RESUMO
Single molecule fluorescence tracking provides information at nanometer-scale and millisecond-temporal resolution about the dynamics and interaction of individual molecules in a biological environment. While the dynamic behavior of isolated molecules can be characterized well, the quantitative insight is more limited when interactions between two indistinguishable molecules occur. We address this aspect by developing a theoretical foundation for a spectroscopy of interaction times, i.e., the inference of interaction from imaging data. A non-trivial crossover between a power law to an exponential behavior of the distribution of the interaction times is highlighted, together with the dependence of the exponential term upon the microscopic reaction affinity. Our approach is validated with simulated and experimental datasets.
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Microscopic imaging at high spatial-temporal resolution over long time scales (minutes to hours) requires rapid and precise stabilization of the microscope focus. Conventional and commercial autofocus systems are largely based on piezoelectric stages or mechanical objective actuators. Objective to sample distance is either measured by image analysis approaches or by hardware modules measuring the intensity of reflected infrared light. We propose here a truly all-optical microscope autofocus taking advantage of an electrically tunable lens and a totally internally reflected infrared probe beam. We implement a feedback-loop based on the lateral position of a totally internally reflected infrared laser on a quadrant photodetector, as an indicator of the relative defocus. We show here how to treat the combined contributions due to mechanical defocus and deformation of the tunable lens. As a result, the sample can be kept in focus without any mechanical movement, at rates up to hundreds of Hertz. The device requires only reflective optics and can be implemented at a fraction of the cost required for a comparable piezo-based actuator.
RESUMO
Inflammation of cardiac tissue is generally associated with an activation of the host's immune system. On the one hand, this activation is mandatory to protect the heart by fighting the invading microbial agents or toxins and by engaging myocardial reparation and healing processes. On the other hand, uncontrolled activation of the immune defense has the risk of an arousal of auto- or cross-reactive immune cells, which in some cases bring more harm than good. Dependent on the individual genetic predisposition, such heart-directed autoimmune reactions most likely occur as a result of myocyte apoptosis or necrosis and subsequent liberation of self-antigens previously hidden to the immune system. During the past two decades, evidence for a pathogenic relevance of autoimmunity in human heart disease has substantially increased. Conformational cardiac (auto)antibodies affecting cardiac function and, in particular, (auto)antibodies that target G protein-coupled cardiac membrane receptors are thought to play a key role in the development of heart failure. Clinical pilot studies even suggest that such antibodies negatively affect survival in heart failure patients. However, the true prevalence and clinical impact of many cardiac (auto)antibodies in human heart diseases are still unclear, as are the events triggering their formation, their titer course, and their patterns of clearance and/or persistence. The present article summarizes current knowledge in the field of cardiac receptor (auto)antibodies including recent efforts to address some of the aforementioned gaps of knowledge, thereby attempting to pave the way for novel, more specific therapeutic approaches.
Assuntos
Autoanticorpos/imunologia , Cardiomiopatia Dilatada/imunologia , Modelos Imunológicos , Miocardite/imunologia , Miócitos Cardíacos/imunologia , Receptores de Superfície Celular/imunologia , Animais , HumanosRESUMO
The barrier function of the endothelium is controlled by the second messengers Ca2+ and cAMP that differentially regulate the permeability of endothelial cells. The Ca2+-elevating agent thrombin has been demonstrated to increase endothelial permeability and to decrease cAMP levels as detected via enzyme immunoassays. To study the effects of thrombin on cAMP with high temporal resolution, we utilised the FRET-based cAMP sensor Epac1-camps in single intact human umbilical vein endothelial cells (HUVECs). In these cells, thrombin induced a delayed increase in [cAMP], initiating after about 40 s, with maximum cAMP levels after 130 s of thrombin application. This increase of cAMP levels was Ca2+-dependent, but did not require calmodulin (CaM). Pharmacological approaches revealed that phospholipase A2 (PLA2) activity and cyclooxygenase (COX)-mediated synthesis of prostaglandins was required for the thrombin-induced elevation of [cAMP]. Furthermore, preincubation of HUVECs with a prostacyclin-receptor antagonist significantly reduced the thrombin-induced increase in [cAMP]. We conclude that thrombin causes the synthesis of prostacyclin in endothelial cells and that the subsequent stimulation of Gs-coupled prostacyclin receptors then results in an increase in [cAMP].
Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Epoprostenol/metabolismo , Trombina/metabolismo , Técnicas Biossensoriais , Permeabilidade Capilar , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Humanos , Fosfolipases A2/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores de Epoprostenol , Receptores de Prostaglandina/metabolismo , Transdução de Sinais , Fatores de Tempo , Regulação para CimaRESUMO
Several lines of evidence suggest that G-protein-coupled receptors can adopt different active conformations, but their direct demonstration in intact cells is still missing. Using a fluorescence resonance energy transfer (FRET)-based approach we studied conformational changes in alpha(2A)-adrenergic receptors in intact cells. The receptors were C-terminally labeled with cyan fluorescent protein and with fluorescein arsenical hairpin binder at different sites in the third intracellular loop: N-terminally close to transmembrane domain V (I3-N), in the middle of the loop (I3-M), or C-terminally close to transmembrane domain VI (I3-C). All constructs retained normal ligand binding and signaling properties. Changes in FRET between the labels were determined in intact cells in response to different agonists. The full agonist norepinephrine evoked similar FRET changes for all three constructs. The strong partial agonists clonidine and dopamine induced partial FRET changes for all constructs. However, the weak partial agonists octopamine and norphenephrine only induced detectable changes in the construct I3-C but no change in I3-M and I3-N. Dopamine-induced FRET-signals were approximately 1.5-fold slower than those for norepinephrine in I3-C and I3-M but >3-fold slower in I3-N. Our data indicate that the different ligands induced conformational changes in the receptor that were sensed differently in different positions of the third intracellular loop. This agrees with X-ray receptor structures indicating larger agonist-induced movements at the cytoplasmic ends of transmembrane domain VI than V and suggests that partial agonism is linked to distinct conformational changes within a G-protein-coupled receptor.
Assuntos
Agonistas Adrenérgicos/farmacologia , Agonistas de Receptores Adrenérgicos alfa 2 , Receptores Adrenérgicos alfa 2/química , Agonistas Adrenérgicos/metabolismo , Animais , Linhagem Celular , Clonidina/metabolismo , Dopamina/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Ligantes , Camundongos , Norepinefrina/metabolismo , Octopamina/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Conformação Proteica/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/metabolismoRESUMO
The crosstalk between Ca(2+) and cAMP signals plays a significant role for the regulation of the endothelial barrier function. The Ca(2+)-elevating agent thrombin was demonstrated to increase endothelial permeability and to decrease cAMP levels. Since Ca(2+) and cAMP signals are highly dynamic, we aimed to study the temporal resolution between thrombin-evoked Ca(2+) signals and subsequent changes of cAMP levels. Here we conduct the first real-time monitoring of thrombin-mediated regulation of cAMP signals in intact human umbilical vein endothelial cells (HUVECs) by utilising the Ca(2+)-sensitive dye Fluo-4 and the fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac1-camps. We calibrated in vitro FRET responses of Epac1-camps to [cAMP] in order to estimate changes in intracellular [cAMP] evoked by thrombin treatment of HUVECs. After increasing [cAMP] to 1.2 +/- 0.2 microm by stimulation of HUVECs with isoproterenol (isoprenaline), we observed a transient decrease of cAMP levels by 0.4 +/- 0.1 microm which reached a minimum value 30 s after thrombin application and 15 s after the thrombin-evoked Ca(2+) peak. This transient decrease in [cAMP] was Ca(2+)-dependent and independent of a G(i)-mediated inhibition of adenylyl cyclases (ACs). Instead the knock down of the predominant subtype AC6 in HUVECs provided the first direct evidence that the Ca(2+)-mediated inhibition of AC6 accounts for the thrombin-induced decrease in cAMP levels.
