CaMKⅣ Signal Affects Muscle Fiber Immune Characteristics Via UPR Response In Vitro Under Inflammatory Environment

BackgroundCalmodulin-dependent protein kinase 4(CaMKⅣ)is one of the intracellular Ca2+ downstream signaling molecules that interfere with Ca2+-dependent muscle gene expression,affects skeletal muscle regeneration,remodeling,and determines muscle fiber heterogeneity.In addition,CaMKⅣ is an essential molecule that regulates peripheral immunity and inflammation,interfering with lymphocyte activation,proliferation,and terminal differentiation.Our previous studies have shown that after an acute muscle injury,regenerative muscle fibers secrete multiple immune molecules and significantly unregulated CaMKⅣ levels.Artificially unregulated CaMKⅣ in vivo will aggravate intramuscular inflammation.Under physiological conditions,changes in the internal environment of skeletal muscle tissue(such as continuous exercise,high-fat diet,etc.)can initiate muscle cell ER stress(Endoplasmic reticulum Stress)and activate UPR(unfolded protein response).This adaptation mechanism helps the sarcoplasmic reticulum(endoplasmic reticulum)to restore homeostasis.Pathological muscle damage(such as idiopathic autoimmune myositis,IIMs)may induce excessive sustained ER Stress,leading to apoptosis of muscle cells and aggravation of intramuscular inflammation.Sustained pro-inflammatory stimuli activates muscle fiber ER Stress and induces UPR responses,suggesting that muscle fiber immune molecule expression is linked to UPR.Studies have shown that UPR is associated with the proliferation,differentiation and functional expression of immune cells,which contribute to the transcriptional regulation of proinflammatory cytokines in immune cells.So,does the endogenous CaMKⅣ signal affect the muscle fiber immune properties through UPR?There are not any relevant research reports yet.This topic will explore the ER Stress activation of muscle fibers in an inflammatory environment,and the interference effect of CaMKⅣ signal on intramuscular ER Stress,and clarify the possibility of CaMKⅣ-UPR pathway affecting the immune characteristics of muscle fibers.Objective1.Analysis of whether the endogenous CaMKⅣ gene affects the immunological properties of myocytes in an in vitro inflammatory environment.2.Analysis of the possibility that the CaMKⅣ transcriptional signal-UPR pathway affects the immunological properties of muscle fibers.Methods1.After CaMKⅣ shRNA transfection,the expression of CaMKⅣ gene and protein in C2C12 cells was noticeably down-regulated.Knockdown cells have good proliferation in vitro,and horse serum can induce cell differentiation and fusion,which can be used in subsequent experiments.2.CaMKⅣ knockdown/non-knockdown C2C12 cells were cultured in vitro,horse serum was induced to differentiate,and IFN-γ(interferon-y)induced inflammation.The expression of myoblast/differentiated muscle fiber immune molecules was analyzed by RT-qPCR,Western Blot and immunofluorescence staining.Flow cytometry analysis of changes in CD40,CD86,and ICM-1(cell adhesion molecules)molecules in CaMKⅣ knockdown/non-knockdown myoblasts and differentiated myotubes.3.CaMKⅣ knockdown/non-knockdown C2C12 cells were cultured in vitro,horse serum was induced to differentiate,and IFN-γ induced inflammation.Adding thapsigargin(Tg)or 4-Phenyl butyric acid(4-PBA)to agonize/inhibit ER Stress in muscle fibers;RT-qPCR,Western Blot and immunofluorescence staining were used to detect the activation of C2C12 muscle fiber UPR pathway and the expression of immune molecules.Results1.RT-qPCR and Western Blot showed that CaMKⅣ shRNA inhibited the expression of CaMKⅣ gene in C2C12 cells.Under the microscope,knockdown cells were observed to have mild proliferation in vitro,and horse serum can induce cell differentiation,which can be employed in all subsequent experiments.2.IFN-y can induce the expression of myoblasts and differentiated myotubes or up-regulate MHC-Ⅰ(Major histocompatibility-I,(H-2Kb)),MHC-Ⅱ(H2-Ea),TLR3(Toll-like receptors-3),CD40,CD86,ICM-1,and Some myogenic factors(myokines).Within the CaMKⅣ gene knockout cells,the levels of the above inflammatory molecules were significantly down-regulated.This suggests that endogenous CaMKⅣ signaling is involved in up-regulating the expression of myocyte immune molecules.3.IFN-γ pro-inflammatory stimulation can induce the differentiation of myotubes to up-regulate the levels of eIF2α(eukaryotic translation initiation factor 2α)and IRE1α(inositol-requiring enzyme-1α)in the UPR pathway.However,when Tg induced sustained ER Stress,the expression of muscle fiber immune molecules was significantly down-regulated.After CaMKⅣ gene knockdown,IFN-y-treated C2C12 muscle fibers further up-regulated the UPR pathway molecule,but the expression level of muscle fiber immune molecules was significantly down-regulated opposed to uninfected cells.This indicates that the CaMKⅣ signal regulates myocyte immune molecule expression through the UPR response.ConclusionIn an in vitro inflammatory environment,endogenous CaMKⅣ signaling may regulate the immune function of muscle fibers via the UPR response.