RESUMEN
Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.
Asunto(s)
Drosophila melanogaster/fisiología , Modelos Animales , Animales , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Femenino , Estudio de Asociación del Genoma Completo , Corazón/embriología , Corazón/fisiología , Humanos , Masculino , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Interferencia de ARNRESUMEN
RATIONALE: Hypoxia favors stem cell quiescence, whereas normoxia is required for stem cell activation, but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown. OBJECTIVE: A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal. METHODS AND RESULTS: Here, we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot re-enter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy. CONCLUSIONS: Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggest that a pool of functionally competent CSCs persists in the senescent heart and that this stem cell compartment can promote myocyte regeneration effectively, partly correcting the aging myopathy.
Asunto(s)
Envejecimiento/efectos de los fármacos , Cardiomiopatías/metabolismo , Hipoxia/metabolismo , Mioblastos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/farmacología , Nicho de Células Madre , Envejecimiento/metabolismo , Animales , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/patología , Ciclo Celular , Linaje de la Célula , Proliferación Celular , Senescencia Celular/efectos de los fármacos , Hipoxia/patología , Ratones , Ratones Endogámicos C57BL , Mioblastos Cardíacos/efectos de los fármacos , Mioblastos Cardíacos/fisiología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Factor de Células Madre/uso terapéutico , Homeostasis del TelómeroRESUMEN
BACKGROUND: Little is known about the function of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the adult heart experimentally. Moreover, whether these Ca(2+) release channels are present and play a critical role in human cardiomyocytes remains to be defined. IP3Rs may be activated after Gαq-protein-coupled receptor stimulation, affecting Ca(2+) cycling, enhancing myocyte performance, and potentially favoring an increase in the incidence of arrhythmias. METHODS AND RESULTS: IP3R function was determined in human left ventricular myocytes, and this analysis was integrated with assays in mouse myocytes to identify the mechanisms by which IP3Rs influence the electric and mechanical properties of the myocardium. We report that IP3Rs are expressed and operative in human left ventricular myocytes. After Gαq-protein-coupled receptor activation, Ca(2+) mobilized from the sarcoplasmic reticulum via IP3Rs contributes to the decrease in resting membrane potential, prolongation of the action potential, and occurrence of early afterdepolarizations. Ca(2+) transient amplitude and cell shortening are enhanced, and extrasystolic and dysregulated Ca(2+) elevations and contractions become apparent. These alterations in the electromechanical behavior of human cardiomyocytes are coupled with increased isometric twitch of the myocardium and arrhythmic events, suggesting that Gαq-protein-coupled receptor activation provides inotropic reserve, which is hampered by electric instability and contractile abnormalities. Additionally, our findings support the notion that increases in Ca(2+) load by IP3Rs promote Ca(2+) extrusion by forward-mode Na(+)/Ca(2+) exchange, an important mechanism of arrhythmic events. CONCLUSIONS: The Gαq-protein/coupled receptor/IP3R axis modulates the electromechanical properties of the human myocardium and its propensity to develop arrhythmias.
Asunto(s)
Potenciales de Acción/fisiología , Señalización del Calcio/fisiología , Insuficiencia Cardíaca/fisiopatología , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Miocitos Cardíacos/fisiología , Adulto , Animales , Arritmias Cardíacas/fisiopatología , Células Cultivadas , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Insuficiencia Cardíaca/genética , Ventrículos Cardíacos/citología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/fisiología , Transducción de Señal/fisiologíaRESUMEN
The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133(+) cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133(+) cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology.
Asunto(s)
Ensayo de Unidades Formadoras de Colonias/métodos , Células Endoteliales/citología , Células Madre/citología , Antígeno AC133 , Adulto , Animales , Antígenos CD/análisis , Diferenciación Celular , Células Cultivadas , Glicoproteínas/análisis , Células Madre Hematopoyéticas/citología , Humanos , Receptores de Lipopolisacáridos/análisis , Ratones , Ratones Endogámicos BALB C , Péptidos/análisis , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
BACKGROUND: Although therapeutic angiogenesis is a most promising strategy for the treatment of myocardial infarction (MI), it remains unknown if and how endogenous angiogenesis inhibitors, such as endostatin, regulate angiogenesis in MI. In the present study the role of endostatin in left ventricular (LV) remodeling and heart failure was tested in a rat MI model. METHODS AND RESULTS: When exposed to hypoxia, rat cardiomyocytes showed increased expression of endostatin. After MI induction in the rat MI model, endostatin expression was upregulated in cardiomyocytes, and serum endostatin levels were significantly elevated. Anti-endostatin antibody treatment resulted in significantly higher mortality of MI rats than controls. The MI rats with endostatin neutralization displayed adverse LV remodeling and severe heart failure compared with control MI rats. Although angiogenesis was increased, tissue remodeling and interstitial fibrosis were further exaggerated in post-MI hearts by endostatin neutralization. Furthermore, the expression and protease activity of matrix metalloproteinases -2 and -9, and of angiotensin-converting enzyme were markedly elevated by endostatin neutralization. CONCLUSIONS: Neutralization of endostatin worsens the symptoms and outcomes of MI in a rat model. The results imply that endogenous endostatin/collagen XVIII may suppress aberrant LV remodeling and heart failure after MI. (Circ J 2010; 74: 109 - 119).
