RESUMEN
Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.
Asunto(s)
Distrofina/genética , Mutación del Sistema de Lectura , Edición Génica/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , ARN Guía de Kinetoplastida/genética , Animales , Modelos Animales de Enfermedad , Exones , Femenino , Regulación de la Expresión Génica , Terapia Genética , Genoma , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/terapia , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Espectrometría de Masas , Músculo Esquelético/metabolismo , Músculos/metabolismo , Mioblastos/metabolismo , Miocitos Cardíacos/metabolismo , Proteoma , PorcinosRESUMEN
BACKGROUND AND AIMS: Whether gamma-glutamyl transferase (GGT) or alkaline phosphatase (ALP) is a better prognostic marker in patients with coronary heart disease (CHD) remains unknown. The aim of this study was to compare the prognostic value of GGT and ALP in patients with CHD. METHODS AND RESULTS: This study included 3768 patients with CHD. The main study outcome was 3-year all-cause mortality. The median values of GGT and ALP were 36.2 U/L and 69.3 U/L. Patients were divided into subgroups according to GGT or ALP activity > or ≤median. Overall, there were 304 deaths: 195 deaths occurred in patients with GGT >median (n = 1882) and 109 deaths occurred in patients with GGT ≤median (n = 1886); Kaplan-Meier [KM] estimates of all-cause mortality were 11.9% and 6.4% (unadjusted hazard ratio [HR] = 1.85, 95% confidence interval [CI], 1.46 to 2.34]; P < 0.001). According to ALP activity, 186 deaths occurred in patients with ALP >median (n = 1883) and 118 deaths occurred in patients with ALP ≤median (n = 1885); KM estimates of all-cause mortality were 11.4% and 7.1% (unadjusted HR = 1.64 [1.30-2.06]; P < 0.001). After adjustment, GGT (adjusted HR = 1.32 [1.11-1.58]; P = 0.002) but not ALP (adjusted HR = 1.20 [1.00-1.43]; P = 0.051, with both HR calculated per 1 unit increment in logarithmic GGT or ALP scale) remained significantly associated with the risk for mortality. The C statistic of the mortality model with GGT was greater than the C statistic of the model with ALP (0.831 [0.802-0.859] vs. 0.826 [0.793-0.855]; P < 0.001). CONCLUSIONS: In patients with CHD, GGT was a stronger correlate of all-cause mortality than ALP.
Asunto(s)
Fosfatasa Alcalina/sangre , Pruebas Enzimáticas Clínicas , Enfermedad Coronaria/diagnóstico , gamma-Glutamiltransferasa/sangre , Anciano , Biomarcadores/sangre , Causas de Muerte , Enfermedad Coronaria/sangre , Enfermedad Coronaria/mortalidad , Enfermedad Coronaria/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/diagnóstico , Infarto del Miocardio/mortalidad , Intervención Coronaria Percutánea , Valor Predictivo de las Pruebas , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/mortalidad , Factores de TiempoRESUMEN
OBJECTIVES: We aimed to analyze angiographic and clinical results of patients undergoing BRS implantation in a real-world setting. BACKGROUND: Angiographic and clinical outcome data from patients undergoing implantation of drug-eluting bioresorbable stents (BRS) in routine clinical practice is scant. METHODS: Consecutive patients undergoing implantation of everolimus-eluting BRS at two high-volume centers in Munich, Germany were enrolled. Data were collected prospectively. All patients were scheduled for angiographic surveillance 6-8 months after stent implantation. Quantitative coronary angiographic analysis was performed in a core laboratory. Clinical follow-up was performed to 12 months and events were adjudicated by independent assessors. RESULTS: A total of 419 patients were studied. Mean age was 66.6 ± 10.9 years, 31.5% had diabetes mellitus, 76.1% had multivessel disease, and 39.0% presented with acute coronary syndrome; 49.0% of lesions were AHA/ACC type B2/C, 13.1% had treatment of bifurcation lesions. Mean reference vessel diameter was 2.89 ± 0.46 mm. At angiographic follow-up in-stent late loss was 0.26 ± 0.51 mm, in-segment diameter stenosis was 27.5 ± 16.1, and binary angiographic restenosis was 7.5%. At 12 months, the rate of death, myocardial infarction, or target lesion revascularization was 13.1%. Definite stent thrombosis occurred in 2.6%. CONCLUSIONS: The use of everolimus-eluting BRS in routine clinical practice is associated with high antirestenotic efficacy in patients undergoing angiographic surveillance. Overall clinical outcomes at 12 months are satisfactory though stent thrombosis rates are not insignificant. Further study with longer term follow-up and larger numbers of treated patients is required before we can be sure of the role of these devices in clinical practice.
