RESUMEN
Low levels of the molecular inotrope S100A1 are sufficient to rescue post-ischemic heart failure (HF). As a prerequisite to clinical application and to determine the safety of myocardial S100A1 DNA-based therapy, we investigated the effects of high myocardial S100A1 expression levels on the cardiac contractile function and occurrence of arrhythmia in a preclinical large animal HF model. At 2 weeks after myocardial infarction domestic pigs presented significant left ventricular (LV) contractile dysfunction. Retrograde application of AAV6-S100A1 (1.5 × 10(13) tvp) via the anterior cardiac vein (ACV) resulted in high-level myocardial S100A1 protein peak expression of up to 95-fold above control. At 14 weeks, pigs with high-level myocardial S100A1 protein overexpression did not show abnormalities in the electrocardiogram. Electrophysiological right ventricular stimulation ruled out an increased susceptibility to monomorphic ventricular arrhythmia. High-level S100A1 protein overexpression in the LV myocardium resulted in a significant increase in LV ejection fraction (LVEF), albeit to a lesser extent than previously reported with low S100A1 protein overexpression. Cardiac remodeling was, however, equally reversed. High myocardial S100A1 protein overexpression neither increases the occurrence of cardiac arrhythmia nor causes detrimental effects on myocardial contractile function in vivo. In contrast, this study demonstrates a broad therapeutic range of S100A1 gene therapy in post-ischemic HF using a preclinical large animal model.
Asunto(s)
Arritmias Cardíacas/terapia , Terapia Genética/efectos adversos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/terapia , Infarto del Miocardio/complicaciones , Isquemia Miocárdica/complicaciones , Miocardio/metabolismo , Proteínas S100/uso terapéutico , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/fisiopatología , Humanos , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/terapia , Isquemia Miocárdica/fisiopatología , Isquemia Miocárdica/terapia , Miocardio/patología , Proteínas S100/genética , Proteínas S100/metabolismo , Volumen Sistólico/fisiología , PorcinosRESUMEN
Heart failure (HF) is a common pathological end point for several cardiac diseases. Despite reasonable achievements in pharmacological, electrophysiological and surgical treatments, prognosis for chronic HF remains poor. Modern therapies are generally symptom oriented and do not currently address specific intracellular molecular signaling abnormalities. Therefore, new and innovative therapeutic approaches are warranted and, ideally, these could at least complement established therapeutic options if not replace them. Gene therapy has potential to serve in this regard in HF as vectors can be directed toward diseased myocytes and directly target intracellular signaling abnormalities. Within this review, we will dissect the adrenergic system contributing to HF development and progression with special emphasis on G-protein-coupled receptor kinase 2 (GRK2). The levels and activity of GRK2 are increased in HF and we and others have demonstrated that this kinase is a major molecular culprit in HF. We will cover the evidence supporting gene therapy directed against myocardial as well as adrenal GRK2 to improve the function and structure of the failing heart and how these strategies may offer complementary and synergistic effects with the existing HF mainstay therapy of ß-adrenergic receptor antagonism.
Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Marcación de Gen/métodos , Terapia Genética/métodos , Insuficiencia Cardíaca/terapia , Antagonistas Adrenérgicos beta/uso terapéutico , Terapia Combinada , Quinasa 2 del Receptor Acoplado a Proteína-G/fisiología , Insuficiencia Cardíaca/fisiopatología , HumanosRESUMEN
We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy.