Assuntos
Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Cálcio/metabolismo , AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais/fisiologia , Trombina/farmacologia , Células Cultivadas , Sistemas Computacionais , Células Endoteliais/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: cAMP is an ubiquitous second messenger mediating various neuronal functions, often as a consequence of increased intracellular Ca2+ levels. While imaging of calcium is commonly used in neuroscience applications, probing for cAMP levels has not yet been performed in living vertebrate neuronal tissue before. RESULTS: Using a strictly neuron-restricted promoter we virally transduced neurons in the organotypic brainstem slices which contained pre-Bötzinger complex, constituting the rhythm-generating part of the respiratory network. Fluorescent cAMP sensor Epac1-camps was expressed both in neuronal cell bodies and neurites, allowing us to measure intracellular distribution of cAMP, its absolute levels and time-dependent changes in response to physiological stimuli. We recorded [cAMP]i changes in the micromolar range after modulation of adenylate cyclase, inhibition of phosphodiesterase and activation of G-protein-coupled metabotropic receptors. [cAMP]i levels increased after membrane depolarisation and release of Ca2+ from internal stores. The effects developed slowly and reached their maximum after transient [Ca2+]i elevations subsided. Ca2+-dependent [cAMP]i transients were suppressed after blockade of adenylate cyclase with 0.1 mM adenylate cyclase inhibitor 2'5'-dideoxyadenosine and potentiated after inhibiting phosphodiesterase with isobutylmethylxanthine and rolipram. During paired stimulations, the second depolarisation and Ca2+ release evoked bigger cAMP responses. These effects were abolished after inhibition of protein kinase A with H-89 pointing to the important role of phosphorylation of calcium channels in the potentiation of [cAMP]i transients. CONCLUSION: We constructed and characterized a neuron-specific cAMP probe based on Epac1-camps. Using viral gene transfer we showed its efficient expression in organotypic brainstem preparations. Strong fluorescence, resistance to photobleaching and possibility of direct estimation of [cAMP] levels using dual wavelength measurements make the probe useful in studies of neurons and the mechanisms of their plasticity. Epac1-camps was applied to examine the crosstalk between Ca2+ and cAMP signalling and revealed a synergism of actions of these two second messengers.
Assuntos
Tronco Encefálico/citologia , Cálcio/metabolismo , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Neurônios/metabolismo , Adenilil Ciclases/metabolismo , Animais , Técnicas de Cultura de Células , Corantes Fluorescentes/metabolismo , Camundongos , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Homologous or agonist-specific desensitization of beta-adrenergic receptors is thought to be mediated by a specific kinase, the beta-adrenergic receptor kinase (beta ARK). However, recent data suggest that a cofactor is required for this kinase to inhibit receptor function. The complementary DNA for such a cofactor was cloned and found to encode a 418-amino acid protein homologous to the retinal protein arrestin. The protein, termed beta-arrestin, was expressed and partially purified. It inhibited the signaling function of beta ARK-phosphorylated beta-adrenergic receptors by more than 75 percent, but not that of rhodopsin. It is proposed that beta-arrestin in concert with beta ARK effects homologous desensitization of beta-adrenergic receptors.
Assuntos
Antígenos/genética , Proteínas Quinases Dependentes de AMP Cíclico , Proteínas do Olho/genética , Inibidores de Fosfodiesterase/farmacologia , Proteínas Quinases/farmacologia , Receptores Adrenérgicos beta/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antígenos/isolamento & purificação , Antígenos/farmacologia , Arrestina , Northern Blotting , Cromatografia por Troca Iônica , Clonagem Molecular , DNA/genética , Proteínas do Olho/isolamento & purificação , Proteínas do Olho/farmacologia , Regulação da Expressão Gênica , Dados de Sequência Molecular , Fosforilação , RNA Mensageiro/análise , Receptores Adrenérgicos beta/fisiologia , Transfecção , Quinases de Receptores Adrenérgicos betaRESUMO
We have investigated the influence of the neural cell adhesion molecules L1 and N-CAM on second messenger systems using a PC12 rat pheochromocytoma cell line as a model and triggering cell surface receptors by specific antibody binding. Antibodies directed against L1 and N-CAM, but not against other cell surface components, reduce intracellular levels of the inositol phosphates IP2 and IP3, while intracellular levels of cAMP are unaffected. Antibodies against L1 and N-CAM also reduce intracellular pH and increase intracellular Ca2+ by opening Ca2+ channels in a pertussis toxin-inhibitable manner, suggesting the involvement of a G protein in the signal transduction process. Cross-linking of the adhesion molecules on the surface membrane is not required for the effects to occur. Furthermore, adhesion of single PC12 cells to each other elicits effects on intracellular pH and Ca2+ similar to those seen after application, underscoring the physiological significance of the observed changes.