Asunto(s)
Implantes Absorbibles , Síndrome Coronario Agudo/terapia , Fármacos Cardiovasculares/administración & dosificación , Angiografía Coronaria , Enfermedad de la Arteria Coronaria/terapia , Vasos Coronarios/efectos de los fármacos , Stents Liberadores de Fármacos , Everolimus/administración & dosificación , Intervención Coronaria Percutánea/instrumentación , Síndrome Coronario Agudo/diagnóstico por imagen , Síndrome Coronario Agudo/mortalidad , Anciano , Fármacos Cardiovasculares/efectos adversos , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/mortalidad , Reestenosis Coronaria/diagnóstico por imagen , Reestenosis Coronaria/etiología , Reestenosis Coronaria/prevención & control , Trombosis Coronaria/diagnóstico por imagen , Trombosis Coronaria/etiología , Trombosis Coronaria/prevención & control , Vasos Coronarios/diagnóstico por imagen , Everolimus/efectos adversos , Femenino , Alemania , Hospitales de Alto Volumen , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/etiología , Infarto del Miocardio/prevención & control , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/mortalidad , Valor Predictivo de las Pruebas , Diseño de Prótesis , Sistema de Registros , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoAsunto(s)
Complejos de Coordinación/farmacocinética , Imagen por Resonancia Magnética/métodos , Infarto del Miocardio/diagnóstico , Infarto del Miocardio/metabolismo , Péptidos Cíclicos/farmacocinética , Tomografía de Emisión de Positrones/métodos , Receptores CXCR4/metabolismo , Biomarcadores/metabolismo , Humanos , Interpretación de Imagen Asistida por Computador/métodos , Masculino , Persona de Mediana Edad , Imagen Molecular/métodos , Imagen Multimodal/métodos , Miocardio/metabolismo , Radiofármacos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución Tisular , Regulación hacia ArribaRESUMEN
There is limited clinical data comparing different P2Y12-receptor inhibitors in patients with acute myocardial infarction (AMI) complicated by cardiogenic shock. The aim of the ISAR-SHOCK registry was to compare the clinical outcome of patients treated with clopidogrel vs prasugrel in this setting. Patients (n=145) with AMI complicated by cardiogenic shock and undergoing primary PCI in two centres (Deutsches Herzzentrum München and Klinikum rechts der Isar, Technical University Munich) between January 2009 and May 2012 were included in this registry. The use of prasugrel for patients within this registry reflected co-morbidities and platelet function testing results during the acute AMI phase. Early outcome at 30-days was reported with regard to all-cause mortality, myocardial infarction (MI), stent thrombosis (ST) and bleeding events. With regard to antiplatelet treatment in the 145 cardiogenic shock patients, 50 patients were initially treated or immediately switched to prasugrel while 95 patients were treated with clopidogrel. All-cause mortality was lower in prasugrel- vs clopidogrel-treated patients (30 % vs 50.5%, HR: 0.51, 95% CI [0.29-0.92], p=0.025). No significant differences in prasugrel- vs clopidogrel-treated patients were observed for the occurrence of MI (p=0.233), ST (p=0.306) or TIMI major bleedings (p=0.571). Results of the ISAR-SHOCK registry suggest that the use of prasugrel in AMI patients complicated by cardiogenic shock might be associated with a lower mortality risk as compared to clopidogrel therapy without increasing the risk of bleeding. These findings, however, need confirmation from specifically designed randomised studies in this high-risk cohort of patients.
Asunto(s)
Plaquetas/efectos de los fármacos , Infarto del Miocardio/terapia , Intervención Coronaria Percutánea , Piperazinas/uso terapéutico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Receptores Purinérgicos P2Y12/efectos de los fármacos , Choque Cardiogénico/etiología , Tiofenos/uso terapéutico , Ticlopidina/análogos & derivados , Anciano , Anciano de 80 o más Años , Plaquetas/metabolismo , Clopidogrel , Trombosis Coronaria/sangre , Trombosis Coronaria/etiología , Trombosis Coronaria/prevención & control , Femenino , Alemania , Hemorragia/inducido químicamente , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/complicaciones , Infarto del Miocardio/diagnóstico , Infarto del Miocardio/mortalidad , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/mortalidad , Piperazinas/efectos adversos , Inhibidores de Agregación Plaquetaria/efectos adversos , Pruebas de Función Plaquetaria , Clorhidrato de Prasugrel , Valor Predictivo de las Pruebas , Antagonistas del Receptor Purinérgico P2Y/efectos adversos , Receptores Purinérgicos P2Y12/sangre , Recurrencia , Sistema de Registros , Factores de Riesgo , Choque Cardiogénico/sangre , Choque Cardiogénico/diagnóstico , Choque Cardiogénico/mortalidad , Tiofenos/efectos adversos , Ticlopidina/efectos adversos , Ticlopidina/uso terapéutico , Factores de Tiempo , Resultado del TratamientoRESUMEN