Asunto(s)
Procedimientos Quirúrgicos Cardíacos/métodos , Dependovirus/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , Miocardio , Animales , Puente Cardiopulmonar , Citomegalovirus , Proteínas Fluorescentes Verdes/genética , Miocardio/metabolismo , OvinosRESUMEN
Hormones and neurotransmitters may mediate common responses through receptors that couple to the same class of heterotrimeric guanine nucleotide-binding (G) protein. For example, several receptors that couple to Gq class proteins can induce cardiomyocyte hypertrophy. Class-specific inhibition of Gq-mediated signaling was produced in the hearts of transgenic mice by targeted expression of a carboxyl-terminal peptide of the alpha subunit Galphaq. When pressure overload was surgically induced, the transgenic mice developed significantly less ventricular hypertrophy than control animals. The data demonstrate the role of myocardial Gq in the initiation of myocardial hypertrophy and indicate a possible strategy for preventing pathophysiological signaling by simultaneously blocking multiple receptors coupled to Gq.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Hipertrofia Ventricular Izquierda/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Angiotensina II/farmacología , Animales , Factor Natriurético Atrial/genética , Células COS , Diglicéridos/metabolismo , Activación Enzimática , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica , Marcación de Gen , Hipertrofia Ventricular Izquierda/prevención & control , Fosfatos de Inositol/metabolismo , Ratones , Ratones Transgénicos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fenilefrina/farmacología , Transducción de Señal , Transfección , Transgenes , Presión VentricularRESUMEN
Transgenic mice were created with cardiac-specific overexpression of the beta-adrenergic receptor kinase-1 (beta ARK1) or a beta ARK inhibitor. Animals overexpressing beta ARK1 demonstrated attenuation of isoproterenol-stimulated left ventricular contractility in vivo, dampening of myocardial adenylyl cyclase activity, and reduced functional coupling of beta-adrenergic receptors. Conversely, mice expressing the beta ARK inhibitor displayed enhanced cardiac contractility in vivo with or without isoproterenol. These animals demonstrate the important role of beta ARK in modulating in vivo myocardial function. Because increased amounts of beta ARK1 and diminished cardiac beta-adrenergic responsiveness characterize heart failure, these animals may provide experimental models to study the role of beta ARK in heart disease.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Corazón/fisiología , Contracción Miocárdica , Miocardio/enzimología , Receptores Adrenérgicos beta/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Presión Sanguínea , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas de Unión al GTP/metabolismo , Isoproterenol/farmacología , Ratones , Ratones Transgénicos , Contracción Miocárdica/efectos de los fármacos , Fenotipo , Sarcolema/enzimología , Presión Ventricular , Quinasas de Receptores Adrenérgicos betaRESUMEN
Complementary DNAs were isolated and used to deduce the primary structures of the alpha 1 and alpha 2 subunits of the dihydropyridine-sensitive, voltage-dependent calcium channel from rabbit skeletal muscle. The alpha 1 subunit, which contains putative binding sites for calcium antagonists, is a hydrophobic protein with a sequence that is consistent with multiple transmembrane domains and shows structural and sequence homology with other voltage-dependent ion channels. In contrast, the alpha 2 subunit is a hydrophilic protein without homology to other known protein sequences. Nucleic acid hybridization studies suggest that the alpha 1 and alpha 2 subunit mRNAs are expressed differentially in a tissue-specific manner and that there is a family of genes encoding additional calcium channel subtypes.
Asunto(s)
Calcio/metabolismo , ADN , Canales Iónicos , Mapeo Peptídico , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bloqueadores de los Canales de Calcio/farmacología , Clonación Molecular , Enzimas de Restricción del ADN , Dihidropiridinas/farmacología , Canales Iónicos/efectos de los fármacos , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/biosíntesis , Conejos , Homología de Secuencia de Ácido NucleicoRESUMEN
G-protein-coupled receptor signaling and receptor tyrosine kinase (RTK) signaling are two mechanisms of transmembrane communication used by numerous extracellular agents and stimuli. The beta gamma-subunit complex of G proteins mediates many of the functions associated with G-protein-coupled receptor signaling and may even provide a means to link G proteins to RTK-initiated cascades. This connection may be mediated by the pleckstrin homology domain, a modular domain found in many signaling proteins that interact with G beta gamma.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Fosfoproteínas , Animales , Sitios de Unión , Proteínas Sanguíneas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al GTP/química , Humanos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Proteínas ras/metabolismoRESUMEN
Driven, at least in part, by the National Institutes of Health roadmap, an increasing number of studies has bridged the chasm between observations in the basic research laboratory and the clinical bedside. These studies have been an integral part in "translating" new discoveries into therapeutic initiatives. However, "translational medicine" has been used less frequently in the development of cardiovascular drugs or in predicting the potential cardiovascular toxicity of non-cardiac agents. Studies in animal models can provide important clues as to the potential cardiotoxicity of new therapeutic agents, as well as providing a template for the rational design of clinical trials. Three examples of drug development programs that might have been altered by clues available from laboratory studies include the development programs for the anti-cancer drug trastuzumab, the cyclooxygenase inhibitors, and the adenosine-receptor agonists and antagonists. Although mouse models may not always represent the physiology of humans, they provide important information that clinical scientists can utilize in designing safe programs for the evaluation of new pharmacologic agents.
