OP0247 IN MYOSITIS MUSCLE FIBRE PLAYS A DIRECT AND CRITICAL ROLE IN THERAPEUTIC RESPONSE TO GLUCOCORTICOIDS

Background:Myositis are rare autoimmune diseases, affecting more women than men, characterized by chronic inflammation of skeletal muscle causing muscle weakness, decreased quality of life and increased mortality.Glucocorticoids (GC) are potent anti-inflammatory drugs, and are the first line treatment of myositis. They improve muscle strength of myositis patients (therapeutic effect), yet muscle recovery is generally only partial. Moreover, GC have an iatrogenic effect on skeletal muscle fibre leading to steroid myopathy. Thus myositis care has to be improved. Despite the autoimmune terrain of myositis, our team has recently shown that muscle fibres themselves develop immuno-metabolic modifications that participate to muscle weakness and perpetuation of the disease1. GC effects are mediated by the glucocorticoid receptor (GR), which is expressed in various cell types including immune cells and myofibres, but the cells mediating therapeutic responses remain to be determined.Objectives:Unravel the mechanisms underlying the therapeutic effect of GC in myositis, particularly elucidate the role of skeletal muscle fibres.Methods:Experimental myositis was induced in eight to ten week-old C57BL/6J female mice by a single intradermal injection of part of skeletal muscle fast-type C protein along with Freund’s adjuvant and an intraperitoneal (IP) injection of pertussis toxin, as previously described2. Prednisone (PDN) was administered 14 days (D) after the immunization at 1 mg/kg/day for 7 days by gavage. Mice were euthanized 21 days after myositis induction. Muscle strength was assessed by grip test at D 0, before the 1st PDN administration (D 14) and the day before sacrifice (D 20). To investigate whether the PDN effects are mediated by myofibre, we generated transgenic mice carrying two LoxP sites within the GR gene in muscle, expressing the tamoxifen-inducible Cre-ERT2 recombinase selectively in skeletal muscle fibre (HSA-CreERT2/GR L2/L2). Tamoxifen (1 mg/day for 5 days by IP injection) was administered 9 days after immunization to induce GR ablation selectively in skeletal muscle fibres (GR(i)skm-/- mice). Similar treatments were applied to GR L2/L2 that do not express Cre-ER(T2), and served as controls.We compared 4 groups of myositis mice, GR L2/L2 treated by PDN (n=9) or vehicle (n=9) and GR(i)skm-/- treated by PDN (n=10) or vehicle (n=10), by grip test and at the histological level (hematoxylin-eosin (HE) and Gomori trichrome (GT) staining). Moreover, LC3 expression was studied by RTqPCR and western blot.Results:Muscle strength was decreased in both GR L2/L2 and GR(i)skm-/- myositis mice from D 14 to D 20. GR L2/L2 myositis mice recovered muscle strength after PDN treatment; no significant difference compared to D 0 was detected. In contrast, PDN did not improve muscle strength in GR(i)skm-/- myositis mice (Figure 1).HE and GT staining did not reveal quantitative differences in inflammatory infiltrate. Necrotic and degenerative fibres were detected in the 4 groups. At RTqPCR, LC3, an autophagy marker, was upregulated in PDN-treated GR L2/L2 myositis mice compared to untreated GR L2/L2 myositis mice; moreover it was 2-fold more expressed in PDN-treated GR L2/L2 myositis mice compared to PDN-treated GR(i)skm-/- mice.Conclusion:GR in skeletal muscle fibre is crucial to mediate the therapeutic response to GC in a murine model of myositis. Autophagy is one of the candidate pathways controlled by myofibre GR underlying this effect.References:[1]Meyer A et al. IFN-β-induced reactive oxygen species and mitochondrial damage contribute to muscle impairment and inflammation maintenance in dermatomyositis. Acta Neuropathol. 2017 Oct;134(4):655-666.[2]Sugihara T et al. A new murine model to define the critical pathologic and therapeutic mediators of polymyositis. Arthritis Rheum. 2007 Apr;56(4):1304-14.Disclosure of Interests:None declared

The relationship between myonuclear number and protein synthesis in individual rat skeletal muscle fibre

Skeletal muscle cell has numerous nucleus within a cell. The nucleus has been considered as the central organelle for muscle protein synthesis. However, it is unclear whether myonuclear number associate with the MPS capacity within the individual muscle fibre. Therefore, the purpose of the present study was to reveal the relationship between myonuclear number per unit muscle fibre length and MPS under basal and conditions of elevated MPS by high-intensity muscle contraction (HiMC) using in vivo nascent protein labelling technique (SUnSET) in rodent. As the result, myonuclear number positively correlate to the MPS in individual muscle fibre at the basal condition. Similarly, ribosomal protein S6 (rpS6) content, which is a rough estimate of ribosome content, was positively correlated with MPS. However, myonuclear number was not associated with rpS6 content. In contrast to the basal condition, where MPS was increased by acute HiMC, no correlation was observed between myonuclear number and MPS, but the association between rpS6 and MPS was maintained. Importantly, these observations indicate that the number of nuclei in individual myofibers is related only to the MPS at rest. However, the ribosome content in individual fibres is related to the MPS of individual myofibers both at rest and in the elevated MPS, due to HiMC.