Assuntos
Cálcio/metabolismo , Moléculas de Adesão Celular Neuronais/fisiologia , AMP Cíclico/metabolismo , Fosfatos de Inositol/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Animais , Anticorpos , Canais de Cálcio/metabolismo , Adesão Celular , Moléculas de Adesão Celular Neuronais/imunologia , Concentração de Íons de Hidrogênio , Fragmentos Fab das Imunoglobulinas , Toxina Pertussis , Ratos , Transdução de Sinais , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The G-protein-coupled receptors (GPCRs) represent one the largest families of drug targets. Upon agonist binding a receptor undergoes conformational rearrangements that lead to a novel protein conformation which in turn can interact with effector proteins. During the last decade significant progress has been made to prove that different conformational changes occur. Today it is mostly accepted that individual ligands can induce different receptor conformations. However, the nature or molecular identity of the different conformations is still ill-known. Knowledge of the potential functionally selective conformations will help to develop drugs that select specific conformations of a given GPCR which couple to specific signalling pathways and may, ultimately, lead to reduced side effects. In this review we will summarize recent progress in biophysical approaches that have led to the current understanding of conformational changes that occur during GPCR activation.
Assuntos
Receptores Acoplados a Proteínas G/química , Animais , Quelantes/farmacologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Conformação Proteica , Receptor Muscarínico M3/química , Receptor Muscarínico M3/efeitos dos fármacos , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores de Droga/química , Receptores de Droga/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Rodopsina/química , Rodopsina/efeitos dos fármacosRESUMO
G-protein-coupled receptors (GPCRs) are the largest group of cell surface receptors. They are stimulated by a variety of stimuli and signal to different classes of effectors, including several types of ion channels and second messenger-generating enzymes. Recent technical advances, most importantly in the optical recording with energy transfer techniques--fluorescence and bioluminescence resonance energy transfer, FRET and BRET--, have permitted a detailed kinetic analysis of the individual steps of the signalling chain, ranging from ligand binding to the production of second messengers in intact cells. The transfer of information, which is initiated by ligand binding, triggers a signalling cascade that displays various rate-controlling steps at different levels. This review summarizes recent findings illustrating the speed and the complexity of this signalling system.
Assuntos
Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , Animais , Arrestinas/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Humanos , Cinética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Anxiety disorders result from a complex interplay of genetic and environmental factors such as stress. On the level of cellular signaling, regulator of G protein signaling 2 (Rgs2) has been implicated in human and rodent anxiety. However, there is limited knowledge about the role of Rgs2 in fear learning and reactivity to stress. In this study, Rgs2-/- mice showed increased fear learning, male mice displayed increased contextual and cued fear learning, while females showed selectively enhanced cued fear learning. Male Rgs2-/- mice displayed increased long-term-contextual fear memory, but increased cued fear extinction. Learning in spatial non-aversive paradigms was also increased in Rgs2-/- mice. Female, but not male mice show increased spatial learning in the Barnes maze, while male mice showed enhanced place preference in the IntelliCage, rendering enhanced cognitive function non-specific for aversive stimuli. Consistent with the previous results, Rgs2 deletion resulted in increased innate anxiety, including neophobic behavior expressed as hypolocomotion, in three different tests based on the approach-avoidance conflict. Acute electric foot shock stress provoked hypolocomotion in several exploration-based tests, suggesting fear generalization in both genotypes. Rgs2 deletion was associated with reduced monoaminergic neurotransmitter levels in the hippocampus and prefrontal cortex and disturbed corresponding GPCR expression of the adrenergic, serotonergic, dopaminergic and neuropeptide Y system. Taken together, Rgs2 deletion promotes improved cognitive function as well as increased anxiety-like behavior, but has no effect on acute stress reactivity. These effects may be related to the observed disruption of the monoaminergic systems.
