Calcium/Calmodulin-Dependent Protein Kinase IV (CaMKIV) Mediates Acute Skeletal Muscle Inflammatory Response

Abstract

The objective of this study is to investigate the role of Calmodulin-dependent protein kinase IV (CaMKIV) in Cardiotoxin (CTX)-induced mice muscle inflammation. CTX injection i.m. was performed to induce B6 mice acute tibialis anterior (TA) muscle injury. The mice were then injected i.p. with the recombinant CaMKIV protein or its antagonist KN-93. Immunoblotting was used to assess Calmodulin (CaM) and CaMKIV levels. Immunofluorescence was used to detect intramuscular infiltration or major histocompatibility complex (MHC)-I expression in damaged muscle. The extent of infiltration was evaluated by fluorescent intensity analysis. Cytokines/chemokines levels were determined by qPCR. CaMKIV gene knockdown in C2C12 cells was performed in order to evaluate the effects of CaMKIV on immuno-behavior of muscle cells. CTX administration induced a strong up-regulation of CaM and p-CaMKIV levels in infiltrated mononuclear cells and regenerated myofibers. In vivo adding of the recombinant CaMKIV protein enhanced intramuscular infiltration of monocytes/macrophages in damaged muscle and increased the number of proinflammatory Ly-6CF4/80 macrophage cells. CaMKIV protein treatment induced a striking up-regulation of mRNA levels of IL-1, IL-6, MCP-1, and MCP-3 in CD45 cells sorted from damaged muscle; increased the infiltration of CD8 T cells; and induced the up-regulation of MHC-I in partial regenerated myofibers, which was rarely observed in muscle damage alone. Additionally, CaMKIV protein treatment diminished the regulatory T cells (Tregs) number and led to the damaged TA muscle repair delay. In vitro CaMKIV gene knockdown reversed IFN-γ-induced up-regulation of MHC-I/II and TLR3 in the differentiated C2C12 myotubes. CaMKIV can act as an immunostimulation molecule and enhances the acute muscle inflammatory responses.

Calcium/Calmodulin-Dependent Protein Kinase IV Mediates IFN-γ-Induced Immune Behaviors in Skeletal Muscle Cells

“…shRNA lentiviral particles with noneffective scrambled shRNA sequences were used as the control. Consistent with our previous report [19], we found cell clones with stable CaMKIV knockdown survived and proliferated well in puromycin-added medium, and can be successfully induced to form multinucleated myotubes expressed MyoD and Myogenin, under horse serum administration for 72 h. Since CaMKIV is undetectable in normal cultured C 2 C 12 cells, we cultured or differentiated transfected cell clones in presence of IFN-γ about 72 h before the analysis. Western blot were carried out to monitor CaMKIV gene knockdown in transfected cells.…”

“…Muscle fibers are therefore believed to be the primary target of the immune attack in inflammatory myopathies. Since we already reported immuno-molecules expression in C 2 C 12 myoblasts and myotubes, involving MHC-I molecule H-2Kb, MHC-II molecule H2-Ea and TLR3 [19], here we further evaluated the above molecules expression in murine myogenic precursor cells (MPCs). C 2 C 12 cells, or MPCs were cultured in IFN-γ-constructed inflammatory environment, added with or without 2% horse serum.…”

“…Recently we found regenerated new myofibers in CTX-damaged muscle highly expressed CaMKIV [19], hence, it is possible that CaMKIV expression is inducible in myocytes under inflammatory environment. To definitively establish this point, in this study, we investigated CaMKIV protein levels in C 2 C 12 cells and MPCs by immunoblot analysis and evidenced the results by the extra CaMKIV antibody competition test.…”

“…Using CaMKIV-shRNA lentivirus knockdown skill, we already obtained C 2 C 12 cell clones with CaMKIV gene silence [19]. To definitively confirming knockout efficiency of CaMKIV gene in muscle cells, we repeated CaMKIV knockdown experiment and checked up CaMKIV protein absence by the CaMKIV antibody competition supportive-immunoblot analysis.…”

“…However, other studies show a lack of CaMKIV expression in skeletal muscle, and CaMKIV is indicated not required for the maintenance of slow-twitch muscle phenotype and endurance training-induced mitochondrial biogenesis in murine skeletal muscle [18]. In our previous work, the striking up-regulation of CaMKIV protein level was observed in regenerated myofibers in Cardiotoxin (CTX)-damaged the tibialis anterior (TA) muscle, while, CaMKIV knockdown reverses IFN-γ induced up-regulation of MHC class-I/II and TLR3 in myocytes [19], indicating CaMKIV can be induced to express or up-regulate in muscle cells under pro-inflammatory stimuli, and it may have a role on regulating the immune behaviors of myocytes. In this study, we extend our investigations on immune characteristics and CaMKIV expression in C 2 C 12 cells and in murine myogenic precursor cells (MPCs), and demonstrated that, IFN-γ induces the expression or up-regulation of the immuno-related molecules, and of CaMKIV in muscle cells.…”