RESUMEN
Dysfunction of cardiac energy metabolism plays a critical role in many cardiac diseases, including heart failure, myocardial infarction and ischemia-reperfusion injury and organ transplantation. The characteristics of these diseases can be elucidated in vivo, though animal-free in vitro experiments, with primary adult or neonatal cardiomyocytes, the rat ventricular H9c2 cell line or the mouse atrial HL-1 cells, providing intriguing experimental alternatives. Currently, it is not clear how H9c2 and HL-1 cells mimic the responses of primary cardiomyocytes to hypoxia and oxidative stress. In the present study, we show that H9c2 cells are more similar to primary cardiomyocytes than HL-1 cells with regard to energy metabolism patterns, such as cellular ATP levels, bioenergetics, metabolism, function and morphology of mitochondria. In contrast to HL-1, H9c2 cells possess beta-tubulin II, a mitochondrial isoform of tubulin that plays an important role in mitochondrial function and regulation. We demonstrate that H9c2 cells are significantly more sensitive to hypoxia-reoxygenation injury in terms of loss of cell viability and mitochondrial respiration, whereas HL-1 cells were more resistant to hypoxia as evidenced by their relative stability. In comparison to HL-1 cells, H9c2 cells exhibit a higher phosphorylation (activation) state of AMP-activated protein kinase, but lower peroxisome proliferator-activated receptor gamma coactivator 1-alpha levels, suggesting that each cell type is characterized by distinct regulation of mitochondrial biogenesis. Our results provide evidence that H9c2 cardiomyoblasts are more energetically similar to primary cardiomyocytes than are atrial HL-1 cells. H9c2 cells can be successfully used as an in vitro model to simulate cardiac ischemia-reperfusion injury.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Mitocondrias/metabolismo , Oxígeno/farmacología , Adenosina Trifosfato/metabolismo , Adenilato Quinasa/metabolismo , Animales , Western Blotting , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Respiración de la Célula/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Fenotipo , Fosforilación/efectos de los fármacos , Ratas , Factores de Transcripción/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Primary sensory afferents of the dorsal root ganglion (DRG) that innervate the skin detect a wide range of stimuli, such as touch, temperature, pain, and itch. Different functional classes of nociceptors project their axons to distinct target zones within the developing skin, but the molecular mechanisms that regulate target innervation are less clear. Here we report that the Nogo66 receptor homolog NgR2 is essential for proper cutaneous innervation. NgR2(-/-) mice display increased density of nonpeptidergic nociceptors in the footpad and exhibit enhanced sensitivity to mechanical force and innocuous cold temperatures. These sensory deficits are not associated with any abnormality in morphology or density of DRG neurons. However, deletion of NgR2 renders nociceptive nonpeptidergic sensory neurons insensitive to the outgrowth repulsive activity of skin-derived Versican. Biochemical evidence shows that NgR2 specifically interacts with the G3 domain of Versican. The data suggest that Versican/NgR2 signaling at the dermo-epidermal junction acts in vivo as a local suppressor of axonal plasticity to control proper density of epidermal sensory fiber innervation. Our findings not only reveal the existence of a novel and unsuspected mechanism regulating epidermal target innervation, but also provide the first evidence for a physiological role of NgR2 in the peripheral nervous system.