In clopidogrel-treated patients undergoing percutaneous coronary intervention (PCI), high platelet reactivity (HPR) is associated with a higher risk for thrombotic events including stent thrombosis (ST). A personalised therapy with selective intensification of treatment may improve HPR patients´ outcome in this setting although recent randomised trials are against this hypothesis. The aim of the ISAR-HPR registry was to assess whether clopidogrel-treated HPR patients benefit from selective intensification of P2Y12 receptor inhibition. For the registry, outcomes were compared between two cohorts. We identified 428 clopidogrel treated HPR patients (AU x min ≥468 on the Multiplate analyser) between 2007-2008 (historical control cohort) without a change of treatment based on platelet function (PF) testing results. Between 2009-2011, we identified 571 HPR patients (guided therapy cohort) and used this information for guidance and selective intensification of P2Y12 receptor directed treatment (reloading with clopidogrel, switch to prasugrel, re-testing) in a setting of routine PF testing. The primary outcome was the composite of death from any cause or ST after 30 days. Major bleeding according to TIMI criteria was also monitored. The incidence of the primary outcome was significantly lower in the guided vs the control cohort (7 [1.2%] vs 16 [3.7%] events; HR 0.32, 95% CI 0.13-0.79; p=0.009). The incidence of major bleeding was numerically but not statistically higher in the guided vs the control cohort (1.9 vs 0.7%; p=0.10). In conclusion, present findings are in support for a PF testing guided antiplatelet therapy with selective intensification of P2Y12 receptor inhibition. The issue of personalised antiplatelet treatment warrants further investigation in randomized and well-controlled clinical trials.
Asunto(s)
Plaquetas/efectos de los fármacos , Trombosis Coronaria/prevención & control , Intervención Coronaria Percutánea , Piperazinas/uso terapéutico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Medicina de Precisión , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Tiofenos/uso terapéutico , Ticlopidina/análogos & derivados , Anciano , Anciano de 80 o más Años , Plaquetas/metabolismo , Estudios de Casos y Controles , Clopidogrel , Trombosis Coronaria/etiología , Trombosis Coronaria/mortalidad , Resistencia a Medicamentos , Sustitución de Medicamentos , Femenino , Alemania , Hemorragia/inducido químicamente , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/mortalidad , Piperazinas/efectos adversos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/efectos adversos , Pruebas de Función Plaquetaria , Clorhidrato de Prasugrel , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Antagonistas del Receptor Purinérgico P2Y/efectos adversos , Receptores Purinérgicos P2Y12/sangre , Receptores Purinérgicos P2Y12/efectos de los fármacos , Sistema de Registros , Factores de Riesgo , Tiofenos/efectos adversos , Ticlopidina/efectos adversos , Ticlopidina/uso terapéutico , Factores de Tiempo , Resultado del TratamientoRESUMEN
The purpose of this study was to investigate characteristics of critically ill patients with Stenotrophomonas maltophilia (S. maltophilia) isolated from the respiratory tract, to identify risk factors for S. maltophilia-pneumonia and intensive care unit (ICU) mortality and to analyze antibiotic susceptibility of S. maltophilia. This was a retrospective analysis of 64 medical ICU patients with S. maltophilia in the respiratory tract. Thirty-six patients fulfilled the criteria for diagnosis of pneumonia. A significantly higher lung injury score (LIS) was observed in patients with pneumonia compared to patients with colonization (p=0.010). Independent risk factors for S. maltophilia-pneumonia were higher Sequential Organ Failure Assessment (SOFA) score (p=0.009) and immunosuppression (p=0.014). Patients with S. maltophilia-pneumonia had higher ICU mortality within a 28-day follow-up (p=0.040) and higher hospital mortality (p=0.018) than patients with colonization. The highest antibiotic susceptibility rates were observed to trimethoprim-sulfamethoxazole, tigecycline, and moxifloxacin. Higher SOFA score when S. maltophilia was isolated (p=0.001) and development of renal failure (p=0.021) were independent risk factors for ICU mortality. Higher SOFA score and immunosuppression are independent risk factors for S. maltophilia-pneumonia. Patients with S. maltophilia-pneumonia have a significantly higher ICU mortality within a 28-day follow-up, hospital mortality and LIS compared to patients with S. maltophilia-colonization.