Asunto(s)
Colágeno Tipo XVIII/antagonistas & inhibidores , Endostatinas/antagonistas & inhibidores , Insuficiencia Cardíaca/fisiopatología , Infarto del Miocardio/fisiopatología , Remodelación Ventricular/fisiología , Animales , Células Cultivadas , Colágeno Tipo XVIII/inmunología , Colágeno Tipo XVIII/fisiología , Modelos Animales de Enfermedad , Endostatinas/inmunología , Endostatinas/fisiología , Insuficiencia Cardíaca/metabolismo , Inmunoglobulina G/farmacología , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Peptidil-Dipeptidasa A/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
The aging myopathy manifests itself with diastolic dysfunction and preserved ejection fraction. We raised the possibility that, in a mouse model of physiological aging, defects in electromechanical properties of cardiomyocytes are important determinants of the diastolic characteristics of the myocardium, independently from changes in structural composition of the muscle and collagen framework. Here we show that an increase in the late Na(+) current (INaL) in aging cardiomyocytes prolongs the action potential (AP) and influences temporal kinetics of Ca(2+) cycling and contractility. These alterations increase force development and passive tension. Inhibition of INaL shortens the AP and corrects dynamics of Ca(2+) transient, cell contraction and relaxation. Similarly, repolarization and diastolic tension of the senescent myocardium are partly restored. Thus, INaL offers inotropic support, but negatively interferes with cellular and ventricular compliance, providing a new perspective of the biology of myocardial aging and the aetiology of the defective cardiac performance in the elderly.
Asunto(s)
Potenciales de Acción , Envejecimiento/metabolismo , Calcio/metabolismo , Cardiomiopatías/metabolismo , Ventrículos Cardíacos/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Sodio/metabolismo , Animales , Cardiomiopatías/fisiopatología , Colágeno , Modelos Animales de Enfermedad , Corazón/fisiopatología , Ventrículos Cardíacos/fisiopatología , Ratones , Ratones Noqueados , Contracción Miocárdica , Técnicas de Placa-Clamp , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/genética , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/metabolismoRESUMEN
AIMS: Increased expression of several subtypes of prostaglandin E(2) receptors (EP1-4) has recently been described in clinical and experimental myocardial ischaemia/reperfusion (I/R) injury. However, their pathophysiological significance in I/R remains obscure. Thus, we determined whether the activation of the prostanoid receptor, EP4, suppresses myocardial I/R injury. METHODS AND RESULTS: To analyse the role of EP4, we administered an EP4 selective agonist (EP4RAG, 1 or 3 mg/kg) or vehicle to rats with myocardial I/R injury. After 7 days of reperfusion, I/R rats exhibited left ventricular (LV) dilatation and contractile dysfunction with myocyte hypertrophy and interstitial fibrosis. EP4RAG significantly reduced infarction area/ischaemic myocardium (72.4 +/- 0.7 vs. 23.3 +/- 0.6%; P < 0.05) and improved LV contraction and dilatation compared with that of the vehicle. EP4RAG also attenuated the recruitment of inflammatory cells, especially macrophages, and interstitial fibrosis in hearts. Monocyte chemoattractant protein (MCP)-1 and other cytokines were increased in both non-ischaemic (area not at risk, ANAR) and ischaemic (area at risk, AAR) myocardium; however, western blot analysis and RNase protection assay showed that EP4RAG suppressed these changes. Gelatin zymography revealed EP4RAG significantly reduced matrix metalloproteinase-2 and -9 activities in both ANAR and AAR. Chemoattractant assay demonstrated that EP4RAG suppressed the migration of cytokine-stimulated macrophages and decreased the level of MCP-1 production in the supernatant (587.3 +/- 55.3 vs. 171.5 +/- 47.5 pg/mL; P < 0.05). CONCLUSION: The data suggest that the EP4 agonist is effective for attenuation of I/R injury by suppressing MCP-1 and the infiltration of inflammatory cells, especially macrophages.
Asunto(s)
Corazón/efectos de los fármacos , Corazón/fisiopatología , Daño por Reperfusión Miocárdica/fisiopatología , Receptores de Prostaglandina E/agonistas , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Movimiento Celular , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Ecocardiografía , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Masculino , Monocitos/patología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , Ratas , Ratas Sprague-Dawley , Subtipo EP4 de Receptores de Prostaglandina ERESUMEN
Tea catechins have many biological functions; these effects are induced by the suppression of several inflammatory factors. However, the effects of catechins on ventricular remodeling after myocardial ischemia have not been well investigated. To test the hypothesis that catechins can attenuate chronic ventricular remodeling after myocardial ischemia, we performed oral administration of catechins into rat myocardial ischemia models. We analyzed the mechanisms using physiological, pathological and molecular examinations. Although severe myocardial fibrosis with enhancement of inflammatory factors were observed in the non-treated ischemia group on day 28, catechins attenuated these changes with suppressed NF-kappaB and matrix metalloproteinases without systemic adverse effects. Catechins are potent for the suppression of chronic ventricular remodeling after myocardial ischemia because they are critically involved in the suppression of several inflammatory genes.