Department of Anatomy, Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering, School of Basic Medical Science, Southern Medical University, No.1838, GuangZhou Avenue North, GuangZhou, 510515, China.
Perelman School of Medicine and Pennsylvania Muscle Institute, University of Pennsylvania, 422 Curie Blvd. Rm 715, Philadelphia, PA, 19104, USA.
Inflammation. 2018 Feb;41(1):199-212. doi: 10.1007/s10753-017-0678-2.
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.
本研究旨在探讨钙调蛋白依赖性蛋白激酶 IV（CaMKIV）在心肌毒素（CTX）诱导的小鼠肌肉炎症中的作用。通过肌内注射 CTX 诱导 B6 小鼠急性胫骨前肌（TA）肌肉损伤。然后，通过腹腔注射重组 CaMKIV 蛋白或其拮抗剂 KN-93 进行处理。采用免疫印迹法检测钙调蛋白（CaM）和 CaMKIV 水平。免疫荧光法检测损伤肌肉中肌内浸润或主要组织相容性复合体（MHC）-I 的表达。通过荧光强度分析评估浸润程度。通过 qPCR 测定细胞因子/趋化因子水平。在 C2C12 细胞中敲低 CaMKIV 基因，以评估 CaMKIV 对肌肉细胞免疫行为的影响。CTX 给药诱导浸润单核细胞和再生肌纤维中 CaM 和 p-CaMKIV 水平的强烈上调。体内添加重组 CaMKIV 蛋白增强了损伤肌肉中单核细胞/巨噬细胞的肌内浸润，并增加了促炎 Ly-6CF4/80 巨噬细胞的数量。CaMKIV 蛋白处理诱导受损肌肉中 CD45 细胞中 IL-1、IL-6、MCP-1 和 MCP-3 的 mRNA 水平显著上调；增加 CD8 T 细胞的浸润；并诱导部分再生肌纤维中 MHC-I 的上调，而单独的肌肉损伤很少观察到这种现象。此外，CaMKIV 蛋白处理减少了调节性 T 细胞（Tregs）的数量，并导致受损 TA 肌肉修复延迟。体外 CaMKIV 基因敲低逆转 IFN-γ诱导的分化 C2C12 肌管中 MHC-I/II 和 TLR3 的上调。CaMKIV 可作为免疫刺激分子，增强急性肌肉炎症反应。