A highly prevalent SINE mutation in the myostatin (MSTN) gene promoter is associated with low circulating myostatin concentration in Thoroughbred racehorses

Horse racing is a popular and financially important industry worldwide and researchers and horse owners are interested in genetic and training influences that maximise athletic performance. An association has been found between the presence of a short interspersed nuclear element (SINE) mutation in the myostatin (MSTN) gene promoter and optimal race distance in Thoroughbred horses. There is previous laboratory evidence that this mutation reduces MSTN expression in a cell culture model and influences skeletal muscle fibre type proportions in horses. Manipulating MSTN expression has been proposed for illicit gene doping in human and equine athletes and already, researchers have generated homozygous and heterozygous MSTN-null horse embryos following CRISPR/Cas9 editing at the equine MSTN locus and nuclear transfer, aiming artificially to enhance performance. To date however, the role of the naturally-occurring equine MSTN SINE mutation in vivo has remained unclear; here we hypothesised that it reduces, but does not ablate circulating myostatin expression. Following validation of an ELISA for detection of myostatin in equine serum and using residual whole blood and serum samples from 176 Thoroughbred racehorses under identical management, horses were genotyped for the SINE mutation by PCR and their serum myostatin concentrations measured. In our population, the proportions of SINE homozygotes, heterozygotes and normal horses were 27%, 46% and 27% respectively. Results indicated that horses that are homozygous for the SINE mutation have detectable, but significantly lower (p < 0.0001) serum myostatin concentrations (226.8 pg/ml; 69.3–895.4 pg/ml; median; minimum–maximum) than heterozygous (766 pg/ml; 64.6–1182 pg/ml) and normal horses (1099 pg/ml; 187.8–1743 pg/ml). Heterozygotes have significantly lower (p < 0.0001) myostatin concentrations than normal horses. Variation in serum myostatin concentrations across horses was not influenced by age or sex. This is the first study to reveal the direct functional effect of a highly prevalent mutation in the equine MSTN gene associated with exercise performance. Determining the reason for variation in expression of myostatin within SINE-genotyped groups might identify additional performance-associated environmental or genetic influences in Thoroughbreds. Understanding the mechanism by which altered myostatin expression influences skeletal muscle fibre type remains to be determined.

Cytoskeleton-extracellular matrix interaction is required to maintain mitochondrial Ca2+ control in mouse skeletal muscle fibre

Cells rapidly lose their physiological phenotype upon isolation from their native microenvironment. Here, we investigated the role of the extracellular matrix (ECM) for mitochondrial morphology and Ca2+ handling in adult mouse skeletal muscle fibres. Adult skeletal muscle fibres were isolated from mouse toe muscle either by collagenase-induced dissociation of the ECM or by mechanical dissection that leaves the proximate ECM intact. Experiments were generally performed four hours after cell isolation. At this time, the expression of genes encoding for structural proteins was lower in enzymatically dissociated than in mechanically dissected fibres. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria in enzymatically dissociated than in mechanically dissected fibres. The increase in free cytosolic [Ca2+] during repeated tetanic stimulation was similar in the two groups of fibres, but this was accompanied by an excessive mitochondrial Ca2+ uptake only in enzymatically dissociated muscle fibres. The aberrant mitochondrial Ca2+ uptake was partially prevented by the mitochondrial Ca2+ uniporter inhibitor Ru360 and by cyclosporine A and NV556, which inhibit the mitochondrial matrix protein PPIF (also called cyclophilin D). Importantly, inhibition of PPIF with NV556 significantly improved survival of mice with mitochondrial myopathy in which muscle mitochondria take up excessive amounts of Ca2+ also with intact ECM. In conclusion, skeletal muscle fibres isolated by collagenase-induced dissociation of the ECM display aberrant mitochondrial Ca2+ uptake, which involves a PPIF-dependent mitochondrial Ca2+ influx resembling that observed in mitochondrial myopathies.

