether their expression is regionally different. RT-PCR and Northern blotting revealed that all isoforms of PP1 and PP2A presently known are expressed in the human heart. Expression levels of PP1 alpha, delta, and gamma as well as 2A alpha were higher in right ventricles than in right atria. However, there was no such difference for PP2A beta. At the protein level PP1 alpha was unchanged, whereas PP2A was by 56% higher in right ventricles compared to atria. The phosphorylation state of TnI was lower in right ventricle than in right atrium. Thus, lower protein expression of PP2A in atrium could contribute to the faster relaxation by increasing the phosphorylation state of TnI. We conclude that expression of PP1 and PP2A isoforms is regionally regulated in the human heart.
Asunto(s)
Miocardio/metabolismo , Fosfoproteínas Fosfatasas/biosíntesis , Northern Blotting , Western Blotting , Catálisis , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Humanos , Fosfoproteínas Fosfatasas/química , Fosforilación , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , Troponina I/biosíntesisRESUMEN
Stimulation of the cAMP-dependent signalling pathway by beta-adrenergic catecholamines is an important physiological mechanism to increase contractile force in the heart. In addition to this, long-term beta-adrenergic stimulation by elevated catecholamines also influences the expressional control of functionally relevant cardiac regulatory proteins in human heart failure. The regulation of transcription by the cAMP-response element (CRE) is an important mechanism for a cAMP-mediated control of gene expression involved e.g. in spermiogenesis and memory/learning processes. This article discusses recent data leading to the hypothesis that this mechanism also contributes to altered gene regulation in heart failure.
Asunto(s)
AMP Cíclico/fisiología , Regulación de la Expresión Génica , Cardiopatías/fisiopatología , Corazón/fisiología , Transcripción Genética , Animales , Catecolaminas/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Corazón/fisiopatología , Cardiopatías/genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Humanos , Contracción Miocárdica , Receptores Adrenérgicos beta/fisiologíaRESUMEN
We tested the hypothesis that altered phosphorylation of Ca2+ regulatory proteins contributes to contractile anomalies in cardiac hypertrophy. Cardiac hypertrophy was induced in rats by chronic s.c. administration of isoproterenol (Iso, 2.4 mg/kg/day) via osmotic minipumps. On day 2 of Iso treatment the expression of atrial natriuretic factor was increased, time of relaxation in isolated papillary muscles shortened and protein expression of phospholamban (PLB) and sarcoplasmic reticulum Ca2+-ATPase reduced. In addition, the phosphorylation state of PLB at serine-16 and threonine-17 was decreased from (arbitrary units) 2.3+/-0.3 to 1.1+/-0.2 and from 4.1+/-0.6 to 2.1+/-0.2, respectively. This was not accompanied by altered activity of PLB-phosphorylating protein kinases (protein kinase A or Ca2+/calmodulin-dependent protein kinase II), whereas the activity of types 1 and 2A protein phosphatases (PP1 and -2A respectively) was enhanced from 1.1+/-0.08 to 1.71+/-0.13 nmol/mg/min. Iso treatment did not alter the PP1/PP2A activity ratio and 1 nmol/l okadaic acid, a concentration which completely blocks the catalytic subunit of PP2A, inhibited about 40% of total PP activity in all groups studied. These data indicate that the activity of both PP1 and PP2A were increased. All effects of Iso treatment were abolished by co-administration of propranolol (29.7 mg/kg/day). It is concluded that dephosphorylation of PLB is due to enhanced activity of PP1 and PP2A. We suggest that chronic beta-adrenergic stimulation, which occurs in human cardiac hypertrophy and failure, can lead to increased activity of PPs. This may contribute to altered contractile responses in the hypertrophied heart.
