Myokines Regulate The Muscle Regeneration After Chronic Injury By Modulating Fibro/Adipogenic Progenitors

Collagen deposition and fatty infiltration is commonly identified in chronic muscle injury.Fibrosis and fatty degeneration can impair the muscular regeneration,and reduce the quality of muscle and atrophy.Chronic rotator cuff tear(RCT)is a classic chronic muscle-tendon injury.Medium to severe RCT often presents obvious fatty infiltration and fibrosis,that can reduce the elasticity and strength of muscle,thus elevating surgical failure rate and retear rate.Fibro/adipogenic progenitors(FAPs)are considered to give rise to the primary source of fatty infiltration and fibrosis after muscle chronic injury.It has a "double-edged sword" effect.In acute injury,FAPs can promote the proliferation and differentiation of satellite cells in muscle.On the otherwise,it will differentiate into adipocyte and myofibroblast after chronic injury and cause the muscular degeneration.Based on above,it is critical to reserve the ability of regeneration of FAPs while inhibiting the abnormal differentition after chronical injury.Previous studies found microenvironment determines the fate of FAP s after muscle injury.After reciprocally transplating FAPs between regenerating muscle and degenerating muscles,FAPs adapted their fate to the new environment but not the one they were derived from.Thus,the change of microenvironment may be a possible way to modulate the rate of FAPs and furthermore to impact the muscular repair.Muscle currently is regarded as an endocrins organ,it can generate and secerete varietis of cytokines,called "myokines".Myokines can regulate the biological functions of target tissues or organs by paracrine,autocrine or by circulation.Myokines highly express in muscle and form a local microenvironment.Futhermore,myokines can regulate the differentian,proliferation of cells and lipid metabolism.We speculate the expression of myokines will substantially change after chronical muscle injury due to atrophy.This change may have effects on the fate of FAPs,including adipogenesis,fibrosis and muscular regeneration.To verify this hypothesis,we firstly screened the changed myokines after muscle chronical injury and found the level of IL-15 reduced significantly after fatty infiltration.Then we tested the effect of IL-15 on the proliferation and differentiation of FAPs and the signalling pathway both in vivo and in vitro and the impact of IL-15 on muscle regeneration after chronical injury.Finally,we analysed the correlation between the expression of IL-15 and the number of FAPs and the degree of fibrosis in human.These findings supported the potential role of IL-15 as a modulator on fate of FAPs in injured muscle and as a novel therapy for chronic muscle injury.Objectives1.To identify the candidate myokines that may regulate the muscle fatty degeneration.2.To study the regulation of IL-15 on muscular fatty infiltration and its mechanism.3.To investigate the effect of IL-15 on the proliferation of FAPs in vivo and in vitro and its singalling pathway.4.To clarify the impact of IL-15 on muscle regeneration.5.To test correlation between the expression of IL-15 and muscle degeneration in human chronic rotator cuff tear.Methods1.Screening candidate myokines that may regulate the fatty infiltration after muscle chronic injury.Muscle degeneration model was induced by injection of glycerol.The tissue was made into cryosections.Muscular atrophy and fatty infiltration was observed by HE staining,Oil Red O analysis and fluorescence histochemistry assay to determine the time point of the occurrence of fatty infiltration.Then the level change of myokines was compared between the tissue with fatty infiltration and normal control by RT-PCR.2.The regulation of IL-15 on muscular fatty infiltration and its mechanism..Primary muscle-derived FAPs were isolated by flow cytometry.The primary FAPs were cultured and induced into adipocyte.The effect of IL-15 on the adipogenesis of FAPs were tested by Oil Red O analysis in vitro.In vivo,mouse IL-15 recombinant protein was injected into muscle chronic injury model and the tissue was made into cryosections.The severity of fatty infiltration after administration by IL-15 was tested by fluorescence histochemistry assay.The expression of Hedgehog signaling pathway after stimulation by IL-15 was analysed by RT-PCR.3.The effect of IL-15 on the proliferation of FAPs in vivo and in vitro and its singalling pathway.Primary FAPs was pre-treated by IL-15.After transplating GFP-FAPs into glycerol-injured muscle,the tissue was collected and analysed by fluorescence histochemistry assay to assess the number change of FAPs stimulated by IL-15.The activation of JAK-STAT signaling pathway stimulated by IL-15 was tested by Western blot.Then causality of JAK-STAT pathway and proliferation of FAPs was confirmed by loss of function methods using JAK-STAT pathway inhibitor.CCK-8 assay was performed to test the viability of FAPs in vitro.Then the proliferation and apoptosis of FAPs after stimulated by IL-15 in vitro was investigated by BrdU assay and flow