Assuntos
Aprendizagem/fisiologia , Proteínas RGS/genética , Proteínas RGS/metabolismo , Animais , Ansiedade/fisiopatologia , Transtornos de Ansiedade/metabolismo , Comportamento Animal , Condicionamento Psicológico , Sinais (Psicologia) , Medo/fisiologia , Medo/psicologia , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Aprendizagem Espacial/fisiologia , Memória Espacial/fisiologiaRESUMO
Increased sympathetic noradrenergic signaling is crucially involved in fear and anxiety as defensive states. MicroRNAs regulate dynamic gene expression during synaptic plasticity and genetic variation of microRNAs modulating noradrenaline transporter gene (SLC6A2) expression may thus lead to altered central and peripheral processing of fear and anxiety. In silico prediction of microRNA regulation of SLC6A2 was confirmed by luciferase reporter assays and identified hsa-miR-579-3p as a regulating microRNA. The minor (T)-allele of rs2910931 (MAFcases = 0.431, MAFcontrols = 0.368) upstream of MIR579 was associated with panic disorder in patients (pallelic = 0.004, ncases = 506, ncontrols = 506) and with higher trait anxiety in healthy individuals (pASI = 0.029, pACQ = 0.047, n = 3112). Compared to the major (A)-allele, increased promoter activity was observed in luciferase reporter assays in vitro suggesting more effective MIR579 expression and SLC6A2 repression in vivo (p = 0.041). Healthy individuals carrying at least one (T)-allele showed a brain activation pattern suggesting increased defensive responding and sympathetic noradrenergic activation in midbrain and limbic areas during the extinction of conditioned fear. Panic disorder patients carrying two (T)-alleles showed elevated heart rates in an anxiety-provoking behavioral avoidance test (F(2, 270) = 5.47, p = 0.005). Fine-tuning of noradrenaline homeostasis by a MIR579 genetic variation modulated central and peripheral sympathetic noradrenergic activation during fear processing and anxiety. This study opens new perspectives on the role of microRNAs in the etiopathogenesis of anxiety disorders, particularly their cardiovascular symptoms and comorbidities.
Assuntos
Encéfalo/metabolismo , Medo/fisiologia , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Norepinefrina/fisiologia , Transtorno de Pânico/metabolismo , Sistema Nervoso Simpático/fisiopatologia , Adulto , Alelos , Ansiedade/genética , Ansiedade/metabolismo , Encéfalo/fisiopatologia , Mapeamento Encefálico , Condicionamento Clássico , Extinção Psicológica , Feminino , Variação Genética , Humanos , Imageamento por Ressonância Magnética , Masculino , MicroRNAs/genética , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Transtorno de Pânico/genética , Transtorno de Pânico/fisiopatologia , Polimorfismo de Nucleotídeo Único , Regulação para CimaRESUMO
Receptors of the seven transmembrane domain family are coupled to heterotrimeric G proteins [1]. Binding of ligand to these receptors induces dissociation of the heterotrimeric complex into free GTP-Galpha and Gbetagamma subunits, which then interact with their respective effector molecules to stimulate specific cellular responses. In some cases, these cellular responses involve mitogenic signalling [2]. The mitogen-activated protein (MAP) kinase cascade is initiated by the protein kinase cRaf1 and links growth factor receptor signalling to cell growth and differentiation [3]. The main activator of cRaf1 is the small GTP-binding protein Ras [4], and the binding of cRaf1 to GTP-Ras translocates cRaf1 to the plasma membrane, where it is activated [5]. It has been reported that cRaf1 associates directly with the beta subunit of heterotrimeric G proteins in vitro, and with the betagamma subunit complex in vivo [6], but the role of this association is not yet understood. Here, we show that cRaf1 associates with Gbeta1gamma2, and that this association in mammalian cells is significantly enhanced when active p21(Ras) is present or when cRaf1 is otherwise targeted to the membrane. Association with Gbeta1gamma2 has no effect on the kinase activity of cRaf1, but cRaf1 can affect Gbetagamma-mediated signalling events. Thus, membrane-localised cRaf1 inhibits G-protein-coupled receptor (GPCR)-stimulated activation of phospholipase Cbeta (PLCbeta) by sequestration of Gbetagamma subunits, an effect also observed with endogenous levels of cRaf1. Our data suggest that cRaf1 may be an important regulator of signalling by Gbetagamma, particularly in those GPCR systems that stimulate the MAP kinase cascade through the activation of p21(Ras).