Asunto(s)
Investigación Biomédica/organización & administración , Enfermedades Cardiovasculares/inducido químicamente , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Ensayos Clínicos como Asunto , Inhibidores de la Ciclooxigenasa 2/efectos adversos , Inhibidores de la Ciclooxigenasa 2/farmacología , Difusión de Innovaciones , Evaluación Preclínica de Medicamentos , Humanos , Antagonistas de Receptores Purinérgicos P1 , TrastuzumabRESUMEN
Our laboratory has been testing the hypothesis that genetic modulation of the beta-adrenergic signaling cascade can enhance cardiac function. We have previously shown that transgenic mice with cardiac overexpression of either the human beta2-adrenergic receptor (beta2AR) or an inhibitor of the beta-adrenergic receptor kinase (betaARK), an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial inotropy. We now have created recombinant adenoviruses encoding either the beta2AR (Adeno-beta2AR) or a peptide betaARK inhibitor (consisting of the carboxyl terminus of betaARK1, Adeno-betaARKct) and tested their ability to potentiate beta-adrenergic signaling in cultured adult rabbit ventricular myocytes. As assessed by radioligand binding, Adeno-beta2AR infection led to approximately 20-fold overexpression of beta-adrenergic receptors. Protein immunoblots demonstrated the presence of the Adeno-betaARKct transgene. Both transgenes significantly increased isoproterenol-stimulated cAMP as compared to myocytes infected with an adenovirus encoding beta-galactosidase (Adeno-betaGal) but did not affect the sarcolemmal adenylyl cyclase response to Forskolin or NaF. beta-Adrenergic agonist-induced desensitization was significantly inhibited in Adeno-betaARKct-infected myocytes (16+/-2%) as compared to Adeno-betaGal-infected myocytes (37+/-1%, P < 0.001). We conclude that recombinant adenoviral gene transfer of the beta2AR or an inhibitor of betaARK-mediated desensitization can potentiate beta-adrenergic signaling.
Asunto(s)
Adenoviridae/genética , Técnicas de Transferencia de Gen , Ventrículos Cardíacos/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Adenilil Ciclasas/análisis , Agonistas Adrenérgicos beta/farmacología , Animales , Supervivencia Celular , Células Cultivadas , AMP Cíclico/metabolismo , Vectores Genéticos , Ventrículos Cardíacos/citología , Humanos , Isoproterenol/farmacología , Masculino , Conejos , Sarcolema/enzimología , Transducción de Señal , TransgenesRESUMEN
To circumvent the embryonic lethality of a complete deficiency in insulin-like growth factor 1 (IGF-1), we generated mice homozygous for a site-specific insertional event that created a mutant IGF-1 allele (igf1m). These mice have IGF-1 levels 30% of wild type yet survive to adulthood, thereby allowing physiological analysis of the phenotype. Miniaturized catheterization technology revealed elevated conscious blood pressure in IGF-1(m/m) mice, and measurements of left ventricular contractility were increased. Adenylyl cyclase activity was enhanced in IGF-1(m/m) hearts, without an increase in beta-adrenergic receptor density, suggesting that crosstalk between IGF-1 and beta-adrenergic signaling pathways may mediate the increased contractility. The hypertrophic response of the left ventricular myocardium in response to aortic constriction, however, was preserved in IGF-1(m/m) mice. We conclude that chronic alterations in IGF-1 levels can selectively modulate blood pressure and left ventricular function, while not affecting adaptive myocardial hypertrophy in vivo.
