Investigating the use of omega three fish oils as protective agents against cytokine-induced skeletal muscle damage
IntroductionMuscle injury and disease impacts on the lives of adults and children, contributing tosevere morbidity and increased mortality e.g. Muscular dystrophy affects over 70,000people UK wide (Muscular Dystrophy UK). Treatment of muscle disorders contributeto unemployment rates and cost the NHS billions of pounds per year. Growth factorsand cytokines regulate skeletal muscle homeostasis and growth and cytokinedysregulation may cause irreversible damage. Currently, poorly effective therapiesfor muscle injury include non-steroidal/ steroidal anti-inflammatory drugs andphysiotherapy.Previous research indicates omega three fish oils including Eicosapentaenoic acid(ERA), may offer protective anti-fibrotic properties and favoring useful repairprocesses. ERA decreases muscle protein degradation in cancer patients (Smith etal 2005) and increases the rate of protein synthesis in elderly patients (Smith et al2011). In vitro studies show ERA down-regulates chronic inflammation via inhibitionof NFKB activation (Huang et al 2011) and fibrosis via suppression of TransformingGrowth Factor-Pi (TGF-pi,Das 2004). Our group showed ERA protection fromcytokine (TNFa) induced muscular atrophy in a murine cell model (Magee et al2008). Further, ERA may have intracellular signaling component interactions and bedependent on PPARy (Magee et al 2012). ERA and DMA interact with lipid rafts (Liet al 2005) therefore may be able to displace receptors from signaling domains.Development of omega 3 fatty acid for therapeutic uses in fibrosis and tissue repairrequires a deeper understanding of additional intracellular signaling components,which is facilitated by a wider range of in vitro models.ObjectivesTo establish whether ERA, and its relative Docosahexaenoic acid (DMA), couldprevent cytokine-induced damage in models of atrophy and pathological fibrosis andidentify some of the mechanisms involved.MethodsUsing the cell line, C2C12, a well established model of myogenesis, ERA and DMAwere investigated to determine if they could attenuate TGFpi induced muscularfibrosis and TNFa/ TNF-related weak inducer of apoptosis (TWEAK) induced skeletalmuscle inflammation/wasting. Myogenesis was assessed by morphology andmyotube measurements or by qPCR of myogenic genes. Fibrosis was assessed byexpression of fibrotic proteins by immunocytochemistry or fibrotic genes by qPCR.Inflammation (IL-6 release) was assessed by ELISA. Signaling mechanisms wereinvestigated using antagonists to: Peroxisome Proliferator-Activated Receptor-y(PPARv) and the sphingosine kinase pathway. ERA and DMA effects on cytokinereceptors were assessed by flow cytometry.ResultsERA and DMA prevented decreases in myogenesis induced by all three cytokinesthat reduced the myotube diameter and myogenic index of differentiating anddifferentiated C2C12 cells. When used as a co-treatment or 2 hour pre-treatment,ERA and DMA returned myotube diameters to control levels and significantlyincreased myogenic index compared to cytokine alone (p<0.05). ERA alleviated TGFinducedmyogenic gene expression changes. ERA and DMA also prevented TGFinducedincreases in proliferation during low serum conditions. ERA and DMAattenuated TNFa and TWEAK induced IL-6 release and reduced TGFpi inducedexpression of fibrotic markers at both the level of gene and protein expression.PPARy blockade rendered ERA and DMA no longer effective in preventing TNFa andTWEAK-induced decreases in myotube diameter and myogenic index and increasedIL-6 production. Blocking PPARy had no effect on ERA and DHA's ability to attenuateTGF(3-induced decreases in myogenesis. Antagonising PPARa had no effect on ERAand DMAs ability to attenuate TWEAK associated decreases in myogenesis orincreased IL-6 production. ERA and DMA prevented TGFp-induced increases in theratio of Sphingosine-1-phosphate receptor 3 (S1Pr3) to S1Pr1 gene expression andincreased Sphingosine Kinase 1 gene expression There were no significantinteractions of ERA and/or DMA with TGF(3, TNFa and TWEAK receptors (TGFpRII,TNFRI and Fn14 respectively).ConclusionOur findings suggest ERA and DMA are potentially useful 'neutriceuticals' that reducefibrosis and improve muscle repair in vivo. Their mode of action is PPARydependent in inflammatory skeletal damage (TNFa and TWEAK) but PPARyindependent to profibrotic TGF(3. EPA and DMA attenuate inflammation PPARaindependently.Taking this work further into other in vitro models or in vivo models will establishwhether these omega-3 fatty acids have the ability to be used as therapeutic agentsalone or in conjunction with other medicines to alleviate suffering caused by skeletalmuscle damage.
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