Asunto(s)
Infecciones por Bacterias Gramnegativas/epidemiología , Neumonía Bacteriana/epidemiología , Sistema Respiratorio/microbiología , Stenotrophomonas maltophilia/aislamiento & purificación , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/farmacología , Femenino , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/mortalidad , Infecciones por Bacterias Gramnegativas/patología , Humanos , Unidades de Cuidados Intensivos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/mortalidad , Neumonía Bacteriana/patología , Estudios Retrospectivos , Factores de Riesgo , Stenotrophomonas maltophilia/efectos de los fármacos , Análisis de SupervivenciaRESUMEN
BACKGROUND: Drug-eluting stent (DES) platforms devoid of durable polymer have potential to enhance long-term safety outcomes. The ISAR-TEST-3 study was a randomised trial comparing three rapamycin-eluting stents with different coating strategies. The present study examined 2-year outcomes of these patients and is the first large-scale trial to report longer-term outcomes with biodegradable polymer and polymer-free DES. METHODS: Patients with de novo coronary lesions in native vessels were randomly assigned to receive biodegradable polymer (BP; n = 202), permanent polymer (PP; Cypher; n = 202) and polymer-free (PF; n = 201) stents. The 2-year endpoints of interest were target lesion revascularisation (TLR), death/myocardial infarction (MI), stent thrombosis and delayed angiographic late luminal loss (LLL) between 6-8 months and 2 years. RESULTS: There were no significant differences in TLR (8.4%, 10.4% and 13.4% for BP, PP and PF stents, respectively; p = 0.19), death/MI (5.9%, 6.4% and 6.5% with BP, PP and PF respectively; p = 0.97) or stent thrombosis (definite/probable 0.5%, 1.0% and 1.0% with BP, PP and PF, respectively; p = 0.82). Paired angiographic follow-up at 6-8 months and 2 years was available for 302 patients (69.0% of eligible patients). Delayed LLL was significantly different across the treatment groups: 0.17 (0.42) mm, 0.16 (0.41) mm and -0.01 (0.36) mm for BP, PP and PF stents, respectively (p<0.001). CONCLUSION: Clinical antirestenotic efficacy was maintained with all three platforms between 1 and 2 years, although angiographic surveillance showed ongoing delayed LLL with both BP and PP stent platforms. At 2 years there was no signal of a differential safety profile between the three stent platforms.
Asunto(s)
Reestenosis Coronaria/prevención & control , Stents Liberadores de Fármacos , Sirolimus/administración & dosificación , Moduladores de Tubulina/administración & dosificación , Anciano , Angiografía Coronaria , Reestenosis Coronaria/diagnóstico por imagen , Femenino , Estudios de Seguimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Resultado del TratamientoRESUMEN
The LIM-homeodomain transcription factor Islet-1 (Isl1) marks a cell population which makes a substantial contribution to the embryonic heart. Isl1 expression is downregulated as soon as the cells adopt a differentiated phenotype, suggesting that this transcription factor delineates a cardiogenic progenitor cell population. Taking advantage of this developmental lineage marker, we have identified in the postnatal heart a novel cardiac cell type, which is capable of self-renewal and readily differentiates into mature cardiomyocytes. Utilization of embryonic stem (ES) cells that harbour knock-ins of reporter genes into the endogenous Isl1 locus will enable us to isolate Isl1+ cardiac progenitors from mouse and human ES cell systems during in vitro cardiogenesis. These genetic cell-based systems should allow the direct identification of signalling pathways which guide formation, renewal and diversification of Isl1+ cardiogenic progenitors into distinct heart cell lineages, and would complement in vitro studies in the mouse embryo during cardiac development.
Asunto(s)
Enfermedad , Corazón/embriología , Proteínas de Homeodominio/metabolismo , Células Madre/citología , Animales , Diferenciación Celular , Linaje de la Célula , Humanos , Proteínas con Homeodominio LIM , Factores de TranscripciónRESUMEN
Myocardial overexpression of the C-terminus of beta-adrenergic receptor kinase (betaARKct) has been shown to result in a positive inotropic effect or an improvement of survival in heart failure. However, it is not clear whether this beneficial effect is mainly because of dominant-negative inhibition of betaARK1, and a consecutive resensitization of beta-adrenergic receptors (betaAR), or rather due to inhibition of other Gbetagamma-mediated effects. In this study, we tested whether overexpression of N-terminally truncated phosducin (nt-del-phosducin), another Gbetagamma-binding protein that does not resensitize betaARs owing to simultaneous inhibition of GDP release from Galpha subunits, shows the same effects as betaARKct. Adenoviral gene transfer was used to express nt-del-phosducin and betaARKct in isolated ventricular cardiomyocytes and in myocardium of rabbits, which suffered from heart failure because of rapid ventricular pacing. BetaAR-stimulated cAMP formation was increased by betaARKct, but not by nt-del-phosducin, whereas both proteins inhibited Gbetagamma-mediated effects. Both transgenes also increased contractility of normal and failing isolated cardiomyocytes and improved contractility in rabbits with heart failure after gene transfer in vivo. In conclusion, overexpression of nt-del-phosducin enhances the contractility of cardiomyocytes to the same extent as betaARKct, suggesting that the effects of betaARKct might be owing to inhibition of Gbetagamma rather than to betaAR resensitization.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas del Ojo/genética , Terapia Genética/métodos , Insuficiencia Cardíaca/terapia , Miocardio/metabolismo , Fosfoproteínas/genética , Adenoviridae/genética , Animales , Estimulación Cardíaca Artificial , AMP Cíclico/metabolismo , Reguladores de Proteínas de Unión al GTP , Vectores Genéticos/administración & dosificación , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Modelos Animales , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Conejos , Fosfolipasas de Tipo C/metabolismo , Quinasas de Receptores Adrenérgicos betaRESUMEN