Asunto(s)
Agonistas Adrenérgicos beta/toxicidad , Proteínas de Unión al Calcio/metabolismo , Cardiomegalia/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas/metabolismo , Análisis de Varianza , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/enzimología , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Isoproterenol/antagonistas & inhibidores , Isoproterenol/toxicidad , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Ácido Ocadaico/farmacología , Fosforilación/efectos de los fármacos , Propranolol/farmacología , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The mechanism(s) underlying contractile dysfunction in cardiac stunning is not completely understood. The expression and/or the phosphorylation state of cardiac Ca(2+) homoeostasis-regulating proteins might be altered in stunning. We tested this hypothesis in a well-characterized model of stunning. Conscious dogs were chronically instrumented, and the left anterior descending artery (LAD) was occluded for 10 min. Thereafter, reperfusion of the LAD was initiated. Tissues from reperfused LAD (stunned) and Ramus circumflexus (control) areas were obtained when left ventricular regional wall thickening fraction had recovered by 50%. Northern and Western blotting revealed no differences in the expression of the following genes: phospholamban, calsequestrin, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, and the inhibitory subunit of troponin I (TnI). However, the phosphorylation state of TnI and phospholamban were reduced in the LAD area. Fittingly, cAMP levels were reduced by 28% (P < 0.05). It is concluded that the contractile dysfunction in cardiac stunning might be mediated in part by decreased levels of cAMP and subsequently a reduced phosphorylation state of phospholamban and TnI.
Asunto(s)
Vasos Coronarios/metabolismo , Regulación de la Expresión Génica , Isquemia Miocárdica/metabolismo , Aturdimiento Miocárdico/metabolismo , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , ATPasas Transportadoras de Calcio/genética , Calsecuestrina/genética , Estado de Conciencia , AMP Cíclico/metabolismo , Perros , Femenino , Hemodinámica , Masculino , Contracción Miocárdica , Isquemia Miocárdica/fisiopatología , Reperfusión Miocárdica , Aturdimiento Miocárdico/genética , Aturdimiento Miocárdico/fisiopatología , Retículo Sarcoplasmático/enzimología , Troponina I/genéticaRESUMEN
In this study we characterized the effects of the protein phosphatase (PP) type 1 and type 2A inhibitor cantharidin (Cant) and its structural analogs cantharidic acid and endothall on PP activity, force of contraction, and myosin light chain phosphorylation in rat aorta. All compounds inhibited PP activity in homogenates of rat aorta with a rank order of potency of Cant = cantharidic acid > endothall. However, only Cant increased force of contraction and myosin light chain phosphorylation in intact isolated rat aortic rings. Based on these findings, we investigated the effects of Cant on alpha-adrenoceptor-mediated vasoconstriction. Cant (1 and 3 microM) enhanced norepinephrine-induced contraction in endothelium-intact rat aorta. In contrast, Cant did not affect norepinephrine-induced contraction in endothelium-denuded rat aorta. We suggest that inhibition of PP1 and/or PP2A activities by Cant enhances vascular contractility in endothelium-intact rat aorta by increasing the phosphorylation state of endothelial regulatory proteins.
Asunto(s)
Cantaridina/farmacología , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Norepinefrina/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/enzimología , Aorta Torácica/fisiología , Catálisis , Sinergismo Farmacológico , Endotelio Vascular/enzimología , Endotelio Vascular/fisiología , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/fisiología , Cadenas Ligeras de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Ratas , Ratas Wistar , Vasoconstricción/fisiologíaRESUMEN
Overexpression of calsequestrin (CSQ) induces severe cardiac hypertrophy, whereas overexpression of Na(+)-Ca(2+) exchanger (NCX) does not affect cardiac weight. To investigate a possible beneficial effect of NCX in hypertrophy, we produced transgenic mice overexpressing both NCX and CSQ (NCX/CSQ). Surprisingly, these mice developed severe heart failure. The heart/body weight ratio was enhanced and the mRNA expression of ANF, as a marker of hypertrophy, was highest in double transgenic mice. In isolated muscle strips, the basal relaxation time was prolonged in CSQ and NCX/CSQ mice. Moreover, in the presence of caffeine, force of contraction was increased only in CSQ and NCX/CSQ and was accompanied by elevated diastolic tension. In some respects, however, additional overexpression of NCX altered the CSQ phenotype into the wild-type phenotype. The expression of sarcoplasmic reticulum (SR)-Ca(2+)-ATPase and phospholamban, proteins involved in the Ca(2+) uptake of the SR, were only increased in CSQ, indicating a possible influence of NCX in the regulation of SR-Ca(2+) uptake proteins. The Ca(2+) transients and the L-type Ca(2+) currents in the presence of caffeine were very large in CSQ, but smaller increases were noted in double transgenic mice. Therefore, the successful co-overexpression of CSQ and NCX in these mice provides a novel model in which to investigate the interaction of proteins tightly linked to maintain Ca(2+) homeostasis.