KCa3.1在压力超负荷所致心脏纤维化中的作用与机制

Cardiac fibrosis is a core pathologic process associated with heart failure. The uptake and differentiation of primitive fibroblast precursor cells of bone marrow origin play a critical role in pathological interstitial cardiac fibrosis. However, the specific cellular and molecular mechanisms of governing this process are still not well defined. The intermediate-conductance Ca2+-activated K+ (KCa3.1) channels express in both of ventricular fibroblasts and circulating mononuclear cells in rats, and is upregulated by angiotensin II (Ang II). Primal study from our group indicated that inhibition of KCa3.1 channels alleviated cardiac fibrosis induced by pressure-overload. We hypothesize that KCa3.1 mediates the recruitment of bone marrow-derived circulating mononuclear cells into heart and inflammatory microenvironment in heart, promotes the infiltrated mononuclear cells to differentiate into myofibroblasts, and results in cardiac fibrosis. The present project is designed to establish pressure-overload induced cardiac fibrosis models in rats by Ang II infusion and abdominal aortic constriction, and explore the effect of inhibiting KCa3.1 on cardiac fibrosis through treatment with specific KCa3.1 blocker and adenovirus-mediated short hairpin RNA (shRNA), respectively. By separating circulating mononuclear cells and CD+ T cells from adult healthy volunteer and culturing human umbilical vein endothelial cells, we will study the role of KCa3.1 in transendothelial migration of circulating mononuclear cells and their differentiation into myofibroblast. The results will elucidate the role and mechanism of KCa3.1 in the development and progression of cardiac fibrosis, and also provide valuable insights for therapeutic approaches to prevent cardiac fibrosis.

心脏纤维化是导致心力衰竭的关键病理过程，骨髓源性纤维细胞前体的摄取与分化在其中起重要作用，但机制尚不清楚。循环单核细胞和心室成纤维细胞表达KCa3.1，且受Ang II刺激上调。预实验发现阻断KCa3.1明显减轻压力超负荷引起的心脏纤维化。假设：KCa3.1介导骨髓源性循环单核细胞向心脏募集和心脏炎症微环境，促进浸润的单核细胞分化为肌成纤维细胞，导致心脏纤维化。本项目分别通过Ang II灌输和腹主动脉缩窄建立大鼠压力超负荷致心脏纤维化模型，分别应用KCa3.1特异性阻断剂和腺病毒载体介导的短发夹RNA探索阻断KCa3.1对心脏纤维化的影响；通过分离成年健康志愿者循环单核细胞、CD4+ T细胞和培养人脐静脉内皮细胞，研究KCa3.1在循环单核细胞跨内皮迁移并向肌成纤维细胞分化中的作用。研究结果不仅可以阐明KCa3.1在心脏纤维化发生发展中的作用与机制，也将为研发防治心脏纤维化的药物提供线索。

非适应性心脏纤维化是导致心力衰竭和恶性心律失常的关键病理过程，其发生机制尚未完全阐明。本项目通过腹主动脉缩窄和皮下埋置微渗透泵恒速灌输血管紧张素II（Ang II，0.8 mg/kg/d）建立了两种大鼠压力超负荷心脏纤维化模型，发现应用TRAM-34（80 mg/kg/d，分别腹腔注射2或4周）特异性阻断KCa3.1可从多个环节阻止心脏纤维化的发生和发展，包括逆转血浆和心肌组织促纤维化因子（Ang II、CTGF和TGF-β）、促炎（TNF-α）和趋化因子（MCP-1）以及Th2型细胞因子（IL-4和IL-13）的上升，抑制炎症细胞（表达CD3、CD45、F4/80或CD68）向心肌浸润和骨髓源性纤维细胞向心脏募集并分化为成纤维细胞（表达CD34、CD45、CD11b和DDR2），抑制心脏成纤维细胞活化和I、III型胶原的表达与分泌，降低左心室重量和心重/体重比，改善大鼠心脏舒张与收缩功能（左心室射血分数和缩短分数的降低、左心室舒张末期内径和收缩末期内径的增加）。在心肌细胞限制性高表达人β2肾上腺素受体基因（β2-TG）引起的小鼠纤维化性心肌病模型上，TRAM-34（120 mg/kg/d腹腔注射1或2个月）显著抑制血清半乳凝素3（Gal-3）、MCP-1和TNF-α的升高和心肌Gal-3、MCP-1、TNF-α、CTGF、TGF-β和KCa3.1的表达，改善心脏纤维化。而Gal-3 基因缺失仅降低β2-TG小鼠心脏炎症细胞而非成纤维细胞KCa3.1的表达，对心肌纤维化无影响。TRAM-34抑制Ang II 和Gal-3诱导的心室成纤维细胞KCa3.1与瞬时受体电位通道（TRPV4和TRPC6）的相互作用及成纤维细胞的激活。该研究首次揭示了免疫细胞和成纤维细胞上Gal-3-KCa3.1-TRPV4/TRPC6相互作用导致趋化因子和细胞因子的合成与释放，促进心脏纤维化，并确立了KCa3.1为防治心脏纤维化的有效靶点，为药物研发打下了基础。