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Humanos , Rim , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Receptor B2 da Bradicinina , Receptores da Bradicinina/genética , Receptores da Bradicinina/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , TransfecçãoRESUMO
The aim of this study was to assess the capability of MRI to characterize systolic and diastolic function in normal and chronically failing mouse hearts in vivo at rest and during inotropic stimulation. Applying an ECG-gated FLASH-cine sequence, MRI at 7 T was performed at rest and after administration of 1.5 microgram/g IP dobutamine. There was a significant increase of heart rate, cardiac output, and ejection fraction and significant decrease of end-diastolic and end-systolic left ventricular (LV) volumes (P<0.01 each) in normal mice during inotropic stimulation. In mice with heart failure due to chronic myocardial infarction (MI), MRI at rest revealed gross LV dilatation. There was a significant decrease of LV ejection fraction in infarcted mice (29%) versus sham mice (58%). Mice with MI showed a significantly reduced maximum LV ejection rate (P<0.001) and LV filling rate (P<0.01) and no increase of LV dynamics during dobutamine action, indicating loss of contractile and relaxation reserve. In 4-month-old transgenic mice with cardiospecific overexpression of the beta(1)-adrenergic receptor, which at this early stage do not show abnormalities of resting cardiac function, LV filling rate failed to increase after dobutamine stress (transgenic, 0.19+/-0.03 microL/ms; wild type, 0.36+/-0.01 microL/ms; P<0.01). Thus, MRI unmasked diastolic dysfunction during dobutamine stress. Dobutamine-stress MRI allows noninvasive assessment of systolic and diastolic components of heart failure. This study shows that MRI can demonstrate loss of inotropic and lusitropic response in mice with MI and can unmask diastolic dysfunction as an early sign of cardiac dysfunction in a transgenic mouse model of heart failure.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Dobutamina/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Imageamento por Ressonância Magnética , Animais , Pressão Sanguínea/efeitos dos fármacos , Feminino , Ventrículos do Coração/fisiopatologia , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/fisiopatologia , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Volume Sistólico/efeitos dos fármacos , Função Ventricular , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Esquerda/fisiologiaRESUMO
Creatine kinase (CK) plays a crucial role in myocardial energy metabolism. Alterations in CK gene expression are found in hypertrophied and failing heart, but the mechanisms behind these changes are unclear. This study tests the hypothesis that increased adrenergic stimulation, which is observed in heart failure, induces changes of myocardial CK-activity, -isoenzyme distribution and -gene expression that are characteristic of the failing and hypertrophied heart. Isolated rat hearts were perfused (constant pressure of 80 mmHg) with red cell suspensions. Following a 20-min warm-up period, perfusion for 3 h with 10(-8) M (iso 3 h) or without (control 3 h) isoproterenol was started or experiments were immediately terminated (control 0 h). Left ventricular tissue was analyzed for total CK-activity, CK-isoenzyme distribution and, by use of quantitative RT-PCR, for B-CK, M-CK, mito-CK and GAPDH- (as internal standard) mRNA. After beta-adrenergic stimulation (iso 3 h) but not after control perfusion (control 3 h) a roughly threefold increase in B-CK mRNA levels and a decrease in M-CK mRNA levels by 18% was found. There were no significant differences among the three groups in total CK-activity and in distribution of CK-MM, CK-BB, CK-MB and mito-CK. Thus, beta-adrenergic stimulation induces a switch in CK gene expression from M-CK to B-CK, which is characteristic for the hypertrophied and failing heart. This may be interpreted as an adaptive mechanism making energy transduction via CK more efficient at times of increased metabolic demand.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Creatina Quinase/genética , Isoproterenol/farmacologia , Miocárdio/enzimologia , Animais , Cardiomegalia/metabolismo , Creatina Quinase/análise , Creatina Quinase/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Isoenzimas/análise , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Modelos Animais , Perfusão , RNA Mensageiro/análise , Ratos , Ratos Wistar , Função Ventricular EsquerdaRESUMO
