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 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

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

Implementación metodológica fundamentada en la combinación de biosensores de especies reactivas del oxígeno y nitrógeno (RONS) y análisis cuantitativo de imagen de microscopía de fluorescencia para el estudio del papel señalizador y modulador de las RONS en la captación de glucosa en el músculo esquelético

Tesis por compendio de publicaciones[ES] La investigación a la que se refiere este registro hace referencia a una tesis elaborada por compendio de publicaciones. El objetivo general de este trabajo consistió en implementar nuevas técnicas basadas en la utilización de biosensores intracelulares en el músculo esquelético que permitan la detección específica de peróxido de hidrógeno, óxido nítrico o del estado redox celular. Investigar si la aplicación de diferentes estímulos aplicados a la fibra muscular (i.e. exposición hormonal u otros compuestos químicos) provoca cambios en la concentración de peróxido de hidrógeno y óxido nítrico, y si afecta a la captación de glucosa y de esta manera, a la resistencia a insulina que se manifestaría en el envejecimiento o en la diabetes tipo 2. El objetivo general se divide en los siguientes: Desarrollar y validar las técnicas para internalizar los vectores, en los que se encuentra la secuencia codificante de los biosensores de peróxido de hidrógeno, óxido nítrico y estado redox del glutatión, en células de músculo esquelético: línea celular de mioblastos / miotubos C2C12 y fibras musculares individuales aisladas del músculo flexor digitorum brevis, y conseguir la expresión de estos biosensores de RONS en estas células. Validación funcional de los biosensores de RONS para la detección y cuantificación del flujo intracelular de RONS que pueda ser ocasionado por diferentes intervenciones experimentales que puedan producir cambios en la homeostasis intracelular de las RONS. Desarrollo y optimización de una metodología basada en el análogo de glucosa 6-NBDG y microscopia de fluorescencia cuantitativa, que permita la determinación de la captación de glucosa en células de músculo esquelético: línea celular de mioblastos/miotubos C2C12 y fibras musculares individuales aisladas del músculo flexor digitorum brevis. Ensayar diferentes agentes que pueden afectar a la homeostasis de las RONS, de forma específica a la concentración intracelular de peróxido de hidrógeno o al óxido nítrico, y también al estado redox y comprobar en qué medida estos cambios afectan a la captación de glucosa por las células musculares: mioblastos/miotubos C2C12 y fibras musculares individuales aisladas del músculo flexor digitorum brevis

Genetically Encoded Biosensors to Monitor Intracellular Reactive Oxygen and Nitrogen Species and Glutathione Redox Potential in Skeletal Muscle Cells

Reactive oxygen and nitrogen species (RONS) play an important role in the pathophysiology of skeletal muscle and are involved in the regulation of intracellular signaling pathways, which drive metabolism, regeneration, and adaptation in skeletal muscle. However, the molecular mechanisms underlying these processes are unknown or partially uncovered. We implemented a combination of methodological approaches that are funded for the use of genetically encoded biosensors associated with quantitative fluorescence microscopy imaging to study redox biology in skeletal muscle. Therefore, it was possible to detect and monitor RONS and glutathione redox potential with high specificity and spatio-temporal resolution in two models, isolated skeletal muscle fibers and C2C12 myoblasts/myotubes. Biosensors HyPer3 and roGFP2-Orp1 were examined for the detection of cytosolic hydrogen peroxide; HyPer-mito and HyPer-nuc for the detection of mitochondrial and nuclear hydrogen peroxide; Mito-Grx1-roGFP2 and cyto-Grx1-roGFP2 were used for registration of the glutathione redox potential in mitochondria and cytosol. G-geNOp was proven to detect cytosolic nitric oxide. The fluorescence emitted by the biosensors is affected by pH, and this might have masked the results; therefore, environmental CO2 must be controlled to avoid pH fluctuations. In conclusion, genetically encoded biosensors and quantitative fluorescence microscopy provide a robust methodology to investigate the pathophysiological processes associated with the redox biology of skeletal muscle

Biosensor HyPer2 to monitor intracellular hydrogen peroxide induced by insulin and interleukin 1 beta stimulation in isolated skeletal muscle fibres

Resumen póster de Congres

Effect of RONS-Induced Intracellular Redox Homeostasis in 6-NBDG/Glucose Uptake in C2C12 Myotubes and Single Isolated Skeletal Muscle Fibres

The glucose uptake in skeletal muscle is essential to produce energy through ATP, which is needed by this organ to maintain vital functions. The impairment of glucose uptake compromises the metabolism and function of skeletal muscle and other organs and is a feature of diabetes, obesity, and ageing. There is a need for research to uncover the mechanisms involved in the impairment of glucose uptake in skeletal muscle. In this study, we adapted, developed, optimised, and validated a methodology based on the fluorescence glucose analogue 6-NBDG, combined with a quantitative fluorescence microscopy image analysis, to determine the glucose uptake in two models of skeletal muscle cells: C2C12 myotubes and single fibres isolated from muscle. It was proposed that reactive oxygen and nitrogen species (RONS) and redox homeostasis play an important role in the modulation of intracellular redox signalling pathways associated with glucose uptake. In this study, we prove that the prooxidative intracellular redox environment under oxidative eustress produced by RONS such as hydrogen peroxide and nitric oxide improves glucose uptake in skeletal muscle cells. However, when oxidation is excessive, oxidative distress occurs, and cellular viability is compromised, although there might be an increase in the glucose uptake. Based on the results of this study, the determination of 6-NBDG/glucose uptake in myotubes and skeletal muscle cells is feasible, validated, and will contribute to improve future research

Monitoring intracellular hydrogen peroxide changes in skeletal muscle fibres by HyPer, a genetically encoded hydrogen peroxide biosensor

[EN] Suplemento de la revista Free Radical Biology and Medicine: Monitoring intracellular hydrogen peroxide changes in skeletal muscle fibres by HyPer, a genetically encoded hydrogen peroxide biosensor

Aislamiento de fibras musculares del músculo Flexor Digirorum Brevis (FDB) de ratón y recolección de las fibras aisladas en cultivo, utilizando equipamiento patch-clamp, para su posterior procesamiento y análisis

[ES]Vídeo que muestra el procedimiento para aislar las fibras musculares del músculo FDB de ratón y recolección de las fibras aisladas en cultivo, utilizando equipamiento patch-clamp, para su posterior análisi