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
The lower esophageal sphincter (LES) is a specialized region of the esophageal smooth muscle that allows the passage of a swallowed bolus into the stomach. Nitric oxide (NO) plays a major role in LES relaxation. Nicorandil possesses dual properties of a NO donor and an ATP-sensitive potassium channel (KATP channel) agonist, and is expected to reduce LES tone. This study investigated the mechanisms underlying the effects of nicorandil on the LES. Rat LES tissues were placed in an organ bath, and activities were recorded using an isometric force transducer. Carbachol-induced LES contraction was significantly inhibited by KATP channel agonists in a concentration-dependent manner; pinacidil >> nicorandil ≈ diazoxide. Nicorandil-induced relaxation of the LES was prevented by pretreatment with glibenclamide, whereas N(G)-nitro-l-arginine methyl ester (l-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and iberiotoxin were ineffective at preventing nicorandil-induced LES relaxation. Furthermore, nicorandil did not affect high K(+)-induced LES contraction. Reverse-transcription polymerase chain reaction analysis and immunohistochemistry revealed expression of KCNJ8 (Kir6.1), KCNJ11 (Kir6.2), ABCC8 (SUR1) and ABCC9 (SUR2) subunits of the KATP channel in the rat lower esophagus. These findings indicate that nicorandil causes LES relaxation chiefly by activating the KATP channel, and that it may provide an additional pharmacological tool for the treatment of spastic esophageal motility disorders.
Asunto(s)
Carbacol/farmacología , Esfínter Esofágico Inferior/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Nicorandil/farmacología , Animales , Diazóxido/farmacología , Relación Dosis-Respuesta a Droga , Gliburida/farmacología , Técnicas In Vitro , Canales KATP/agonistas , Canales KATP/biosíntesis , NG-Nitroarginina Metil Éster/farmacología , Oxadiazoles/farmacología , Péptidos/farmacología , Pinacidilo/farmacología , Potasio/farmacología , Quinoxalinas/farmacología , RatasRESUMEN
Dexmedetomidine is a selective α2 adrenergic agonist. Although dexmedetomidine is widely used for sedation and analgesia, it frequently produces hypotension and bradycardia. The present study aimed to evaluate the effects of dexmedetomidine on cardiac function and coronary circulation using Langendorff-perfused guinea pig hearts. Coronary perfusion pressure (CPP) and left ventricular pressure (LVP) were continuously monitored, and electric field stimulation (EFS) was applied to stimulate sympathetic nerve terminals. Dexmedetomidine almost completely inhibited the EFS-induced increase in LVP at all ages. The effect of dexmedetomidine on coronary artery resistance varied according to postnatal age, i.e., dexmedetomidine had little effect on CPP in young hearts (<4 weeks) but increased CPP by 10 mmHg at 4-8 weeks and by 15 mmHg at >8 weeks. The increase in CPP in adult hearts was inhibited by imiloxan, an α2B antagonist, and prazosin, an α1 antagonist. The results suggest that dexmedetomidine acts on α2 adrenergic receptors at sympathetic nerve terminals to suppress the release of norepinephrine. In addition, the findings suggest that dexmedetomidine directly affects α1 adrenoceptors and/or α2B adrenoceptors on coronary smooth muscles to increase CPP. The age-related changes in α adrenoceptor subtypes may be linked to the cardiodepressant effects of dexmedetomidine.