Cardiac myocyte apoptosis has been demonstrated in end-stage failing human hearts. The therapeutic utility of blocking apoptosis in congestive heart failure (CHF) has not been elucidated. This study investigated the role of caspase activation in cardiac contractility and sarcomere organization in the development of CHF. In a rabbit model of heart failure obtained by rapid ventricular pacing, we demonstrate, using in vivo transcoronary adenovirus-mediated gene delivery of the potent caspase inhibitor p35, that caspase activation is associated with a reduction in contractile force of failing myocytes by destroying sarcomeric structure. In this animal model gene transfer of p35 prevented the rise in caspase 3 activity and DNA-histone formation. Genetically manipulated hearts expressing p35 had a significant improvement in left ventricular pressure rise (+dp/dt), decreased end-diastolic chamber pressure (LVEDP), and the development of heart failure was delayed. To better understand this benefit, we examined the effects of caspase 3 on cardiomyocyte dysfunction in vitro. Microinjection of activated caspase 3 into the cytoplasm of intact myocytes induced sarcomeric disorganization and reduced contractility of the cells. These results demonstrate a direct impact of caspases on cardiac function and may lead to novel therapeutic strategies via antiapoptotic regimens.
Asunto(s)
Apoptosis , Inhibidores de Caspasas , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/patología , Contracción Miocárdica , Miocardio/enzimología , Miocardio/patología , Adenoviridae/genética , Animales , Peso Corporal , Caspasa 3 , Caspasas/administración & dosificación , Caspasas/metabolismo , Caspasas/farmacología , Células Cultivadas , Inhibidores de Cisteína Proteinasa/uso terapéutico , Fragmentación del ADN , Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/terapia , Ventrículos Cardíacos/enzimología , Ventrículos Cardíacos/fisiopatología , Proteínas Luminiscentes , Masculino , Miocardio/metabolismo , Tamaño de los Órganos , Marcapaso Artificial , Conejos , Ratas , Sarcómeros/enzimología , Sarcómeros/metabolismo , Sarcómeros/patología , Taquicardia/fisiopatología , Factores de Tiempo , Transgenes/genéticaRESUMEN
In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to beta-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective beta-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V(2) vasopressin receptors (rV(2)-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant beta(2)-adrenergic receptors (rbeta(2)-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V(2)-Rs are uniquely coupled to Gs, PTH1-Rs and beta(2)-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rbeta(2)-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV(2)-R-expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of beta(2)-ARS: In the absence of receptor agonists, rPTH1-Rs and rbeta(2)-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV(2)-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V(2)-Rs might be more suited to test the effects of cAMP-stimulating receptors in heart failure than that of PTH1-Rs or beta(2)-ARS:
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Hormona Paratiroidea/metabolismo , Adenoviridae/genética , Toxina de Adenilato Ciclasa , Animales , Arginina Vasopresina/metabolismo , Medios de Cultivo Condicionados/metabolismo , AMP Cíclico/metabolismo , Desamino Arginina Vasopresina/farmacología , Relación Dosis-Respuesta a Droga , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Proteínas Fluorescentes Verdes , Insuficiencia Cardíaca/metabolismo , Proteínas Luminiscentes/genética , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/genética , Miocardio/citología , Hormona Paratiroidea/farmacología , Proteína Relacionada con la Hormona Paratiroidea , Fragmentos de Péptidos/farmacología , Proteínas/metabolismo , Conejos , Ensayo de Unión Radioligante , Receptores de Superficie Celular/genética , Receptores de Hormona Paratiroidea/genética , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Transgenes/genética , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
BACKGROUND: Systemic levels of arginine vasopressin (AVP) are increased in congestive heart failure, resulting in vasoconstriction and reduced cardiac contractility via V(1) vasopressin receptors. V(2) vasopressin receptors (V2Rs), which promote activation of adenylyl cyclase, are physiologically expressed only in the kidney and are absent in the myocardium. Heterologous expression of V2Rs in the myocardium could result in a positive inotropic effect by using the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: We tested gene transfer with a recombinant adenovirus for the human V2R (Ad-V2R) to stimulate contractility of rat or rabbit myocardium in vivo. Ultrasound-guided direct injection or transcoronary delivery of adenovirus in vivo resulted in recombinant receptor expression in the myocardial target area, leading to a substantial increase in [(3)H]AVP binding. In 50% of the cardiomyocytes isolated from the directly injected area, single-cell shortening measurements detected a significant increase in contraction amplitude after exposure to AVP or the V2R-specific desmopressin (DDAVP). Echocardiography of the target myocardial area documented a marked increase in local fractional shortening after systemic administration of DDAVP in V2R-expressing animals but not in control virus-treated hearts. Simultaneous measurement of global contractility (dP/dt(max)) confirmed a positive inotropic effect of DDAVP on left ventricular function in the Ad-V2R-injected animals. CONCLUSIONS: Adenoviral gene transfer of the V2R into the myocardium increases cardiac contractility in vivo. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in the therapy of congestive heart failure by bypassing the desensitized beta-adrenergic receptor-signaling cascade.