Asunto(s)
Epidermis/inervación , Ganglios Espinales/citología , Regulación del Desarrollo de la Expresión Génica/genética , Receptores de Superficie Celular/metabolismo , Células Receptoras Sensoriales/metabolismo , Versicanos/metabolismo , Animales , Animales Recién Nacidos , Células CHO , Péptido Relacionado con Gen de Calcitonina/metabolismo , Cricetulus , Proteínas F-Box , Glicoproteínas/metabolismo , Hiperalgesia/fisiopatología , Ratones , Ratones Noqueados , Proteínas de Neurofilamentos/metabolismo , Nociceptores/metabolismo , Proteína NgR2 , Umbral del Dolor/fisiología , Estimulación Física/efectos adversos , Unión Proteica/genética , Receptores de Superficie Celular/genética , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/metabolismo , Células Receptoras Sensoriales/clasificación , Células Receptoras Sensoriales/citología , Canales Catiónicos TRPV/metabolismo , Tubulina (Proteína)/metabolismo , Versicanos/química , Versicanos/genéticaRESUMEN
BACKGROUND: Many diseases and pathological conditions are characterized by transient or constitutive overproduction of reactive oxygen species (ROS). ROS are causal for ischemia/reperfusion (IR)-associated tissue injury (IRI), a major contributor to organ dysfunction or failure. Preventing IRI with antioxidants failed in the clinic, most likely due to the difficulty to timely and efficiently target them to the site of ROS production and action. IR is also characterized by changes in the activity of intracellular signaling molecules including the stress kinase p38MAPK. While ROS can cause the activation of p38MAPK, we recently obtained in vitro evidence that p38MAPK activation is responsible for elevated mitochondrial ROS levels, thus suggesting a role for p38MAPK upstream of ROS and their damaging effects. RESULTS: Here we identified p38MAPKα as the predominantly expressed isoform in HL-1 cardiomyocytes and siRNA-mediated knockdown demonstrated the pro-oxidant role of p38MAPKα signaling. Moreover, the knockout of the p38MAPK effector MAPKAP kinase 2 (MK2) reproduced the effect of inhibiting or knocking down p38MAPK. To translate these findings into a setting closer to the clinic a stringent kidney clamping model was used. p38MAPK activity increased upon reperfusion and p38MAPK inhibition by the inhibitor BIRB796 almost completely prevented severe functional impairment caused by IR. Histological and molecular analyses showed that protection resulted from decreased redox stress and apoptotic cell death. CONCLUSIONS: These data highlight a novel and important mechanism for p38MAPK to cause IRI and suggest it as a potential therapeutic target for prevention of tissue injury.
Asunto(s)
Apoptosis , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Estrés Oxidativo , Animales , Línea Celular , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Proteína Quinasa 14 Activada por Mitógenos/genética , Miocitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Endogámicas LewRESUMEN
Lipocalin-2 (Lcn2) expression contributes to ischemia and reperfusion injury (IRI) by enhancing pro-inflammatory responses. The aim of this work was to elucidate the regulation of Lcn2 during hypoxia and its effects on the expression of key chemokines and adhesion molecules. Lcn2 wt and Lcn2(-/-) mice were used in a heterotopic heart transplantation model. Quantitative RT-PCR was applied for chemokine gene expression analysis. Reporter gene studies were used to elucidate the regulation of the Lcn2 promoter by hypoxia. HIF-1ß expression led to a 2.4-fold induction of the Lcn2 promoter. Apart from an earlier onset of granulocyte infiltration in the Lcn2 wt setting after 2 h of reperfusion compared with the Lcn2(-/-) setting (P < 0.013), exogenous application of recombinant Lcn2 revealed a trend toward increase of granulocyte infiltration. Analyzed chemokines were expressed significantly higher in the Lcn2 wt setting at 2 h of reperfusion (P ≤ 0.05). The number of apoptotic cells observed in Lcn2(-/-) grafts was significantly higher than in the Lcn2 wt setting. Our results indicate that Lcn2 affects granulocyte infiltration in the reperfused graft by modulating the expression of chemokines, their receptors and the apoptotic rate.
Asunto(s)
Proteínas de Fase Aguda/fisiología , Quimiocinas/genética , Trasplante de Corazón , Lipocalinas/fisiología , Daño por Reperfusión Miocárdica/inmunología , Infiltración Neutrófila , Proteínas Oncogénicas/fisiología , Proteínas de Fase Aguda/genética , Animales , Apoptosis , Células COS , Moléculas de Adhesión Celular/genética , Chlorocebus aethiops , Lipocalina 2 , Lipocalinas/genética , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/patología , Proteínas Oncogénicas/genética , Regiones Promotoras Genéticas , Receptores de Quimiocina/genéticaRESUMEN
Suppressor of cytokine signalling 2 (SOCS-2), a dual effector of growth hormone signalling, was found to be heterogeneously expressed in murine liver parenchyma. Data from Affymetrix gene arrays, confirmed by quantitative RT-PCR using preparations of periportal and pericentral hepatocyte subpopulations as well as immunohistochemical detection, showed a preferential expression of SOCS-2 in pericentral hepatocytes. Stimulation of cultured periportal and pericentral hepatocyte subpopulations by different concentrations of growth hormone for 1 h resulted at 100 ng mL(-1) in a 1.6-fold and 4.3-fold increase of SOCS-2 mRNA, respectively. Likewise, insulin-like growth factor-1, another physiological target of growth hormone, was stimulated preferentially in pericentral hepatocytes. As growth hormone receptor was found to be homogeneously expressed in mouse liver parenchyma, our data indicate that growth hormone signalling downstream of growth hormone receptor is more sensitive and/or effective in pericentral than in periportal hepatocytes. Presumably, the heterogeneous distribution of SOCS-2 may contribute to the pericentral preference of growth hormone action via differential feedback.