A Highly Prevalent SINE Mutation in The Myostatin (MSTN) Gene Promoter Is Associated With Low Circulating Myostatin Concentration in Thoroughbred Racehorses

Horse racing is a popular and financially important industry worldwide and researchers and horse owners are interested in genetic and training influences that maximise athletic performance. An association has been found between the presence of a short interspersed nuclear element (SINE) mutation in the myostatin (MSTN) gene promoter and optimal race distance in Thoroughbred horses. There is previous laboratory evidence that this mutation reduces MSTN expression in a cell culture model and influences skeletal muscle fibre type proportions in horses. Manipulating MSTN expression has been proposed for illicit gene doping in human and equine athletes and already, researchers have generated homozygous and heterozygous MSTN-null horse embryos following CRISPR/Cas9 editing at the equine MSTN locus and nuclear transfer, aiming artificially to enhance performance. To date however, the role of the naturally-occurring equine MSTN SINE mutation in vivo has remained unclear; here we hypothesised that it reduces, but does not ablate circulating myostatin expression. Following validation of an ELISA for detection of myostatin in equine serum and using residual whole blood and serum samples from 176 Thoroughbred racehorses under identical management, horses were genotyped for the SINE mutation by PCR and their serum myostatin concentrations measured. In our population, the proportions of SINE homozygotes, heterozygotes and normal horses were 27%, 46% and 27% respectively. Results indicated that horses that are homozygous for the SINE mutation have detectable, but significantly lower (p<0.0001) serum myostatin concentrations (226.8pg/ml; 69.3-895.4pg/ml; median; minimum-maximum) than heterozygous (766pg/ml; 64.6-1182pg/ml) and normal horses (1099pg/ml; 187.8-1743pg/ml). Heterozygotes have significantly lower (p<0.0001) myostatin concentrations than normal horses. Variation in serum myostatin concentrations across horses was not influenced by age or sex. This is the first study to reveal the direct functional effect of a highly prevalent mutation in the equine MSTN gene associated with exercise performance. Determining the reason for variation in expression of myostatin within SINE-genotyped groups might identify additional performance-associated environmental or genetic influences in Thoroughbreds. Understanding the mechanism by which altered myostatin expression influences skeletal muscle fibre type remains to be determined.

Role of Sphingosine 1-Phosphate Signalling Axis in Muscle Atrophy Induced by TNFα in C2C12 Myotubes

Skeletal muscle atrophy is characterized by a decrease in muscle mass causing reduced agility, increased fatigability and higher risk of bone fractures. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), are strong inducers of skeletal muscle atrophy. The bioactive sphingolipid sphingosine 1-phoshate (S1P) plays an important role in skeletal muscle biology. S1P, generated by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK1/2), exerts most of its actions through its specific receptors, S1P1–5. Here, we provide experimental evidence that TNFα induces atrophy and autophagy in skeletal muscle C2C12 myotubes, modulating the expression of specific markers and both active and passive membrane electrophysiological properties. NMR-metabolomics provided a clear picture of the deep remodelling of skeletal muscle fibre metabolism induced by TNFα challenge. The cytokine is responsible for the modulation of S1P signalling axis, upregulating mRNA levels of S1P2 and S1P3 and downregulating those of SK2. TNFα increases the phosphorylated form of SK1, readout of its activation. Interestingly, pharmacological inhibition of SK1 and specific antagonism of S1P3 prevented the increase in autophagy markers and the changes in the electrophysiological properties of C2C12 myotubes without affecting metabolic remodelling induced by the cytokine, highlighting the involvement of S1P signalling axis on TNFα-induced atrophy in skeletal muscle.

Heme-Induced Oxidation of Cysteine Groups of Myofilament Proteins Leads to Contractile Dysfunction of Permeabilized Human Skeletal Muscle Fibres

Heme released from red blood cells targets a number of cell components including the cytoskeleton. The purpose of the present study was to determine the impact of free heme (20–300 µM) on human skeletal muscle fibres made available during orthopedic surgery. Isometric force production and oxidative protein modifications were monitored in permeabilized skeletal muscle fibre segments. A single heme exposure (20 µM) to muscle fibres decreased Ca2+-activated maximal (active) force (Fo) by about 50% and evoked an approximately 3-fold increase in Ca2+-independent (passive) force (Fpassive). Oxidation of sulfhydryl (SH) groups was detected in structural proteins (e.g., nebulin, α-actinin, meromyosin 2) and in contractile proteins (e.g., myosin heavy chain and myosin-binding protein C) as well as in titin in the presence of 300 µM heme. This SH oxidation was not reversed by dithiothreitol (50 mM). Sulfenic acid (SOH) formation was also detected in the structural proteins (nebulin, α-actinin, meromyosin). Heme effects on SH oxidation and SOH formation were prevented by hemopexin (Hpx) and α1-microglobulin (A1M). These data suggest that free heme has a significant impact on human skeletal muscle fibres, whereby oxidative alterations in structural and contractile proteins limit contractile function. This may explain and or contribute to the weakness and increase of skeletal muscle stiffness in chronic heart failure, rhabdomyolysis, and other hemolytic diseases. Therefore, therapeutic use of Hpx and A1M supplementation might be effective in preventing heme-induced skeletal muscle alterations.