Calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) acts as a molecular switch regulating cardiovascular Ca²⁺ handling and contractility in health and disease. Activation of CaMKIIδ is also known to regulate cardiovascular inflammation and is reported to be required for pro-inflammatory NF-κB signalling. In this study the aim was to characterise how CaMKIIδ interacts with and modulates NF-κB signalling and whether this interaction exists in non-contractile cells of the heart. Recombinant or purified CaMKIIδ and the individual inhibitory -κB kinase (IKK) proteins of the NF-κB signalling pathway were used in autoradiography and Surface Plasmon Resonance (SPR) to explore potential interactions between both components. Primary adult rat cardiac fibroblasts were then used to study the effects of selective CaMKII inhibition on pharmacologically-induced NF-κB activation as well as interaction between CaMKII and specific IKK isoforms in a cardiac cellular setting. Autoradiography analysis suggested that CaMKIIδ phosphorylated IKKβ but not IKKα. SPR analysis further supported a direct interaction between CaMKIIδ and IKKβ but not between CaMKIIδ and IKKα or IKKγ. CaMKIIδ regulation of IκΒα degradation was explored in adult cardiac fibroblasts exposed to pharmacological stimulation. Cells were stimulated with agonist in the presence or absence of a CaMKII inhibitor, autocamtide inhibitory peptide (AIP). Selective inhibition of CaMKII resulted in reduced NF-κB activation, as measured by agonist-stimulated IκBα degradation. Importantly, and in agreement with the recombinant protein work, an interaction between CaMKII and IKKβ was evident following Proximity Ligation Assays in adult cardiac fibroblasts. This study provides new evidence supporting direct interaction between CaMKIIδ and IKKβ in pro-inflammatory signalling in cardiac fibroblasts and could represent a feature that may be exploited for therapeutic benefit.

钙/钙调蛋白依赖性蛋白激酶 IIδ (CaMKIIδ) 作为调节心血管 Ca ²⁺的分子开关健康和疾病中的处理和收缩。CaMKIIδ 的激活也被称为调节心血管炎症，据报道它是促炎 NF-κB 信号传导所必需的。在这项研究中，目的是表征 CaMKIIδ 如何与 NF-κB 信号相互作用并调节 NF-κB 信号，以及这种相互作用是否存在于心脏的非收缩细胞中。重组或纯化的 CaMKIIδ 和 NF-κB 信号通路的单个抑制性 -κB 激酶 (IKK) 蛋白用于放射自显影和表面等离子体共振 (SPR)，以探索两种成分之间的潜在相互作用。然后使用原代成年大鼠心脏成纤维细胞研究选择性 CaMKII 抑制对药理学诱导的 NF-κB 活化的影响以及 CaMKII 与心脏细胞环境中特定 IKK 同工型之间的相互作用。放射自显影分析表明 CaMKIIδ 磷酸化了 IKKβ 而不是 IKKα。SPR 分析进一步支持 CaMKIIδ 和 IKKβ 之间的直接相互作用，但不支持 CaMKIIδ 和 IKKα 或 IKKγ 之间的直接相互作用。在暴露于药理刺激的成年心脏成纤维细胞中探索了 CaMKIIδ 对 IκΒα 降解的调节。在存在或不存在 CaMKII 抑制剂 autocamtide 抑制肽 (AIP) 的情况下，用激动剂刺激细胞。CaMKII 的选择性抑制导致 NF-κB 活化减少，如激动剂刺激的 IκBα 降解所测量的那样。重要的，并且与重组蛋白的工作一致，在成人心脏成纤维细胞中进行邻近连接试验后，CaMKII 和 IKKβ 之间的相互作用很明显。这项研究提供了新的证据，支持 CaMKIIδ 和 IKKβ 在心脏成纤维细胞促炎信号传导中的直接相互作用，并且可能代表一个可用于治疗益处的特征。