钙/钙调蛋白依赖性蛋白激酶II(CaMKII)已被证明在病毒性心肌炎(VMC)中异常激活,但其亚型CaMKIIδ在VMC中的作用尚不清楚。通过柯萨奇病毒B3(CVB3)处理诱导VMC小鼠和心肌细胞模型。接受假手术的小鼠和盐水处理的心肌细胞作为对照。体重,生存,左心室射血分数(LVEF),测量缩短分数(LVFS),并进行HE染色以评估VMC小鼠模型和假对照的心功能。ELISA法检测血清或细胞上清液中的炎症因子。CaMKIIδ的表达,含Toll/白介素-1受体结构域的衔接蛋白(TIRAP),胰岛素样生长因子2mRNA结合蛋白2(IGF2BP2),核因子NF-κB(NF-κB)信号,通过定量实时聚合酶链反应(qRT-PCR)或Westernblot检查炎症因子。CCK-8,EdU,和流式细胞术用于评估细胞行为。免疫共沉淀(Co-IP),RNA免疫沉淀(RIP),和RNA下拉来验证分子相互作用。进行甲基化RNA免疫沉淀(MeRIP)以测量特定分子的N6-甲基腺苷(m6A)水平。CaMKIIδ在VMC小鼠和CVB3处理的原代心肌细胞中上调,其中敲除提高了细胞活力,扩散,并在体外抑制细胞凋亡,从而减轻体内心肌炎。CaMKIIδ的稳定性归因于通过m6A修饰的IGF2BP2的存在。CaMKIIδ的缺失通过负向和直接调节TIRAP来抑制NF-κB途径,从而参与炎性损伤。CaMKIIδ,由m6A阅读器IGF2BP2稳定,通过与TIRAP相互作用调节NF-κB途径以改变细胞活力,扩散,和细胞凋亡,从而影响VMC结果。
Calcium/calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to be aberrantly activated in viral myocarditis (VMC), but the role of its subtype
CaMKIIδ in VMC remains unclear.VMC mice and cardiomyocytes models were induced by Coxsackievirus B3 (CVB3) treatment. Mice that underwent sham surgery and saline-treated cardiomyocytes served as controls. Body weight, survival, left ventricular ejection fraction (LVEF), and fractional shortening (LVFS) were measured, and HE staining was performed to evaluate heart function in VMC mice model and sham control. Inflammation factors in serum or cell supernatant were detected by ELISA. Expressions of
CaMKIIδ, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), nuclear factor NF-kappaB (NF-κB) signals, and inflammation factors were examined by quantitative real time polymerase chain reaction (qRT-PCR) or western blot. CCK-8, EdU, and flow cytometry were used to evaluate cell behaviors. Co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down were utilized to validate molecule interaction. Methylated RNA immunoprecipitation (MeRIP) was performed to measure N6-methyladenosine (m6A) level of specific molecule.CaMKIIδ was upregulated in VMC mice and CVB3-treated primary cardiomyocytes, of which knockdown improved cell viability, proliferation, and suppressed cell apoptosis in vitro, thereby alleviating myocarditis in vivo. The stability of
CaMKIIδ was attributed to the presence of IGF2BP2 through m6A modification. Loss of
CaMKIIδ repressed NF-κB pathway via negatively and directly regulating TIRAP to be involved in inflammatory damage.CaMKIIδ, stabilized by m6A reader IGF2BP2, modulated NF-κB pathway via interacting with TIRAP to alter cell viability, proliferation, and apoptosis, thereby affecting VMC outcome.