Effect of RONS in 6-NBDG/glucose uptake in C2C12 myotubes and single isolated skeletal muscle fibres

[ES] Suplemento de la revista Free Radical Biology and Medicine: Effect of RONS in 6-NBDG/glucose uptake in C2C12 myotubes and single isolated skeletal muscle fibres

H2O2 biosensors HyPer2, HyPer3 and GFP2-Orp1 detect rapid pH changes due to environmental CO2 fluctuations, in addition to intracellular H2O2, in isolated skeletal muscle fibres

[EN] Hydrogen peroxide (H2O2) is one of the Reactive Oxygen Species (ROS) that seems to play an essential role in pathophysiological processes. H2O2 might act as a signaling molecule and modulate different crucial cellular signaling pathways, such as the glucose uptake in skeletal muscle, where H2O2 has been proposed to play an important role. HyPer2, HyPer3 and GFP2-Orp1 are hydrogen peroxide biosensors. We use these biosensors to monitor intracellular H2O2 in single skeletal muscle fibres isolated from the flexor digitorum brevis (FDB) mouse muscle. Previously, the coding sequences of these biosensors were microinjected and electroporated in FDB. Isolated fibres in culture that expressed one of the biosensors were settled incubation chamber coupled to the fluorescence microscope. The chamber maintains temperature (37ºC), environmental CO2 (5%) and humidity. Different time course experimental conditions were performed where fibres were exposed to different agents (insulin, interleukin 1β, H2O2, DTT) and intracellular H2O2 flux was registered in real time using fluorescence microscopy imaging analysis. We observed that when there were environmental CO2 (5%) fluctuations, due to initial medium stabilization or occasional interruption of CO2 supply, the biosensors showed changes in the fluorescence emission, which were registered. The main consequence of CO2 fluctuations is the change in the pH of medium. The main part of the biosensor structure is a fluorescent protein, YFP in de case of HyPer2 and HyPer3, and GFP2 in GFP2-Orp1. It has been reported that these fluorescent proteins are sensitive to pH and this might be a disadvantage for the biosensors. However, we believe that this pH sensitivity should be considered as an additional property of this biosensors, since they provide information in real time about the rapid changes of pH due to environmental fluctuation of CO2 and likely other gases such as O2 or N2

Effect of aging in functional redox state of single isolated skeletal muscle fibres

[EN] Skeletal muscle constantly produces reactive oxygen species (ROS). During contractile activity ROS are generated in skeletal muscle fibres. There is considerable support for an involvement of ROS in the process of aging. Several studies indicate that adaptive responses of skeletal muscle that are activated and regulated by ROS are disrupted during aging. The aim of this study was to monitor, in real time, intracellular ROS production in single skeletal muscle fibres from old and young mice and study the effect of contractile activity in these cells. Following evaluate and correlate the potential changes in intracellular ROS production with glutathione redox state and antioxidant enzymatic activities in muscle. Single skeletal muscle fibres were isolated from the Flexor Digitorus Brevis muscle from young (2-4 monthold) and old (26-28 month-old) C57BL/6 mice. Fibres were loaded with DCFH-DA, a fluorophore probe that allows the quantification of intracellular ROS generation by fluorescence microscopy imaging. Contractile activity was induced in fibres by electrical stimulation. Glutathione redox state and activity of antioxidant enzymes were analysed in gastrocnemious muscle. Intracellular basal level of ROS was higher in fibres from old mice. Contractile activity induced increase of ROS generation in fibres from young mice. However, this response was attenuated in fibres from old mice. Glutathione redox state was significant different, in favour of oxidized glutathione, in muscles from old mice. Glutathione peroxidase and catalase activities were significantly augmented in muscles from old mice. In conclusion, the process of aging modifies the basal redox status in skeletal muscle fibres in favour of oxidation and induces adaptation mechanisms of antioxidant defences. These are not able to neutralize the increase of basal oxidation, but they might lead to the attenuation of ROS produced by contractile activity observed in fibres from old mice

HyPer biosensor to monitor intracellular hydrogen peroxide in skeletal muscle cells

[EN] Suplemento de la revista Free Radical Biology and Medicine: HyPer biosensor to monitor intracellular hydrogen peroxide in skeletal muscle cells