Asunto(s)
Técnicas de Transferencia de Gen , Contracción Miocárdica/fisiología , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/fisiología , Adenoviridae/genética , Infecciones por Adenoviridae/fisiopatología , Animales , Arginina Vasopresina/farmacología , Cateterismo Cardíaco , Desamino Arginina Vasopresina/farmacología , Ecocardiografía , Corazón/fisiopatología , Humanos , Inyecciones/métodos , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocardio/citología , Conejos , Ratas , Ratas Wistar , Ultrasonido , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
BACKGROUND: In congestive heart failure, high systemic levels of the hormone arginine vasopressin (AVP) result in vasoconstriction and reduced cardiac contractility. These effects are mediated by the V1 vasopressin receptor (V1R) coupled to phospholipase C beta-isoforms. The V2 vasopressin receptor (V2R), which promotes activation of the Gs/adenylyl cyclase system, is physiologically expressed in the kidney but not in the myocardium. Expression of a recombinant V2R (rV2R) in the myocardium could result in a positive inotropic effect via the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: A recombinant adenovirus encoding the human V2R (Ad-V2R) was tested for its ability to modulate the cardiac Gs/adenylyl cyclase system and to potentiate contractile force in rat ventricular cardiomyocytes and in H9c2 cardiomyoblasts. Ad-V2R infection resulted in a virus concentration-dependent expression of the transgene and led to a marked increase in cAMP formation in rV2R-expressing cardiomyocytes after exposure to AVP. Single-cell shortening measurements showed a significant agonist-induced contraction amplitude enhancement, which was blocked by the V2R antagonist, SR 121463A. Pretreatment of Ad-V2R-infected cardiomyocytes with AVP led to desensitization of the rV2R after short-term agonist exposure but did not lead to further loss of receptor function or density after long-term agonist incubation, thus demonstrating resistance of the rV2R to downregulation. CONCLUSIONS: Adenoviral gene transfer of the V2R in cardiomyocytes can modulate the endogenous adenylyl cyclase-signal transduction cascade and can potentiate contraction amplitude in cardiomyocytes. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in congestive heart failure by bypassing the desensitized beta-adrenergic receptor signaling.
Asunto(s)
Adenoviridae/genética , Técnicas de Transferencia de Gen , Contracción Miocárdica/fisiología , Receptores de Vasopresinas/genética , Infecciones por Adenoviridae/metabolismo , Adenilil Ciclasas/metabolismo , Animales , AMP Cíclico/biosíntesis , Regulación hacia Abajo , Ventrículos Cardíacos , Humanos , Membranas Intracelulares/metabolismo , Masculino , Miocardio/citología , Ratas , Ratas Wistar , Receptores Adrenérgicos beta/metabolismo , Receptores de Vasopresinas/metabolismo , Sarcolema/enzimología , beta-Galactosidasa/genéticaRESUMEN
BACKGROUND: Platelet/endothelium interaction plays an important role in the pathophysiology of inflammation and atherosclerosis. The role of platelets for monocyte chemotactic protein-1 (MCP-1) secretion and surface expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells has been assessed. METHODS AND RESULTS: Monolayers of human umbilical vein endothelial cells were incubated with nonstimulated or ADP-activated platelets for 6 hours, and secretion of MCP-1 and surface expression of ICAM-1 were determined by ELISA and flow cytometry, respectively. In the presence of ADP-activated platelets, both MCP-1 secretion and ICAM-1 surface expression were significantly increased compared with nonstimulated platelets (P<0.02). Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) determined by electrophoretic mobility shift assay and kappaB-dependent transcriptional activity was enhanced in the presence of activated platelets. In addition, ADP-activated platelets induced MCP-1 and ICAM-1 promoter-dependent transcription. Liposomal transfection of a double-stranded kappaB phosphorothioate oligonucleotide, but not of the mutated form, inhibited MCP-1 secretion and surface expression of ICAM-1 on activated endothelium (P<0.05). CONCLUSIONS: The present study indicates that activated platelets modulate chemotactic (MCP-1) and adhesive (ICAM-1) properties of endothelial cells via an NF-kappaB-dependent mechanism. Platelet-induced activation of the NF-kappaB system might contribute to early inflammatory events in atherogenesis.