Asunto(s)
Adenilil Ciclasas/metabolismo , Presión Sanguínea , Hormona del Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/deficiencia , Factor I del Crecimiento Similar a la Insulina/genética , Mutagénesis Insercional , Contracción Miocárdica , Hipófisis/metabolismo , Receptores Adrenérgicos beta/metabolismo , Alelos , Animales , Glucemia/metabolismo , Cartilla de ADN , Femenino , Hormona del Crecimiento/sangre , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Masculino , Ratones , Ratones Mutantes , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Caracteres SexualesRESUMEN
While an age-associated diminution in myocardial contractile response to beta-adrenergic receptor (beta-AR) stimulation has been widely demonstrated to occur in the context of increased levels of plasma catecholamines, some critical mechanisms that govern beta-AR signaling must still be examined in aged hearts. Specifically, the contribution of beta-AR subtypes (beta1 versus beta2) to the overall reduction in contractile response with aging is unknown. Additionally, whether G protein-coupled receptor kinases (GRKs), which mediate receptor desensitization, or adenylyl cyclase inhibitory G proteins (Gi) are increased with aging has not been examined. Both these inhibitory mechanisms are upregulated in chronic heart failure, a condition also associated with diminished beta-AR responsiveness and increased circulatory catecholamines. In this study, the contractile responses to both beta1-AR and beta2-AR stimulation were examined in rat ventricular myocytes of a broad age range (2, 8, and 24 mo). A marked age-associated depression in contractile response to both beta-AR subtype stimulation was observed. This was associated with a nonselective reduction in the density of both beta-AR subtypes and a reduction in membrane adenylyl cyclase response to both beta-AR subtype agonists, NaF or forskolin. However, the age-associated diminutions in contractile responses to either beta1-AR or beta2-AR stimulation were not rescued by inhibiting Gi with pertussis toxin treatment. Further, the abundance or activity of beta-adrenergic receptor kinase, GRK5, or Gi did not significantly change with aging. Thus, we conclude that the positive inotropic effects of both beta1- and beta2-AR stimulation are markedly decreased with aging in rat ventricular myocytes and this is accompanied by decreases in both beta-AR subtype densities and a reduction in membrane adenylate cyclase activity. Neither GRKs nor Gi proteins appear to contribute to the age-associated reduction in cardiac beta-AR responsiveness.
Asunto(s)
Envejecimiento/metabolismo , Proteínas de Unión al GTP/metabolismo , Miocardio/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Toxina de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Membrana Celular/metabolismo , Colforsina/farmacología , Etanolaminas/farmacología , Proteínas de Unión al GTP/fisiología , Imidazoles/farmacología , Immunoblotting , Miocardio/citología , Norepinefrina/farmacología , Toxina del Pertussis , Reacción en Cadena de la Polimerasa , ARN/análisis , ARN/metabolismo , Ratas , Ratas Wistar , Fluoruro de Sodio/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including beta-adrenergic receptor (betaAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in betaAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the beta(2)AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the betaAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.
Asunto(s)
Señalización del Calcio , Cardiomiopatía Hipertrófica/fisiopatología , Receptores Adrenérgicos beta 2/metabolismo , Actinas/genética , Animales , Factor Natriurético Atrial/genética , Biomarcadores , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Expresión Génica , Insuficiencia Cardíaca/patología , Masculino , Ratones , Ratones Transgénicos , Actividad Motora , Miocardio/metabolismo , Miocardio/patología , Cadenas Pesadas de Miosina/genética , Receptores Adrenérgicos beta 2/genética , Quinasas de Receptores Adrenérgicos betaRESUMEN
Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.
Asunto(s)
