RESUMEN
AIMS: Apoptosis of cardiomyocytes significantly contributes to the development of post-ischaemic cardiomyopathy. Although mitochondria have been suggested to play a crucial role in this process, the precise mechanisms controlling the mitochondria-dependent apoptosis in cardiomyocytes under ischaemia/reperfusion are still poorly understood. Here we aimed to analyse the role of the soluble adenylyl cyclase (sAC). METHODS AND RESULTS: Adult rat cardiomyocytes were subjected to simulated in vitro ischaemia (SI) consisting of glucose-free anoxia at pH 6.4. Apoptosis was detected by DNA laddering, chromatin condensation, and caspases cleavage. SI led to the translocation of sAC to the mitochondria and mitochondrial depolarization followed by cytochrome c release, caspase-9/-3 cleavage and apoptosis during simulated reperfusion (SR). Pharmacological inhibition of sAC during SI, but not during SR, significantly reduced the SI/SR-induced mitochondrial injury and apoptosis. Similarly, sAC knock-down mediated by an adenovirus coding for shRNA targeting sAC prevented the activation of the mitochondrial pathway of apoptosis. Analysis of the link between sAC and apoptosis revealed a sAC and protein kinase A-dependent Bax phosphorylation at Thr(167) and its translocation to mitochondria during SI, which subsequently caused mitochondrial oxygen radical formation followed by cytochrome c release and caspase-9 cleavage during SR. CONCLUSION: These results suggest a key role of sAC in SI-induced mitochondrial Bax translocation and activation of the mitochondrial pathway of apoptosis in adult cardiomyocytes.
Asunto(s)
Adenilil Ciclasas/fisiología , Apoptosis , Mitocondrias Cardíacas/metabolismo , Reperfusión Miocárdica , Miocitos Cardíacos/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Células Cultivadas , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Masculino , Miocitos Cardíacos/citología , Transporte de Proteínas , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.
Asunto(s)
Calcio/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/fisiología , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Adenosina Trifosfato/metabolismo , Animales , Ciclosporina/farmacología , Fluoresceínas/análisis , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Poro de Transición de la Permeabilidad Mitocondrial , Necrosis , Ratas , Ratas Wistar , Compuestos de Rutenio/farmacología , Rianodina/farmacología , Tapsigargina/farmacología , Tiopronina/farmacologíaRESUMEN
AIMS: Ischaemia-reperfusion provokes barrier failure of the coronary microvasculature, impeding functional recovery of the heart during reperfusion. The aim of the present study was to investigate whether the stimulation of cGMP signalling by activation of soluble guanylyl cyclase (sGC) can reduce reperfusion-induced endothelial intercellular gap formation and to determine whether this is due to an influence on endothelial cytosolic Ca(2+) homeostasis during reperfusion. METHODS AND RESULTS: Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 micromol/L) or DEAnonoate (0.5 micromol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca(2+) sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca(2+)]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca(2+) pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca(2+) and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development. CONCLUSION: sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.
Asunto(s)
Vasos Coronarios/efectos de los fármacos , GMP Cíclico/metabolismo , Células Endoteliales/efectos de los fármacos , Activadores de Enzimas/farmacología , Uniones Comunicantes/efectos de los fármacos , Daño por Reperfusión Miocárdica/prevención & control , Receptores Citoplasmáticos y Nucleares/agonistas , Sistemas de Mensajero Secundario , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Carbazoles/farmacología , Hipoxia de la Célula , Células Cultivadas , Vasos Coronarios/citología , Vasos Coronarios/enzimología , Vasos Coronarios/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Citosol/metabolismo , Edema Cardíaco/metabolismo , Edema Cardíaco/prevención & control , Retículo Endoplásmico/metabolismo , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Uniones Comunicantes/metabolismo , Guanilato Ciclasa/metabolismo , Homeostasis , Hidrazinas/farmacología , Masculino , Daño por Reperfusión Miocárdica/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Guanilil Ciclasa Soluble , Sulfonamidas/farmacología , Factores de Tiempo , ortoaminobenzoatos/farmacologíaRESUMEN