Asunto(s)
Arteriosclerosis/etiología , Plaquetas/fisiología , Quimiocina CCL2/metabolismo , Endotelio Vascular/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , FN-kappa B/fisiología , Arteriosclerosis/terapia , Células Cultivadas , Endotelio Vascular/citología , Humanos , Molécula 1 de Adhesión Intercelular/genética , Activación Plaquetaria , Regiones Promotoras GenéticasRESUMEN
Chemoelectrical signal transduction in olfactory neurons appears to involve intracellular reaction cascades mediated by heterotrimeric GTP-binding proteins. In this study attempts were made to identify the G protein subtype(s) in olfactory cilia that are activated by the primary (odorant) signal. Antibodies directed against the alpha subunits of distinct G protein subtypes interfered specifically with second messenger reponses elicited by defined subsets of odorants; odor-induced cAMP-formation was attenuated by Galphas antibodies, whereas Galphao antibodies blocked odor-induced inositol 1,4, 5-trisphosphate (IP3) formation. Activation-dependent photolabeling of Galpha subunits with [alpha-32P]GTP azidoanilide followed by immunoprecipitation using subtype-specific antibodies enabled identification of particular individual G protein subtypes that were activated upon stimulation of isolated olfactory cilia by chemically distinct odorants. For example odorants that elicited a cAMP response resulted in labeling of a Galphas-like protein, whereas odorants that elicited an IP3 response led to the labeling of a Galphao-like protein. Since odorant-induced IP3 formation was also blocked by Gbeta antibodies, activation of olfactory phospholipase C might be mediated by betagamma subunits of a Go-like G protein. These results indicate that different subsets of odorants selectively trigger distinct reaction cascades and provide evidence for dual transduction pathways in olfactory signaling.
Asunto(s)
Cilios/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Odorantes , Mucosa Olfatoria/efectos de los fármacos , Acetatos/farmacología , Aldehídos/farmacología , Secuencia de Aminoácidos , Animales , Benzaldehídos/farmacología , Cilios/metabolismo , AMP Cíclico/biosíntesis , Ciclohexenos , Ciclopentanos/farmacología , Eugenol/farmacología , Inositol 1,4,5-Trifosfato/biosíntesis , Datos de Secuencia Molecular , Nitrilos/farmacología , Mucosa Olfatoria/metabolismo , Oxilipinas , Etiquetas de Fotoafinidad , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVES: beta-Adrenergic receptor kinase (beta ARK) phosphorylates and thereby inactivates agonist-occupied beta-adrenergic receptors (beta AR). beta ARK is thought to play an important role in the regulation of cardiac function. Therefore, we studied beta ARK activation and its inhibition in intact smooth muscle cells and in cardiomyoblasts. METHODS AND RESULTS: beta AR agonist-stimulated translocation of beta ARK was monitored by immunofluorescence labelling with specific antibodies and confocal laser scanning microscopy in DDT-MF 2 hamster smooth muscle cells and in H9c2 rat cardiomyoblasts. In unstimulated cells. beta ARK was mainly located in the cytosol. After beta AR agonist stimulation, the beta ARK signal was partially translocated to the membranes. Liposomal gene transfer of the COOH-terminus of beta ARK ('beta ARKmini') as a beta ARK inhibitor led to functional expression of this protein in both cell lines with high efficiency. Western blots with beta ARK antibodies showed a gene concentration-dependent immunoreactivity of the 'beta ARKmini' protein. 'beta ARKmini'-transfected myocytes demonstrated reduced membrane targeting of the beta ARK immuno-fluorescence signal. Additionally, the effect of 'beta ARKmini' on beta AR-induced desensitization of myocytic cAMP accumulation was investigated. In control cells, desensitization with isoproterenol led to a subsequent reduction of beta AR-induced cAMP accumulation. In 'beta ARKmini'-transfected myocytes, this beta AR-induced desensitization was significantly diminished, whereas normal beta AR-induced cAMP accumulation was unaffected. A gene concentration of 2 micrograms 'beta ARKmini' DNA/100,000 cardiomyoblasts, and of 0.7 microgram 'beta ARKmini' DNA/100,000 DDT-MF2 smooth muscle cells led to approximately 5.9- and approximately 5.6-fold overexpressions of 'beta ARKmini' vs. native beta ARK, respectively. These gene doses proved sufficient to attenuate beta-adrenergic desensitization significantly. CONCLUSIONS: (1) beta ARK translocation was evidenced in DDT-MF2 smooth muscle cells and in cardiomyoblasts by confocal laser scanning microscopy. (2) Feasibility of 'beta ARKmini' gene transfer to myocytes was demonstrated, and necessary gene doses for beta ARK inhibition were titered. (3) Overexpression of 'beta ARKmini' functionally interacted with endogenous beta-adrenergic signal transduction, leading to sustained cAMP accumulation after prolonged beta-adrenergic stimulation.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Músculo Liso/enzimología , Miocardio/enzimología , Agonistas Adrenérgicos beta/farmacología , Animales , Western Blotting , Línea Celular , Membrana Celular/enzimología , Cricetinae , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Citosol/enzimología , Activación Enzimática , Citometría de Flujo , Isoproterenol/farmacología , Microscopía Confocal , Músculo Liso/efectos de los fármacos , Miocardio/metabolismo , Transducción de Señal/fisiología , Estimulación Química , Translocación Genética , Quinasas de Receptores Adrenérgicos betaRESUMEN