Adenoviridae/genética , Terapia Genética , Vectores Genéticos/genética , Insuficiencia Cardíaca/terapia , Miocardio/metabolismo , Receptores Adrenérgicos beta 2/genética , Agonistas Adrenérgicos beta/farmacología , Agonistas Adrenérgicos beta/uso terapéutico , Animales , Cateterismo Cardíaco , Células Cultivadas , Vasos Coronarios , Regulación de la Expresión Génica , Insuficiencia Cardíaca/tratamiento farmacológico , Pruebas de Función Cardíaca , Humanos , Inyecciones Intraarteriales , Isoproterenol/farmacología , Isoproterenol/uso terapéutico , Masculino , Conejos , Receptores Adrenérgicos beta 2/efectos de los fármacos , Receptores Adrenérgicos beta 2/fisiología , Transducción de SeñalRESUMEN
S100A1, a Ca superset2+-binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been shown to enhance cardiac contractile performance by regulating both sarcoplasmic reticulum (SR) Ca superset2+-handling and myofibrillar Ca superset2+-responsiveness. In cardiac disease, the expression of S100A1 is dynamically altered as it is significantly down-regulated in end stage human heart failure (HF), and it is up-regulated in compensated hypertrophy. Therefore, the delivery of a transgene encoding for S100A1 to the myocardium might be an attractive strategy for improving cardiac function in HF by replacing lost endogenous S100A1. In this study we sought to test whether exogenous S100A1 gene delivery to alter global cardiac function is feasible in the normal rabbit heart. An adenoviral S100A1 transgene (AdvS100A1) also containing the green fluorescent protein (GFP) was delivered using an intracoronary injection method with a dose of 5 x 10 superset11 total virus particles (tvp) (n = 8). Rabbits treated with either a GFP-only adenovirus (AdvGFP) or saline were used as control groups (n = 11 each). Seven days after global myocardial in vivo gene delivery hemodynamic parameters were assessed. S100A1 overexpression as a result of the intracoronary delivery of AdvS100A1 significantly increased left ventricular (LV) +dP/dt subsetmax, -dP/dt subsetmin and systolic ejection pressure (SEP) compared to both control groups after administration of isoproterenol (0.1, 0.5 and 1.0 microg/kgBW/min), while contractile parameters remained unchanged under basal conditions. These results demonstrate that global myocardial in vivo gene delivery is possible and that myocardial S100A1 overexpression can increase cardiac performance. Therefore, substitution of down-regulated S100A1 protein expression levels may represent a potential therapeutic strategy for improving the cardiac performance of the failing heart.
Asunto(s)
Técnicas de Transferencia de Gen , Contracción Miocárdica/genética , Miocardio/metabolismo , Proteínas S100/genética , Adenoviridae , Animales , Vectores Genéticos , Proteínas Fluorescentes Verdes , Inyecciones , Masculino , Conejos , Proteínas S100/metabolismo , Función Ventricular IzquierdaRESUMEN
BACKGROUND: Understanding the cellular signals that initiate cardiac hypertrophy is of critical importance in identifying the pathways that mediate heart failure. The family of mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated kinases (ERKs), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPKs, may play specific roles in myocardial growth and function. METHODS AND RESULTS: To determine the mechanism of activation of MAPK pathways during the development of cardiac hypertrophy, we evaluated the induction of MAPK activity after aortic constriction in wild-type and in 2 types of cardiac gene-targeted mice: one overexpressing a carboxyl-terminal peptide of Galpha(q) that inhibits G(q)-mediated signaling (TG GqI mouse) and another overexpressing a carboxyl-terminal peptide of beta-adrenergic receptor kinase-1 that inhibits Gbetagamma signaling (TG betaARKct mouse). Wild-type mice with pressure overload showed an acute induction of JNK, followed by the induction of p38/p38beta at 3 days and ERK at 7 days. Both JNK and p38 activity remained elevated at 7 days after banding. In TG GqI mice, hypertrophy was significantly attenuated, and induction of ERK and JNK activity was abolished, whereas the induction of p38 and p38beta was robust, but delayed. By contrast, all 3 MAPK pathways were activated by aortic constriction in the TG betaARKct hearts, suggesting a role for Galpha(q), but not Gbetagamma. CONCLUSIONS: Taken together, these data show that the induction of ERK and JNK activity in in vivo pressure-overload hypertrophy is mediated through the stimulation of G(q)-coupled receptors and that non-G(q)-mediated pathways are recruited to activate p38 and p38beta.
