Calmodulin/CaMKIV Signal Mediates With Acute Myoinjury-induced Inflammatory Response

BackgroundThe Calcium/Calmodulin(Ca2+/CaM)signalling is essential for immune and inflammatory responses in peripheral tissues and organs.The Ca2+/CaM-dependent kinases ?(CaMKIV),one of the key downstream molecules of activation of intracellular Ca2+signal,contributes to initiate calcium signaling related to transcriptional response.CaMKIV has a role in regulating lymphocyte activity and function,and in affecting inflammatory process of peripheral tissues.Also well,CaMKIV is a key molecule that regulates muscle gene expression and assists in muscle regeneration and remodeling.However,whether CaM/CAMKIV signaling directly interferes with myoinjury induced-inflammatory response remains mostly unknown.Objective1.To analyse the change of activity of calmodulin(CaM)and CaMKIV in damaged muscle.2.To investigate the role of CaM and CaMKIV in mice muscle inflammation and to explore the effects of CaM and CaMKIV on immuno-capacities of myofibers.Methods1.CTX was injected intramuscularly into tibialis anterior(TA)of the B6 mice to induce acute muscle injury.CaM levels in mice were interfered by the intraperitoneally injection of CaM inhibitor R24571,or CaM agonist Calcium-like peptide 1(CALP1),respectively.Or the mice were injected intraperitoneally with recombinant CaMKIV protein or its antagonist KN-93 to interfere the CaMKIV levels.2.ELISA was used to assess the concentration of PDE1(Phosphodiesterase type 1)and immunoblotting(WB)was used to evaluate the levels of CaM and CaMKIV in damaged muscle.qRT-PCR and western blotting were applied to detect the muscle autoantigens(Mi-2,HRS and Ku-70)and TLR3 mRNA and protein levels,despectively.Immunofluorescence was used to observe intramuscular infiltration in damaged muscle or MHC expression in regenerated myofiber.The degree of CD45+cells infiltration and the proportion of pro-inflammatory F4/80+Ly-6C-macrophages were detected by FACS in injured muscle.Cytokines/chemokines levels were determined by qPCR.3.C2C12 cells were thawed and generally cultured,with horse serum to induce differentiation and were treated with IFN-?.For CaM activity interference,R24571 or CALP1 were added to the corresponding medium supplemented with IFN-?,respectively.CaMKIV gene was knocked-down in C2C12 myoblasts using specific ShRNA,then the cells were differentiated with horse serum and were treated with IFN-y.Cells were analyzed after 48h.qRT-PCR or WB were applied to detect the expression of MHC and TLRs in myoblasts or differentiated myotubes.Results1.CTX intramuscularly injection can induced acute TA muscle injury successfully.The level of CaM was significantly upregulated in damaged muscle;the level of CaM-dependent phosphodiesterase 1(PDE1)and CaM in damaged muscle were significantly changed after CALP1 and R24571 treatment.2.We observed the enhanced intramuscular infiltration of monocytes/macrophages in the damaged muscle treated with CALP1 to upregulate the level of CaM or with administration of the recombinant CaMKIV to upregulate the activity of CaMKIV,as compared to untreatment.As well,CALP1 treatment up-regulated levels of muscle autoantigens(Mi-2,HARS,Ku70)and TLR3 in damaged muscle.CALP1 or CaMKIV treatment also up-regulated levels of proinflammatory cytokines and chemokines in damaged muscle.Intramuscular T cell infiltration increased,part of the regenerated muscle fibers showed MHC-I molecule H-2Kb,MHC-II molecule H2-Ea positively expression in the damaged muscle treated with CALP1 or CaMKIV.In contrast,treatment with R24571 and KN-93 resulted in inhibition of intramuscular inflammatory response.3.In vitro,R24571 treatment or CaMKIV gene knockdown induced down-regulation of expression levels of immune molecules(H-2Kb,H2-Ea and TLR3)induced by IFN-y in C2C12 myoblasts or the differenciated myotubes.ConclusionThe expression of CaM and CaMKIV were up-regulated in acute injured skeletal muscle.The continuous activation of the CaM-CaMKIV signal may aggravate intramuscular inflammation and affect the regeneration of injured muscle tissue.