急性心肌梗死小鼠单核/巨噬细胞钾离子通道介导心肌重塑机制研究

Inflammation sequentially recruits circulating Ly-6Chi and Ly-6Clo monocytes subsets to infarcting myocardium in myocardial infarction (MI), which commit to functions of inflammatory removal and myocardial healing and remodelling, respectively. Conversion and dysfunction of monocytes/macrophages subsets plays significant role impacting cardiac remodeling in MI. Kv1.3 and KCa3.1 channel could regulate the function of monocytes/macrophages system (MPS). It is still unknown whether change of expression and function of Kv1.3 and KCa3.1 current induced the conversion of subsets of MPS and whether modulation of potassium channels could influence cardiac remodelling through regulating immunological function. Applying the techniques such as patch clamps, immunoblotting, confocal laser, flow cytometry, pathology and immunohistochemical and so on, this research would illustrate whether signal of myocardial infarction initiates change of expression and function of Kv1.3 and KCa3.1 channel of MPS, and induces it to differentiate into distinct subsets, then regulates the state and function of MPS in different phase of myocardial infarction, and influences chemotaxis of monocytes, secretion of cytokine, angiogenesis, myofi broblast accumulation, and finally mediates the processing of cardiac remodelling through the control effect with administration of specific potassium channel blockers in mice with myocardial infarction, which could provide new therapeutic immunoregulatory target.for myocardial remodelling of MI.

急性心肌梗死后炎症依次募集Ly-6Chi和Ly-6Clo两种单核细胞亚型至梗死部位分别发挥炎症清除和修复重塑作用。单核/巨噬细胞系统（MPS）亚型转换和功能失调在心肌重塑中发挥重要作用。MPS细胞膜Kv1.3和KCa3.1钾通道可调节其功能。心肌梗死后MPS细胞亚型转换是否由于膜Kv1.3和KCa3.1电流及表达的改变？调节钾通道功能是否可调节细胞免疫功能而影响心肌重塑，国内外尚无研究。本课题通过选择性通道阻断剂作用前后效应，运用膜片钳、免疫印迹、激光共聚焦、流式细胞术、病理和免疫组化等技术，研究小鼠心肌梗死信号是否启动了MPS细胞膜Kv1.3和KCa3.1钾离子通道表达和功能的改变，使细胞分化为不同亚型，通过胞内Ca2+信号通路，调节心肌梗死后不同时期MPS细胞状态和功能，影响单核细胞趋化、细胞因子分泌、新生血管生成和成纤维细胞的沉积，介导心肌重塑进程，为心肌重塑免疫调节治疗提供新靶点。

心肌重塑是心肌梗死后心力衰竭进展和恶性心律失常发生的重要原因。调节心肌梗死后的炎症通路一定程度上可以减少心肌损害和心肌的不良重塑。单核巨噬细胞、B淋巴细胞、调节性T细胞、辅助性T细胞及效应T细胞等免疫细胞贯穿着整个炎症反应的始末，而这些炎症细胞的激活、增殖、趋化、吞噬等功能均与其细胞膜上Kv1.3和KCa3.1等离子通道的活动密切相关。本项目在前期工作的基础上，通过以下研究：分离急性心肌梗死小鼠外周血单核细胞记录其膜离子通道表达的改变；结合使用选择性离子通道阻断剂TRAM-34，研究心肌梗死后单核细胞的增殖与离子通道表达的改变的相关性；离子通道的改变对细胞内ERK信号通路和NF-κB信号通路的影响；对Kv1.3和KCa3.1离子通道的阻断是否改善梗死后心肌重塑和心功能。现已证实Kv1.3和KCa3.1离子通道参与单核巨噬细胞及B淋巴细胞的增殖趋化及其所引发的一系列炎症反应、促进心室重塑、降低心功能,KCa3.1离子通道阻断剂TRAM-34能抑制心肌梗死后的过度炎症反应及心室重塑，增强心功能）。此外，在此基础上进一步研究了小鼠及大鼠单核/巨噬细胞表型（M1表型、M2表型）分化与KCa3.1离子通道的相关性，证实TRAM-34阻断KCa3.1离子通道后，能减少单核/巨噬细胞向M1表型的转化，从而减轻心肌梗死早期的过度炎症反应及不良心室重塑、促进组织的有效修复从而增强心功能.另外，本实验室也对心肌细胞及成纤维细胞上KCa3.1离子通道在心肌梗死后心室重塑的相关作用做深入研究，证实阻断KCa3.1通道能够通过抑制心脏成纤维细胞的促纤维化作用减轻心肌梗死后心室重塑，提高心功能。我们不仅证实KCa3.1等离子通道在心肌梗死后炎症反应及心室重塑中所起到的关键作用，同时其选择性阻断剂也为临床上心肌梗死病人提供了一个新的治疗靶点。