BACKGROUND: Angiotensin II activates 2 distinct G protein-coupled receptors, the AT(1) and AT(2) receptors. Most of the known cardiovascular effects of angiotensin II are mediated by the AT(1) receptor subtype. The aim of the present study was to test whether deletion of the AT(2) receptor gene in mice (AT(2)-KO mice) leads to long-term functional or structural alterations in the cardiovascular system. METHODS AND RESULTS: In vivo pressure responses to angiotensin II or the alpha(1)-adrenergic receptor agonist phenylephrine were greatly enhanced in AT(2)-KO mice. Deletion of the angiotensin AT(2) receptor did not lead to a compensatory increase of the activity of the circulating renin-angiotensin system, and arterial blood pressure was identical in wild-type control mice (WT) and AT(2)-KO mice. Cardiac contractility as assessed by LV catheterization and by rapid MRI also did not differ between AT(2)-KO and WT mice. Isolated femoral arteries from AT(2)-KO mice, however, showed enhanced vasoconstriction to angiotensin II, norepinephrine, and K(+) depolarization compared with WT. Morphometric analysis of large and small femoral arteries revealed a significant hypertrophy of media smooth muscle cells. Phospho-P70S6 kinase levels were significantly increased in aortas from AT(2)-KO mice compared with WT mice. Treatment of mice with an ACE inhibitor for 8 weeks abolished the increased pressure responsiveness, vascular hypertrophy, and enhanced P70S6 kinase phosphorylation in AT(2)-KO mice. CONCLUSIONS: These results indicate that vascular AT(2) receptors inhibit the activity and, hence, hypertrophic signaling by the P70S6 kinase in vivo and thus are important regulators of vascular structure and function.
Assuntos
Receptores de Angiotensina/genética , Receptores de Angiotensina/fisiologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Doenças Vasculares/etiologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Pressão Sanguínea , Captopril/farmacologia , Técnicas de Cultura , Coração/fisiopatologia , Hemodinâmica , Hipertrofia/etiologia , Hipertrofia/patologia , Hipertrofia/fisiopatologia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Miografia , Fosforilação , Receptor Tipo 2 de Angiotensina , Transdução de Sinais , Doenças Vasculares/patologia , Doenças Vasculares/fisiopatologia , VasoconstriçãoRESUMO
OBJECTIVES: This study investigated the relation between the severity of heart failure and the extent of the reduction of beta 1-adrenergic receptor messenger ribonucleic acid (mRNA) levels in biopsy specimens from the ventricular septum obtained during cardiac catheterization of patients with various degrees of heart failure. BACKGROUND: Heart failure is accompanied by desensitization of the beta-adrenergic receptor system, which is in part due to downregulation of beta 1-adrenergic receptors. Downregulation of beta 1-adrenergic receptors has been suggested to be caused by reductions in mRNA levels. METHODS: Because biopsy specimens were small and receptor mRNAs not abundant, mRNA levels were determined by quantitative reverse transcription/polymerase chain reactions. This method was validated by measuring synthetic ribonucleic acid (RNA) standards and samples from explanted hearts by solution hybridization assays. Both methods yielded similar results, but the polymerase chain reaction method was approximately 1,000-fold more sensitive. Sources of variations in the polymerase chain reaction were quantitated and found to be best controlled for by determination of the glyceraldehyde phosphate dehydrogenase mRNA as an endogenous control. RESULTS: Beta 1-adrenergic receptor mRNA levels in the biopsy specimens were decreased by 7% in mild (New York Heart Association functional class II), 26% in moderate (functional class III) and > 50% in severe heart failure (functional class IV). There was a good correlation between hemodynamic indicators of heart failure and beta 1-adrenergic receptor mRNA levels. In contrast, beta 2-adrenergic receptor mRNA levels were apparently unaffected by heart failure. CONCLUSIONS: Reduced beta 1-adrenergic receptor mRNA levels occur early in heart failure and can be detected in septal biopsy specimens during right heart catheterization. The reduction in beta 1-adrenergic receptor expression may contribute to further loss of cardiac function.