Asunto(s)
Envejecimiento/fisiología , Circulación Coronaria/efectos de los fármacos , Dexmedetomidina/farmacología , Corazón/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Analgésicos no Narcóticos/farmacología , Animales , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/fisiología , Cobayas , Corazón/fisiología , Frecuencia Cardíaca/efectos de los fármacos , Hipnóticos y Sedantes/farmacología , Técnicas In Vitro , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Previously, we showed that in adult rat cardiomyocytes, nitric oxide (NO) donors stimulate mitochondrial cGMP production, followed by cytochrome c release, independently of the mitochondrial permeable transition pore. We investigated whether mitochondrial cGMP-induced cytochrome c release from cardiac mitochondria is Ca(2+)-sensitive. Mitochondria and primary cultured cardiomyocytes were prepared from left ventricles of male Wistar rats. The cytosolic Ca(2+) concentration was adjusted with Ca(2+)-EGTA buffers. Cytochrome c released from mitochondria was measured by Western blotting. Cardiomyocyte apoptosis was assessed by Annexin V staining. Cytochrome c release from cardiac mitochondria was evoked by buffered Ca(2+) (1 µM); this was inhibited by NO-cGMP pathway inhibitors such as N(G)-monomethyl-l-arginine monoacetate (inhibitor of NO synthase), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NO scavenger), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, NO-sensitive guanylyl cyclase inhibitor) and voltage-dependent anion channel (VDAC) inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, but not by cyclosporin A (mitochondrial permeable transition pore inhibitor). Furthermore, this release was significantly and dose dependently inhibited by 0.3-3 µM KT5823 (protein kinase G inhibitor). At the cellular level, intracellular perfusion of cardiomyocytes with buffered Ca(2+) (1 µM) also induced apoptosis, which was inhibited in the presence of ODQ. A membrane-permeable cGMP analog, 8-Br-cGMP, but not cGMP itself, mimicked buffered Ca(2+) actions in both cardiac mitochondria and cardiomyocytes. We further confirmed an increase in protein kinase G activity by adding cGMP in mitochondrial protein fraction. Our results suggest that mitochondrial NO-cGMP pathway-induced cytochrome c release from cardiac mitochondria, triggered by increased cytosolic Ca(2+), occurs through VDAC via the stimulation of an undiscovered mitochondrial protein kinase G.
Asunto(s)
Apoptosis/efectos de los fármacos , Calcio/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Citosol/fisiología , Mitocondrias Cardíacas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Óxido Nítrico/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Separación Celular , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Depuradores de Radicales Libres , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Dilatación Mitocondrial/efectos de los fármacos , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Heart rate (HR) of mammalian species changes postnatally, i.e., HR of large animals including humans decreases, while HR in small animals such as mice and rats increases. To clarify cellular mechanisms underlying the postnatal HR changes, we performed in vivo HR measurement and electrophysiological analysis on sinoatrial node (SAN) cells in mice. The in vivo HR was ~320 beats min(-1) (bpm) immediately after birth, and increased with age to ~690 bpm at postnatal day 14. Under blockage of autonomic nervous systems, HR remained constant until postnatal day 5 and then increased day by day. The spontaneous beating rate of SAN preparation showed a similar postnatal change. The density of the L-type Ca(2+) current (LCC) was smaller in neonatal SAN cells than in adult cells, accompanied by a positive shift of voltage-dependent activation. Thus, the postnatal increase in HR is caused by both the increased sympathetic influence and the intrinsic activity of SAN cells. The different conductance and kinetics of LCC may be involved in the postnatal increase in pacemaker activity.
Asunto(s)
Relojes Biológicos , Frecuencia Cardíaca , Nodo Sinoatrial/fisiología , Potenciales de Acción , Factores de Edad , Animales , Animales Recién Nacidos , Relojes Biológicos/efectos de los fármacos , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Femenino , Regulación de la Expresión Génica , Frecuencia Cardíaca/efectos de los fármacos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Activación del Canal Iónico , Cinética , Masculino , Moduladores del Transporte de Membrana/farmacología , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Nodo Sinoatrial/citología , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/inervación , Nodo Sinoatrial/metabolismo , Sistema Nervioso Simpático/fisiologíaRESUMEN
To elucidate the physiological importance of neuronal (N)-type calcium channels in sympathetic controls, we analyzed N-type channel-deficient (NKO) mice. Immunoprecipitation analysis revealed increased interaction between beta3 (a major accessory subunit of N-type channels) and R-type channel-forming CaV2.3 in NKO mice. R-R intervals in NKO ECG recordings were elongated and fluctuating, suggesting disturbed sympathetic tonus. N-type channel inhibitors elongated the R-R interval in control mice, whereas R-type channel blocking with SNX-482 significantly affected NKO but not control mice, indicating a compensatory role for R-type channels. Echocardiography and Langendorff heart analysis confirmed a major role for R-type channels in NKO mice. Combined, our biochemical and physiological analyses strongly suggest that the remaining sympathetic tonus in NKO mice is dependent on R-type calcium channels.