Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB183

Poster Communications

Examining the behaviour of reactive oxygen species in specific intracellular locations of skeletal muscle fibres in inflamed and non-inflamed conditions.

M. Kalakoutis1, M. M. Steinz1, Z. Liu1, T. Mader1, P. Tavi2, L. T. Johanna1

1. Molecular muscle physiology and pathophysiology group, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden. 2. Pasi Tavi group, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Finland, United Kingdom.

Skeletal muscle weakness is a feature of diseases associated with inflammation, such as cancer and rheumatoid arthritis. Our previous work proposed that peroxynitrite-mediated 3-nirotyrosine accumulation on actin contributes to muscle weakness in rodent models of rheumatoid arthritis (Yamada et al., 2014). Furthermore, an increased protein expression of NADPH oxidase 2 (NOX2) in a rat model of arthritis suggested that reactive oxygen species (ROS) from NOX2 could be increased in inflammatory conditions, contributing to peroxynitrite formation (Yamada et al., 2015). Genetically encoded, redox sensitive green fluorescent proteins (roGFPs) enable the measurement of changes in ROS levels in specific intracellular locations, in live cells, in real time. Our aim was to use roGFPs to determine whether the ROS production from known intracellular sources differs between skeletal muscle fibres from inflammatory and healthy conditions. The sources of ROS assessed were the mitochondria (Mito-roGFP), NOX2 (p47-roGFP), and the cytosolic environment (PLPCX-Orp1). A protocol was developed in order to demonstrate that the fluorescent roGFP signals were reversible and could be measured and quantified based on a calibration, and that the impact of muscle contraction could be tested. Mouse flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation (mice were anaesthetised with 4.8% isofluorane) with one of three roGFPs: p47-roGFP, Mito-roGFP, or PLPCX-roGFP. FDB muscles were enzymatically dissociated into single fibres using collagenase type I. Confocal images were taken while fibres were perfused with standard Tyrode solution. Perfusion with dithiothreitol (DTT) and then hydrogen peroxide (H2O2) enabled measurement of maximum and minimum fluorescent signals, respectively. Muscle contraction was induced via electrical stimulation. Our pilot data clearly demonstrate that roGFPs respond dynamically to changes in intracellular ROS concentrations in real time, which can be quantified relative to the minimum and maximum signals. Furthermore, ROS concentrations were different in different intracellular sources in resting skeletal muscle fibres, being ~8 and ~5 fold higher in the mitochondrial networks compared to NOX2 located near the t-tubules, and the cytosolic environment, respectively. At the time of writing this abstract, the aforementioned protocol is being applied in an adjuvant-induced arthritis model, in order to study ROS behaviour in an inflamed and non-inflamed muscle condition at rest and following muscle contraction. A more precise understanding of the intracellular sources of ROS in inflammatory conditions will aid in the identification of a therapeutic target.

Where applicable, experiments conform with Society ethical requirements