Molecular mechanisms of cerebral vasospasm after subarachnoid hemorrhage (SAH) include specific modes of cell signaling like activation of nuclear factor (NF)-kappaB and vascular cell adhesion molecules (VCAM)-1 expression. The study's hypothesis is that cisternal cerebral spinal fluid (CSF) from patients after SAH may cause Ca(2+) oscillations which induce these modes of vascular inflammation in an in vitro model of human cerebral endothelial cells (HCECs). HCECs were incubated with cisternal CSF from 10 SAH patients with confirmed cerebral vasospasm. The CSF was collected on days 5 and 6 after hemorrhage. Cytosolic Ca(2+) concentrations and cell contraction as an indicator of endothelial barrier function were examined by fura-2 microflurometry. Activation of NF-kappaB and VCAM-1 expression were measured by immunocytochemistry. Incubation of HCEC with SAH-CSF provoked cytosolic Ca(2+) oscillations (0.31+/-0.09 per min), cell contraction, NF-kappaB activation, and VCAM-1 expression, whereas exposure to native CSF had no significant effect. When endoplasmic reticulum (ER) Ca(2+)-ATPase and ER inositol trisphosphate (IP3)-sensitive Ca(2+) channels were blocked by thapsigargin or xestospongin, the frequency of the Ca(2+) oscillations was reduced significantly. In analogy to the reduction of Ca(2+) oscillation frequency, the blockers impaired HCEC contraction, NF-kappaB activation, and VCAM-1 expression. Cisternal SAH-CSF induces cytosolic Ca(2+) oscillations in HCEC that results in cellular constriction, NF-kappaB activation, and VCAM-1 expression. The Ca(2+) oscillations depend on the function of ER Ca(2+)-ATPase and IP3-sensitive Ca(2+) channels.
Asunto(s)
Señalización del Calcio , Citosol/metabolismo , Células Endoteliales/metabolismo , Hemorragia Subaracnoidea/metabolismo , Adulto , Anciano , Canales de Calcio/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Quimiocinas CXC , Retículo Endoplásmico/metabolismo , Femenino , Homeostasis , Humanos , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismoRESUMEN
OBJECTIVE: The autonomous proliferative response of endothelial cells to hypoxia has been shown to be dependent on activation of NAD(P)H oxidase, on the cytosolic Ca2+ load, and, consequently, on nuclear translocation of extracellular signal-regulated kinase (ERK)1/2 during transient hypoxia. The aim of the present study was to investigate whether poly(ADP-ribose) polymerase (PARP) is a downstream signal of NAD(P)H oxidase, mediating cytosolic Ca2+ load and hence nuclear translocation of ERK1/2 and endothelial cell proliferation. METHODS: Porcine aortic endothelial cells were incubated under hypoxic conditions for 40 min. Cytosolic [Ca2+] and reactive oxygen species (ROS) formation were measured in fura-2- and DCF-loaded cells, respectively. PARP activation was detected by immunocytochemistry, and endothelial cell proliferation was determined 24 h after 60 min of transient hypoxia. RESULTS: Inhibition of NAD(P)H oxidase with antisense oligonucleotide against the p22(phox) subunit, MEK/ERK signalling with UO 126 (30 microM), or PARP with PJ 34 (10 microM) leads to a marked reduction in hypoxia-induced cytosolic Ca2+ load and activation of PARP. Hypoxia-induced translocation of ERK1/2 and endothelial cell proliferation were also prevented when NAD(P)H oxidase or PARP were inhibited; however, hypoxic ROS formation was not affected in the presence of PARP inhibitor. CONCLUSION: PARP represents a downstream effector of NADP(H) oxidase and acts as a necessary intermediate step for the hypoxic proliferative response of endothelial cells.
Asunto(s)
Células Endoteliales/metabolismo , Endotelio Vascular , Sistema de Señalización de MAP Quinasas , Poli(ADP-Ribosa) Polimerasas/fisiología , Animales , Butadienos/farmacología , Calcio/análisis , Calcio/metabolismo , Hipoxia de la Célula/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citosol/química , Citosol/metabolismo , Células Endoteliales/citología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/análisis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Peróxido de Hidrógeno/metabolismo , Inmunohistoquímica , Microscopía Fluorescente , NADPH Oxidasas/genética , Nitrilos/farmacología , Oligonucleótidos Antisentido/farmacología , Fenantrenos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Especies Reactivas de Oxígeno/metabolismo , PorcinosRESUMEN
OBJECTIVE: Ischemia-reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we tested whether adenosine 5'-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect. METHODS: Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used. RESULTS: After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP (ARL 67156; 100 microM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 microM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 microM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 microM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT. CONCLUSION: ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.