cytometry,respectively.The expression of Ki67 in FAPs in vivo was analysed to clarify the effect of IL-15 on proliferation of FAPs in vivo.The effect on fibrosis by IL-15 was tested in CTX induced acute muscle injury induced and glycerol induced chronic muscle injury by fluorescence histochemistry assay and RT-PCR.4.The impact of IL-15 on muscle regenerationThe glycerol induced chronic muscle injury model was established and stimulated by IL-15.The average cross-sectional area,percentages of number of myofibers with centrally located nuclei and number of nuclei present in myofibers were analysed by fluorescence histochemistry assay.5.The correlation between the expression of IL-15 and muscle degeneration in human chronic rotator cuff tearSamples were collected from patients performed by arthroscopy.The number of FAP,collagen deposition and fatty infiltration were investigated by fluorescence histochemistry assay.The mRNA expression of IL-15 was analysed by RT-PCR.The correlation between the expression of IL-15 and number of FAPs and area of fibrosis were anaylsed by Pearson correlation analysis.Results1.IL-15 downregulated after occurrence of fatty infiltration in muscle chronic injurySignificant atrophy can be observed at 3 day post injury,myotubes can be identified at 7 day post injury and fatty infiltration occurs.The mRNA level of IL-15 downregulated at 7 day post injury comparing with normal muscle.The data indicated IL-15 may participate the fatty infiltration after muscle chronic injury.2.IL-15 can inhibit adipogenesis of FAPs and attenuate fatty degenerationIL-15 can inhibit the adipogenesis of FAPs in vitro,the mRNA expression of classic adipogenic markers,C/EBP? ? PPAR? ? FABP4,downregulated significantly.After administrating IL-15 recombinate protein,the severity of fatty infiltration was signifcantly attenuated.Furthemore,the mRNA level of two important genes which regulating the adipogenisis of FAPs,DHH and TIMP3,was upregulated obviously.These results showed IL-15 can attenuete the fatty infiltration in chronic muscle injury by directly inhibit the adipogenesis of FAPs and this is related to hedgehog pathway.3.IL-15 promotes the prolifertion of FAPs via JAK-STAT pathway and enhances the fibrosis after chronic muscle injuryThe viability of FAPs can be promoted by IL-15.Moreover,IL-15 can stimulate the proliferation of FAPs through JAK-STAT pathway while have no effect on apoptosis.Muscle fibrosis was enhanced after administration of IL-15 and can be inhibited by JAK-STAT pathway inhibitor,the expression of Fn1 and Collagen I overexpressed.These results showed IL-15 can cause proliferation of FAPs via JAK-STAT pathway and enhance the fibrosis after muscle injury.4.IL-15 faciliatates muscle regenerationThe myotubes after injection with IL-15 was significantly larger than control group.Quantification analysis showed cross sectional area of myofibers injection with IL-15 was nearly 2-folds larger than control.In addition,the proportion of myofibers with centrally located nuclei was significantly increased in muscle injection with IL-15.The number of myofiber with two or three central nuclei was also more in sample injection with IL-15 compared with control.Our results showed after injecting IL-15,the regeneration of myofibers were stimulated.5.The expression of IL-15 correlates positively with numbers of FAPs and collagen deposition in patients with chronic rotator cuff tearThe number of FAPs in lesion were much more than in normal tissue.The area of deposition of collagen ? showed nearly 5-folders larger than in normal tissue.RT-PCR analysis showed mRNA level of IL-15 upregulated significantly in lesion.Moreover,the expression of IL-15 in rotator cuff was positively correlated with the number FAPs and deposition of collagen ? by Pearson correlation test.The results showed IL-15 upregulated in patients with chronic RCT,and had a positive correlation with the number of FAP s and the severity of fibrosis.Conclusion1.The level of IL-15 downregulated at the occurence of fatty infiltration after muscle chronic injury.2.IL-15 can inhibit the adipogenesis of FAPs and attenuate fatty infiltration in vivo.3.IL-15 promoted the proliferation of FAPs through JAK-STAT pathway and enhanced the fibrosis.4.IL-15 faciliated the muscle regeneration.5.IL-15 had positive correlation with number of FAPs and collagen deposition in patients with chronic RCT.SignificanceIn this research,we demonstrated that IL-15 can stimulate the proliferation of FAPs and enhance the fibrosis in early stage through JAK-STAT pathway while inhibit adipogenesis both in vivo and in vitro.Thus,IL-15 can facilitate the muscle regeration after chronic injury.Moreover,the level of IL-15 expressed highly in chronic teared rotator cuff and had a positive correlation with the number of FAPs and the area of collagen deposition.Our research further explained how the microenvironment effected FAPs and found a n ew mechanism for regulating the fate of FAPs.Furthermore,it may be a possible therapeutical target for preventing fatty infiltration and promoting muscle regeneration after chronic muscle injury.