We investigated, in dog thyroid membranes, the ability of the dog thyrotropin (TSH) receptor to interact with the endogenous G proteins expressed in this tissue. Activation of the receptor led to increased incorporation of the photoreactive GTP analog [alpha-(32)P]GTP azidoanilide into immunoprecipitated alpha subunits of three G protein families: G(s), G(q/11), G(i/o). This effect was not due to a general loss of receptor G protein specificity since carbamylcholine, in the same membrane preparations, only stimulated the binding of the GTP analog to the alpha subunits of G(q/11) proteins. To investigate the multiple coupling of the dog TSH receptor in intact cells, cyclic AMP accumulation, IP(3) formation and (45)Ca2+ efflux experiments were performed. When thyrocytes were pretreated with pertussis toxin (PTX), the TSH receptor-mediated accumulation of cAMP increased by approximately 45% with TSH at 1 mU/ml, suggesting that the TSH receptor coupled to both G(s) and G(i) in vivo. On the other hand, no increase in IP(3) accumulation nor Ca2+ efflux was observed in the presence of thyrotropin. These data in intact cells are thus in contradiction with those obtained in membranes, suggesting that receptor-mediated transmembrane signalling may implicate a specificity which itself may reflect a localization and organization of the different components (receptors, G proteins, ...) in the plasma membrane of intact cells. As in some cells, G(i) activates mitogenesis by hormone activated G-protein-coupled receptors, we tested its role in the stimulation by TSH of the proliferation of thyrocytes. This was not affected by PTX, suggesting that the mitogenic effect of TSH does not involve G(i)-proteins.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de Tirotropina/metabolismo , Glándula Tiroides/metabolismo , Tirotropina/metabolismo , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , Perros , Immunoblotting , Transducción de SeñalAsunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Comunicación Celular , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Humanos , Oligonucleótidos Antisentido/farmacología , Conformación Proteica , Transducción de SeñalRESUMEN
The wasp venom, mastoparan (MP), is a direct activator of reconstituted pertussis toxin-sensitive G-proteins and of purified nucleoside diphosphate kinase (NDPK) [E.C. 2.6.4.6.]. In HL-60 membranes, MP activates high-affinity GTPase [E.C. 3.6.1.-] and NDPK-catalyzed GTP formation, but not photolabeling of G-protein alpha-subunits with GTP azidoanilide; this suggests that the venom activates G-proteins in this system indirectly via stimulation of NDPK. Moreover, the MP analogue, mastoparan 7 (MP 7), is a much more effective activator of reconstituted G-proteins than MP, whereas with regard to NDPK and GTPase in HL-60 membranes, the two peptides are similarly effective. In our present study, we investigated NDPK- and G-protein activation by MP in membranes of the human neuroblastoma cell line, SH-SY5Y, the human erythroleukemia cell line, HEL, the rat basophilic leukemia cell line, RBL 2H3, and the hamster ductus deferens smooth muscle cell line, DDT1MF-2. All these membranes exhibited high NDPK activities that were increased by MP. Compared to basal GTP formation rates, basal rates of high-affinity GTP hydrolysis in cell membranes were low. MP activated high-affinity GTP hydrolysis in cell membranes but did not enhance incorporation of GTP azidoanilide into G-protein alpha-subunits. As with HL-60 membranes, MP and MP 7 were similarly effective activators of NDPK and GTPase in SH-SY5Y membranes. Pertussis toxin inhibited MP-stimulated GTP hydrolyses in SH-SY5Y- and HEL membranes, whereas NDPK activations by MP were pertussis toxin-insensitive. Our data suggest that indirect G-protein activation via NDPK is not restricted to HL-60 membranes but is a more general mechanism of MP action in cell membranes. Pertussis toxin-catalyzed ADP-ribosylation of alpha-subunits may inhibit the transfer of GTP from NDPK to G-proteins. NDPK may play a much more important role in transmembrane signal transduction than was previously appreciated and, moreover, the GTPase of G-protein alpha-subunits may serve as GDP-synthase for NDPK.