Reactive oxygen species and loss of muscle fibres during ageing

[ES] Sumario de la revista Neuromuscular Disorders: Especies reactivas de oxígeno y pérdida de fibras musculares durante el envejecimiento

Role of S-adenosylmethionine on the hepatobiliary homeostasis of glutathione during cyclosporine A treatment

[EN] The effects of cyclosporine A (CyA) treatment on the hepatic content and biliary output of reduced (GSH) and oxidized (GSSG) glutathione and lipid peroxidation in the liver, and the ability of S-adenosylmethionine (SAMe) to antagonize the CyA-induced alterations were studied in male Wistar rats. To evaluate the efficacy of SAMe, three CyA and SAMe protocols were used: Cotreatment with SAMe plus CyA, pretreatment with SAMe before starting cotreatment, and post-treatment with SAMe after beginning treatment with CyA alone. CyA treatment for one and four weeks depleted liver GSH, decreased the GSH/GSSG ratio and significantly reduced GSH and GSSG biliary concentrations and secretion rates. Additionally, long-term treatment enhanced lipid peroxidation. By contrast, when the rats were treated with CyA plus SAMe using any of the administration protocols, SAMe was seen to be efficient in antagonizing the GSH hepatic depletion, the changes in hepatic GSH/GSSG ratio and the increase induced by CyA in lipid peroxidation. Furthermore, SAMe also abolished the effects of CyA on the biliary secretion rates of GSH and GSSG. The efficacy of SAMe was similar, regardless of the administration protocols used. In conclusion, our results clearly demonstrate that SAMe is good for preventing, antagonizing and reversing the CyA-induced alterations in the hepatobiliary homeostasis of glutathione

Involvement of Reactive Oxygen Species (ROS) inskeletal muscle function during ageing: Study in amodel of isolated single skeletal muscle fibre

[ES] Suplemento de la revista Free Radical Biology and Medicine: Involvement of Reactive Oxygen Species (ROS) inskeletal muscle function during ageing: Study in amodel of isolated single skeletal muscle fibre

Expression and functional analysis of the hydrogen peroxide biosensors HyPer and HyPer2 in C2C12 myoblasts/myotubes and single skeletal muscle fibres

[EN] Hydrogen peroxide (H2O2) is generated in cells and plays an important role as a signalling molecule. It has been reported that H2O2 is involved in physiological and pathological processes in skeletal muscle. However, H2O2 detection in cells with traditional techniques produces frequent artefacts. Currently, the HyPer biosensor detects intracellular H2O2 specifcally in real time using fuorescence microscopy. The aim of this study was to develop and optimize approaches used to express the HyPer biosensor in diferent models of skeletal muscle cells, such as the C2C12 myoblast/myotube cell line and mature skeletal muscle fbres isolated from C57BL/6J mice, and to measure intracellular H2O2 in real time in these cells. The results show that the expression of the HyPer biosensor in skeletal muscle cells is possible. In addition, we demonstrate that HyPer is functional and that this biosensor detects changes and fuctuations in intracellular H2O2 in a reversible manner. The HyPer2 biosensor, which is a more advanced version of HyPer, presents improved properties in terms of sensitivity in detecting lower concentrations of H2O2 in skeletal muscle fbres. In conclusion, the expression of the HyPer biosensor in the diferent experimental models combined with fuorescence microscopy techniques is a powerful methodology to monitor and register intracellular H2O2 specifcally in skeletal muscle. The innovation of the methodological approaches presented in this study may present new avenues for studying the role of H2O2 in skeletal muscle pathophysiology. Furthermore, the methodology may potentially be adapted to yield other specifc biosensors for diferent reactive oxygen and nitrogen species or metabolites involved in cellular functions

El uso de la musicalización por parte de los alumnos como herramienta mnemotécnica de la asignatura de Farmacología del Grado de Enfermería

Memoria ID-163. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2018-2019

Implementación del Estudio de Casos utilizando aplicaciones digitales educativas, como estrategia activa de aprendizaje cooperativo de Farmacología por estudiantes de Medicina

Memoria ID-151. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2019-2020