Assuntos
Insuficiência Cardíaca/patologia , Ventrículos do Coração/química , RNA Mensageiro/análise , Receptores Adrenérgicos beta/análise , Adulto , Sequência de Bases , Biópsia , Cateterismo Cardíaco , Regulação para Baixo , Feminino , Insuficiência Cardíaca/fisiopatologia , Septos Cardíacos/química , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Miocárdio/química , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Receptores Adrenérgicos beta/genética , Sensibilidade e Especificidade , Disfunção Ventricular/patologia , Disfunção Ventricular/fisiopatologiaRESUMO
OBJECTIVES: We investigated whether autoantibodies against the human beta-adrenergic receptor (beta-AR) might be involved in cardiomyopathies secondary to valvular heart disease (VHD) or hypertensive heart disease (HHD). BACKGROUND: Autoimmunity to beta-AR has been proposed as a pathogenic principle in human cardiomyopathy. Recently, by the use of intact recombinant human beta-AR, we were able to confirm the existence of functionally active anti-beta-1-AR autoantibodies in patients with dilated cardiomyopathy (26% prevalence) or ischemic cardiomyopathy (10% prevalence); however, their prevalence in other (secondary) cardiomyopathies remained to be determined. METHODS: Immunoglobulin G (IgG) was prepared from the sera of 28 VHD and 19 HHD patients and first screened by a peptide-based enzyme-linked immunosorbent assay (antigens: aminoterminus, second extracellular loop [ECII] and carboxyterminus of human beta-1- and beta-2-AR). IgG from 108 gender- and age-matched healthy subjects served to define the threshold for positive immunoreactions. Positive sera were further screened for their ability to recognize and activate native human beta-AR situated in a cell membrane. RESULTS: Twenty-five percent (VHD) or 11% (HHD) of the patients and 4% of the healthy controls had IgG antibodies randomly directed against all the three domains tested and both beta-AR subtypes. Only one patient with aortic valve and concomitant coronary heart disease and one healthy subject had functionally active anti-b1-AR (targeting beta-1-ECII). Moreover, one HHD patient with concomitant collagenosis had IgG that was cross-reacting with recombinant beta-AR in immunological assays but was unable to affect receptor function. CONCLUSIONS: Autoimmune reactions against the human beta-AR do not appear to be associated with cardiomyopathies secondary to VHD or HHD.
Assuntos
Autoanticorpos , Cardiomiopatias/imunologia , Doenças das Valvas Cardíacas/complicações , Hipertensão/complicações , Receptores Adrenérgicos beta/imunologia , Idoso , Autoimunidade , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: Our study attempted to gain further understanding of the allosteric effects of human autoantibodies on beta1-adrenergic receptor (beta1-AR) function. BACKGROUND: Recently, we reported on the existence of activating anti-beta1-AR antibodies in patients with dilated cardiomyopathy (DCM 26% prevalence) or ischemic cardiomyopathy (ICM, 10% prevalence); however, their functional effects have not yet been thoroughly characterized. METHODS: In this study we detected functionally active receptor-antibodies in 8 out of 30 DCM patients. Their immunological and functional properties were analyzed using both synthetic receptor-peptides and intact recombinant human beta1-AR, and were compared with those of heterologous antibodies to selected beta1-AR domains generated in rabbits and mice. RESULTS: Rabbit, mouse, and human anti-beta1-AR against the second extracellular domain preferentially bound to a native receptor conformation and impaired radioligand binding to the receptor. However, their functional effects differed considerably: Rabbit and mouse antibodies decreased both basal and agonist-stimulated cAMP production, whereas the patient antibodies (n = 8) increased basal, and six of them also increased agonist-stimulated receptor activity (i.e., acted as receptor-sensitizing agents). Two out of eight human anti-beta1-AR increased basal but decreased agonist-stimulated receptor activity (i.e., acted as partial agonists). CONCLUSIONS: Antibodies against the same small beta1-AR domain can have very divergent allosteric effects, ranging from inhibitory to agonist-promoting activities. Activating autoantibodies were associated with severe cardiac dysfunction and thus might be involved in the development and/or course of human cardiomyopathy.