Calpain调节HDAC3活性介导病毒性扩张型心肌病心肌纤维化发生的机制研究

One of the malignant outcomes of viral myocarditis is its progression into dilated cardiomyopathy (DCM). The prognosis of DCM are poor; however, the treatment of DCM is still lack of specific measures. And myocardial fibrosis is the important pathological changes in DCM. Our previous study has shown that calcium dependent cysteine protease (calpain) induce myocardial fibrosis in the mice with CVB3-induced DCM; however, the detailed mechanism is not clear. Nuclear factor-ΚB (NF-ΚB) is closely related to fibrosis, and acetylation is reported to play an important role on the regulation of NF-ΚB activity. Several investigations have shown that histone deacetylase 3 (HDAC3) is a key to regulate the acetylation of NF-ΚB. Recently, we have found that calpain can increase HDAC3 expression and activate NF-ΚB in the murine model of CVB3-induced DCM. Therefore, we propose the hypothesis that calpain contributed to myocardial fibrosis through increasing acetylation of NF-kB. To explore the potential mechanisms, we will furtherly study the effects of subtypes of calpain on myocardial fibrosis after CVB3 infection in vivo and in vitro, and the mechanisms on calpain-regulated the acetylation of NF-ΚB. The study is expected to reveal the new mechanisms on calpain-induced myocardial fibrosis in DCM and provide the novel ideas for the treatment of DCM.

病毒性心肌炎发病率高，慢性迁延导致的扩张型心肌病(DCM)病死率高，治疗棘手。心肌纤维化是其重要病理改变。我们前期发现钙离子依赖性的半胱氨酸蛋白酶(calpain)可诱导病毒性DCM小鼠心肌纤维化，机制不清。NF-ΚB活化与纤维化密切相关，而乙酰化修饰是其活性调节的重要方式。既往报道组蛋白去乙酰基酶(HDAC3)是调控NF-ΚB乙酰化的关键靶点。新近我们发现在病毒性DCM中，Calpain可上调HDAC3表达，并引起NF-ΚB活化。故提出假说：Calpain通过诱导NF-ΚB乙酰化，促进心肌纤维化。为了弄清其机制，本项目拟进一步在细胞、过表达calpain内源性抑制蛋白小鼠模型上研究CVB3感染后calpain不同亚型活性变化对心肌纤维化的作用，明确calpain特异性亚型对NF-ΚB乙酰化调控的分子机制，有望揭示calpain促进心肌纤维化的新机制，为临床DCM诊治靶标的探寻提供新思路。

心肌纤维化是病毒性扩张型心肌病的重要病理特征，病毒感染后通过其介导的炎症免疫反应是调控心肌炎心肌纤维化发生发展的主要因素。我们既往的研究发现，感染CVB3的calpastain转基因小鼠（TG-CAST小鼠）心肌纤维化程度明显减轻，肌间质纤维结缔组织总量减少。提示，抑制calpain活性可逆转心肌纤维化的发生。为了探讨其可能机制，本项目利用TG-CAST转基因小鼠建立急、慢性病毒性心肌炎模型，针对病毒感染后calpain活化通过影响NF-ΚB/HMGB1乙酰化，进而调控炎症相关因子，导致心肌纤维化的分子网络调控机制进行了研究。结果发现，在病毒性心肌炎发病及向扩张型心肌病转化过程中，IL17是重要炎症因子，病毒感染后calpain活性改变可调节IL17,IFNγ,穿孔素等炎症因子以及纤维化相关因子MMP2和Smad3的表达与功能；HMGB1可以通过RAGE受体上调心脏中IFN-γ,IL-17等炎症因子的水平。利用阻断HMGB1释放的抑制剂丙酮酸乙酯可以明显降低胞外HMGB1水平，同时观察到心肌炎小鼠心脏中炎症浸润面积、病毒含量及 IFN-γ,IL-17等炎症因子水平明显减少，心肌纤维化程度减轻。提示胞外 HMGB1可促进心肌炎小鼠心脏炎症及纤维化发生。. HMGB1是一个非组蛋白，受到各种刺激后HMGB1会从核内释放到胞外，胞外的 HMGB1与其高亲受体结合后能激活下游炎症通路诱导其他炎症因子（如：TNF-α,IL-1,IL-6,IL-17）产生和释放，增加心肌损伤。Calpain对HMGB1的释放是否有调控作用及其机制如何？我们的研究结果显示，病毒感染心肌细胞后，calpain激活能下调HDAC活性，促进HMGB1乙酰化修饰，继而引起心肌细胞核中的HMGB1易位至胞浆，并藉此介导了后续的心脏炎症损伤及纤维化的发生，也从新的角度深度认识了calpain促进病毒性扩张型心肌病心肌纤维化发生的机制。