ATF3与HDAC1结合调控心肌缺血再灌注损伤中自噬流受阻的作用及机制研究

Our previous study have demonstrated that impaired autophagic flux is the main reason that aggravates myocardial ischemia/reperfusion (I/R) injury. However, the exact mechanisms are still unknown. Some reports have indicated that activating transcription factor 3 (ATF3) is closely associated with autophagy, and lysosomal-associated membrane protein 2 (LAMP2) is the key protein of impaired autophagic flux. Our preliminary experiment results have demonstrated that ATF3 could combine with the promoter of Lamp2 and the expression of LAMP2 decreased when the expression of ATF3 reduced. The further experiments revealed that ATF3 could combine with histone deacetylase 1 (HDAC1), and inhibiting the activity of HDACs could increase the expression of LAMP2. Therefore, we hypothesize that there is a combination between ATF3 and HDAC1 during myocardial I/R, and the combination inhibited the effect of ATF3 on promoting the transcription of Lamp2 and result in impaired autophagic flux and aggravating myocardial I/R injury. Our research will demonstrate that ATF3 regulates the autophagic flux during myocardial I/R injury. We will verify that ATF3 combined with the HDAC1 during myocardial ischemia/reperfusion and illuminate the binding site of ATF3 and HDAC1. Through disturbing the combination and using the Atf3 knockout mice to illuminate the effect and mechanism of the combination of ATF3 and HDAC1 on regulating the impaired autophagic flux during myocardial I/R injury. The successful completion of this research will provide new evidence for seeking out the new targets and new ideas of alleviating myocardial I/R injury.

我们前期研究发现自噬流受阻是心肌缺血再灌注（I/R）损伤的重要原因，但其机制尚不明确。文献表明活化转录因子3（ATF3）与自噬关系密切，溶酶体相关膜蛋白2（LAMP2）是自噬流受阻的关键蛋白。我们预实验结果表明：心肌I/R过程中ATF3能与Lamp2启动子结合，沉默ATF3后，LAMP2表达减少；进一步实验发现ATF3能结合HDAC1，抑制HDACs后LAMP2表达增加。因此提出科学假说：心肌I/R过程中ATF3与HDAC1结合，抑制ATF3对Lamp2的转录，使LAMP2表达减少，自噬流受阻进而加重心肌I/R损伤。本课题拟证明心肌I/R过程中ATF3调控自噬流受阻；验证ATF3与HDAC1结合并明确结合位点；通过干扰结合位点并构建Atf3基因敲除小鼠，阐明ATF3与HDAC1结合调控心肌I/R过程中自噬流受阻的作用及具体机制。有望为寻找改善心肌I/R损伤的新靶点及新思路提供科学依据。

研究背景:目前全球心血管疾病的发病率和死亡率连续多年高居首位，在我国也是呈逐年攀升趋势，其中急性心肌梗死是最为凶险的类型之一，及时的再灌注治疗恢复血流通畅，是治疗心肌梗死最为行之有效的救治手段。然而，心肌血流再通引起的缺血再灌注（Ischemia/Reperfusion，I/R）损伤是影响心梗患者预后的重要因素之一。目前临床上对心肌I/R损伤的防治仍处于探索阶段，效果不稳定，防治I/R损伤依然面临瓶颈。因此，寻找新的防治靶点和策略显得尤为重要。. 主要研究内容：在C57BL/6小鼠心肌I/R模型和C57BL/6小鼠乳鼠原代心肌细胞H/R模型的基础上，采用转录组测序（RNA-seq）分析比较不同再灌注时间下小鼠心肌组织中差异表达的基因，筛选出ATF3进行下一步研究。运用基因敲除、回复、过表达等技术，结合RNA-seq、染色质免疫共沉淀（ChIP）等分子生物学手段分析得出铁死亡负调控分子FANCD2可能是ATF3下游直接调控的基因，并进一步阐明ATF3对心肌I/R损伤中心肌细胞及铁死亡的影响。. 主要研究结果.1．ATF3在心肌缺血再灌注早期表达显著上调，且向细胞核转入增加；.2．敲除ATF3可显著加重小鼠心肌I/R损伤，回复ATF3表达可减轻I/R损伤；.3．ATF3可直接结合铁死亡抑制基因FANCD2转录起始位点并促进其转录；.4．ATF3可抑制心肌缺血再灌注/缺氧复氧诱导的铁死亡；.5．ATF3可抑制Erastin和RSL3诱导的铁死亡，增加心肌细胞活力；.6．ATF3对野生型小鼠心肌I/R损伤具有显著的治疗性作用。. 科学意义：在本项研究中，我们基于目前研究的热点和前期高通量测序结果，聚焦在ATF3对铁死亡调控这一方面，着重阐明ATF3对心肌I/R损伤中铁死亡的作用，为I/R诱导的铁死亡机制探究提供科学依据。我们的研究结果不仅揭示了ATF3在调节I/R诱导的铁死亡中的作用和机制，而且也进一步佐证了转录因子在不同类型的细胞中生物过程的调控发挥着不同的作用，对于我们理解I/R反应机制的动态平衡的复杂性也有重要的依据。

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