Asunto(s)
Cardiomegalia/enzimología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/enzimología , Animales , Cardiomegalia/metabolismo , Modelos Animales de Enfermedad , Inducción Enzimática , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Quinasa 11 Activada por Mitógenos , Péptidos , Presión , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
BACKGROUND: Acute cardiac contractile dysfunction is common after cardiopulmonary bypass (CPB). A potential molecular mechanism is enhanced beta-adrenergic receptor kinase (betaARK1) activity, because beta-adrenergic receptor (betaAR) signaling is altered in cardiomyocytes after cardioplegia. Therefore, we examined whether adenovirus-mediated intracoronary delivery of a betaARK1 inhibitor (Adv-betaARKct) could prevent post-CPB dysfunction. METHODS AND RESULTS: Rabbits were randomized to receive 5x10(11) total viral particles of Adv-betaARKct or PBS. After 5 days, hearts were arrested with University of Wisconsin solution, excised, and stored at 4 degrees C for 15 minutes or 4 hours before reperfusion on a Langendorff apparatus. Left ventricular (LV) function measured by end-diastolic pressure response to preload augmentation, contractility (LV dP/dt(max)), and relaxation (LV dP/dt(min)) was assessed by use of increasing doses of isoproterenol and compared with a control group of nonarrested hearts acutely perfused on the Langendorff apparatus. In the PBS-treated hearts, LV function decreased in a temporal manner and was significantly impaired compared with control hearts after 4 hours of cardioplegic arrest. LV function in Adv-betaARKct-treated hearts, however, was significantly enhanced compared with PBS treatment and was similar to control nonarrested hearts even after 4 hours of cardioplegia. Biochemically, several aspects of betaAR signaling were dysfunctional in PBS-treated hearts, whereas they were normalized in betaARKct-overexpressing hearts. CONCLUSIONS: Myocardial gene transfer of Adv-betaARKct stabilizes betaAR signaling and prevents LV dysfunction induced by prolonged cardioplegic arrest. Thus, betaARK1 inhibition may represent a novel target in limiting depressed ventricular function after CPB.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/administración & dosificación , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Terapia Genética/métodos , Paro Cardíaco Inducido , Fragmentos de Péptidos/administración & dosificación , Proteínas Recombinantes , Disfunción Ventricular/prevención & control , Adenoviridae/genética , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Paro Cardíaco Inducido/efectos adversos , Hemodinámica/efectos de los fármacos , Técnicas In Vitro , Masculino , Contracción Miocárdica/efectos de los fármacos , Reperfusión Miocárdica , Miocardio/metabolismo , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , ARN Mensajero/metabolismo , Conejos , Resultado del Tratamiento , Disfunción Ventricular/etiología , Función Ventricular Izquierda/efectos de los fármacos , Quinasas de Receptores Adrenérgicos betaRESUMEN
BACKGROUND: Transgenic mice with constitutive myocardium-targeted expression of a peptide inhibitor of the ss-adrenergic receptor kinase (ssARKct) have increased in vivo cardiac function and enhanced ss-adrenergic receptor (ssAR) responsiveness. METHODS AND RESULTS: In the present study, we created transgenic mice with myocardium-targeted ssARKct transgene expression under control of the CARP (cardiac ankyrin repeat protein) promoter, which is active during cardiac development and inactive in the normal adult mouse heart. Consistent with this, adult CARP-ssARKct transgenic mice have normal in vivo cardiac contractility and ssAR responsiveness indistinguishable from their nontransgenic littermates (NLCs). However, because CARP is in a group of fetal genes activated in the adult ventricle during hypertrophy, we subjected animals to transverse aortic constriction (TAC) to induce pressure overload. Seven days after TAC, CARP-ssARKct hearts had elevations in left ventricular mass similar to those in NLCs; however, TAC did induce demonstrable ssARKct expression in the transgenic hearts. TAC in NLC mice resulted in an upregulation of myocardial ssARK1 and a loss of ssAR-mediated inotropic reserve. Importantly, although ssARK1 was increased in the hypertrophic CARP-ssARKct mice, the in vivo loss of ssAR responsiveness was not seen after induced ssARKct expression. CONCLUSIONS: These results demonstrate that acute ssARK1 inhibition can restore lost myocardial ssAR responsiveness and inotropic reserve in vivo. Furthermore, these mice demonstrate the novel utility of the CARP promoter as an inducible element responsive to pathophysiological conditions in the adult heart.
