室旁核p300介导的HMGB1乙酰化调控在心肌梗死后中枢-心脏交感神经激活中的作用及机制

Ventricular arrhythmia (VAs) is the main cause of death and disability post myocardial infarction (MI). There exists interaction between central and cardiac electrical activity. Our previous study hinted that paraventricular HMGB1 was the key element to central and cardiac sympathetic interaction, moreover, and the nuclear-cytoplasmic transposition of HMGB1 played a potential role in the occurrence of VAs, with the underlying regulating mechanism unclear. Previous study shows that the acetylation medication played an important role in the regulation of HMGB1. Through screening we found histone acetylases p300 increased specificity, and preliminarily confirmed the role of p300 on the translocation of HMGB1. The NMDA receptor (NMDAR) in the paraventricular nucleus played a driving function in the excitation of sympathetic preganglionic fibers. Targeted inhibition of HMGB1 can effectively and specifically reduce the expression and excitatory postsynaptic currents of NMDAR. Therefore, we speculated that the p300 could regulate the process of HMGB1 translocation by the acetylation of certain position, and activate NMDAR on sympathetic preganglionic fibers, thus participating the occurrence of VAs. The present study is designed to explore the specific acetylation sites of HMGB1 and confirm the role of p300-HMGB1 axis in NMDAR mediated sympathetic activation in vivo and in vitro of by site-directed/ dominant mutagenesis, Co-IP , MS assay and other methods, which may provide potential molecule targets and a novel idea for prevention and treatment of VAs post-MI.

室性心律失常（VAs）是心梗（MI）患者致死、致残的主因。中枢-心脏电活动有交互作用，我们前期示室旁核HMGB1是连接中枢-心脏交感对话的关键，核-浆转位后可参与诱发VAs。但目前对HMGB1调控尚不清楚。有研究表明乙酰化修饰对HMGB1的调控起重要作用，我们筛选乙酰化酶发现MI后室旁核p300显著上调，并初步验证了其对HMGB1的转位调控作用。NMDA受体（NMDAR）在室旁核交感节前神经纤维兴奋中起驱动作用，我们预测发现HMGB1与NMDAR结合，抑制HMGB1后NMDAR表达和电流降低，综上提出假说：室旁核p300介导HMGB1乙酰化并通过NMDAR调控MI后心脏交感神经激活，致VAs发生。本课题拟利用基因定点突变、Co-IP质谱检测等方法，明确p300与HMGB1的具体作用靶点，并体内、外逐层验证中枢HMGB1去乙酰化修饰在心脏交感激活中的作用，为防治MI后室性心律失常提供新靶点。

背景：.心脏交感神经激活致室性心律失常（VAs）是心梗（MI）患者致死、致残的主因，我们既往研究发现中枢-心脏存在电交互作用：中枢室旁核是心脏交感神经激活的上游，相关机制不明。.方法与结果：.我们通过结扎大鼠前降支制造心梗模型，后利用PRM测序发现，相较于其他乙酰化酶，p300在MI后室旁核上调最为显著，且与小胶质细胞共染；利用立体定位仪向室旁核靶向干预p300 shRNA可显著降低中枢-周围交感神经活性，最终降低心律失常易感性，下游靶点不明。下一步对单核/巨噬细胞系中p300行乙酰化label-free测序，后进行GO分析、KEGG分析验证发现HMGB1是p300的下游靶蛋白；进一步构建HMGB1进行截短突变及点突变质粒，通过CO-IP实验发现p300乙酰化结合HMGB1 K43位点。功能学验证发现，p300shRNA可逆转 HMGB1乙酰化程度及核-浆转位。最后进行p300-HMGB1的下游机制探讨：NMDA受体（NMDAR）在室旁核交感节前神经纤维兴奋中起驱动作用，Discovery studio软件预测发现HMGB1与NMDAR结合，我们在HEK293中转染p300与HMGB1 WT/HMGB1K43R，后与神经元共培养，联合NMDAR、AMPKR阻断剂Ro 25-6981、YM-90K 行rescue实验，并行兴奋性突触后电位EPSCs检测，发现HMGB1受p300乙酰化修饰发生转位后通过NMDAR介导神经兴奋。.结论与科学意义：.室旁核p300介导小胶质细胞HMGB1乙酰化，后者以旁分泌形式激活神经元NMDAR致MI后VAs发生，从分子层面探索炎症细胞和神经元信息传递的方式，从新视角解读炎症反应致神经激活的原因，为MI后VAs的发生提供新研究方向和治疗靶点。.