Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB184

Poster Communications

Peroxynitrite-induced modifications results in increased open probability of RyR1 in skeletal muscle

M. M. Steinz1, N. Beard2, L. T. Johanna1

1. Department of Physiology and Pharmacology, Karolinska Institutet, Solna, Stockholm, Sweden. 2. Faculty or Science and Technology, University of Canberra, Canberra, Australian Capital Territory, Australia.

Skeletal muscle weakness contributes to decreased quality of life for patients with chronic inflammatory conditions, such as rheumatoid arthritis (RA). We have previously reported that muscle weakness in mice with arthritis (collagen-induced arthritis) was accompanied by increased peroxynitrite-derived nitrosative stress (3-nitrotyrosine, 3-NT) on the ryanodine receptor (RyR1) complex (Yamada T, et al 2015). RyR1 is the major intracellular Ca2+ release channel and essential for apt excitation-contraction coupling in skeletal muscle. Moreover, altered RyR1 Ca2+ release has been linked to muscle dysfunction in several clinical aspects, e.g. in muscle dystrophies (Bellinger et al., 2009; Hernández-Ochoa et al., 2015; Lanner et al., 2012). However, the functional effects of peroxynitrite-induced RyR1 modifications are still unclear. The aim of this study was to induce peroxynitrite-induced RyR1 modifications using 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1) to identify modified amino acid residues with mass spectrometry and examine RyR1 gating properties using single channel recordings. SIN-1 [1mM, 15 min] introduced a significant 1.6-fold increase in 3-NT levels on RyR1 as identified by immunoblotting (t-test, p< 0.05, n=4, SE= 0.05). In the presence of SIN-1, RyR1 single channel recordings in planar bilayers showed a 2.7-fold increase in open probability (t-test, p< 0.05, n=6, SEM= 0.12). Mass spectrometry analysis of SIN-1 treated RyR1 revealed 28 specific amino acid residues with oxidative modifications (15 tyrosine residues carried a 3-NT modification and malondialdehyde (MDA) adducts were identified on 13 residues). The oxidative modifications were located in the linker regions as well as in the pore domain of RyR1. Here we have specifically identified RyR1 residues that undergo peroxynitrite-induced protein modification (3-NT and MDA), which were directly linked to increased open probability of RyR1. The identified 3-NT and MDA-modified RyR1 residues appear to be clustered in regions of RyR1 that may serve as drug targets in the search for novel therapeutics to counteract muscle dysfunction associated with increased oxidative stress and altered RyR1 function.

Where applicable, experiments conform with Society ethical requirements