Asunto(s)
Cardiomegalia/fisiopatología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Receptores Adrenérgicos beta/metabolismo , Transgenes/fisiología , Animales , Cardiomegalia/etiología , Constricción Patológica/complicaciones , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Expresión Génica , Corazón/fisiopatología , Hemodinámica , Masculino , Ratones , Ratones Transgénicos , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Miocardio/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transgenes/genética , Quinasas de Receptores Adrenérgicos betaRESUMEN
BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Insuficiencia Cardíaca/enzimología , Fragmentos de Péptidos/fisiología , Procesamiento Proteico-Postraduccional , Receptores Adrenérgicos beta/fisiología , Proteínas Recombinantes , Adenilil Ciclasas/fisiología , Animales , Cateterismo Cardíaco , Cardiotónicos/farmacología , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Inducción Enzimática , Estudios de Factibilidad , Regulación de la Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Isoproterenol/farmacología , Ratones , Ratones Transgénicos , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/genética , Fragmentos de Péptidos/genética , Fosforilación , Sistemas de Mensajero Secundario , Fluoruro de Sodio/farmacología , Transgenes , Función Ventricular Izquierda/efectos de los fármacos , Quinasas de Receptores Adrenérgicos betaRESUMEN
BACKGROUND: Elevated circulating norepinephrine (NE) has been implicated in causing the profound beta-adrenergic receptor (betaAR) downregulation and receptor uncoupling that are characteristic of end-stage human dilated cardiomyopathy, a process mediated in part by increased levels of beta-adrenergic receptor kinase (betaARK1). To explore whether chronic sustained NE stimulation is a primary stimulus that promotes deterioration in cardiac signaling, we characterized a gene-targeted mouse in which activation of the sympathetic nervous system cannot lead to an elevation in plasma NE and epinephrine. METHODS AND RESULTS: Gene-targeted mice that lack dopamine beta-hydroxylase (dbh-/-), the enzyme needed to convert dopamine to NE, were created by homologous recombination. In vivo contractile response to the beta1AR agonist dobutamine, measured by a high-fidelity left ventricular micromanometer, was enhanced in mice lacking the dbh gene. In unloaded adult myocytes isolated from dbh-/- mice, basal contractility was significantly increased compared with control cells. Furthermore, the increase in betaAR responsiveness and enhanced cellular contractility were associated with a significant reduction in activity and protein level of betaARK1 and increased high-affinity agonist binding without changes in betaAR density or G-protein levels. CONCLUSIONS: Mice that lack the ability to generate NE or epinephrine show increased contractility associated primarily with a decrease in the level of betaARK1 protein and kinase activity. This animal model will be valuable in testing whether NE is required for the pathogenesis of heart failure through mating strategies that cross the dbh-/- mouse into genetically engineered models of heart failure.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Epinefrina/deficiencia , Contracción Miocárdica/fisiología , Norepinefrina/deficiencia , Agonistas Adrenérgicos beta/farmacología , Animales , Dobutamina/farmacología , Dopamina beta-Hidroxilasa/genética , Femenino , Proteínas de Unión al GTP/metabolismo , Marcación de Gen , Hemodinámica/efectos de los fármacos , Hemodinámica/fisiología , Masculino , Ratones , Ratones Mutantes , Contracción Miocárdica/efectos de los fármacos , Receptores Adrenérgicos beta/metabolismo , Sarcolema/metabolismo , Quinasas de Receptores Adrenérgicos betaRESUMEN
BACKGROUND: Cardiac gene therapy offers the possibility of enhancing myocardial performance in the compromised heart. However, current gene delivery techniques have limited myocardial transgene expression and pose the risk of extracardiac expression. Isolation of the coronary circulation during cardiac surgery may allow for more efficient and cardiac-selective gene delivery in a clinically relevant model. Methods and Results-- Neonatal piglets (3 kg) underwent a median sternotomy and cardiopulmonary bypass, followed by aortic cross-clamping with 30 minutes of cardioplegic arrest. Adenoviral vectors containing transgenes for either beta-galactosidase (adeno-beta-gal, n=11) or the human beta(2)-adrenergic receptor (adeno-beta(2)-AR, n=15) were administered through the cardioplegia cannula immediately after arrest and were allowed to dwell in the coronary circulation during the cross-clamp period. After 1 week, the animals were killed, and their heart, lungs, and liver were excised and examined for gene expression. Analysis of beta-galactosidase staining revealed transmural myocardial gene expression among animals receiving adeno-beta-gal. No marker gene expression was detected in liver or lung tissue. beta-AR density in the left ventricle after adeno-beta(2)-AR delivery was 396+/-85% of levels in control animals (P<0.01). Animals receiving adeno-beta(2)-AR and control animals demonstrated similar beta-AR density in both the liver (114+/-8% versus 100+/-9%, P=NS) and lung (114+/-7% versus 100+/-9%, P=NS). There was no evidence of cardiac inflammation. CONCLUSIONS: By using cardiopulmonary bypass and cardioplegic arrest, intracoronary delivery of adenoviral vectors resulted in efficient myocardial uptake and expression. Undetectable transgene expression in liver or lung tissue suggests cardiac-selective expression.