心肌梗死后局部内皮素-1与sema3a相互作在心脏交感神经重构中的作用

Sympathetic nerve remodeling plays an important role in the pathogenesis of lethal ventricular arrhythmias after myocardial infarction (MI). A balanced interaction between ET-1/NGF and Sema3a determines the normal innervation pattern of the cardiac sympathetic nerve. Our previous studies have shown that sympathetic nerve was hyper-innervated in the MI border-zone. Over-expression of Sema3a could reduce sympathetic innervation and decrease the incidence of triggered ventricular tachycardia. However, the mechanism of sympathetic remodeling is unclear. Some studies have shown that miR-1 expression was lower in the infarct border zone and endothelin-1 (ET-1) gene is one of the target genes of miR-1. Our preliminary data indicated that sema3a expression was down-regulated in the endocardium of the animal MI model. Thus, we hypothesize that decreased miR-1 and sema3a expressions increase ET-1/NGF levels in the MI border-zone and result in sympathetic nerve remodeling. In this study, in order to elucidate the molecular mechanism of sympathetic nerve remodeling, we investigate the regulation of mRNA-1 on ET-1/NGF's expression and effect of ET-1/NGF on Sema3a's expression in mouse MI models. We also study the expression changes of mRNA-1 and ET-1/NGF and changes in sympathtic innervation patterns in Sema3a KO mouse MI model.

交感神经重构在心肌梗死后室性心律失常中起重要作用.ET-1/NG与sema3a之间的平衡决定交感神经的正常分布.我们前期研究显示心肌梗死边缘区交感神经异常增生,梗死边缘区高表达sema3a可降低局部交感神经增生并降低室速诱发率.但心梗后交感神经重构机制尚不明确.研究显示心肌梗死后梗死边缘区miR-1表达减低,靶点预测示ET-1基因是miR-1的靶基因.同时内膜缺血受损心肌sema3a表达也降低.故推测miR-1降低可增加局部ET-1浓度，与降低的sema3a共同决定交感神经重构.本研究拟通过实验证明miR-1对ET-1的调控作用;研究梗死后心脏交感神经的分布,miR-1和ET-1/NGF、sema3a之间的关系;通过高表达和敲除miRNA-1研究其对ET-1/NGF、sema3a及心脏交感神经纤维芽生影响；研究miR-1对sema3a敲除小鼠心脏交感神经分布.探讨心梗后交感神经重构的机制。

目的：心肌梗死（MI）后继发交感神经重构，其与缺血性心律失常的发生关系密切。ET-1/NGF 与sema3a 之间的平衡决定交感神经的正常分布. ET-1 基因是miR-1 的靶基因。本研究拟通过实验证明miR-1 对ET-1的调控作用；研究梗死后心脏交感神经的分布，miR-1 和ET-1/NGF、sema3a 之间的关系；通过高表达和敲除miRNA-1 研究其对ET-1/NGF、sema3a 及心脏交感神经纤维芽生影响；探讨心梗后交感神经重构的机制。方法：分离培养野生型/Sema3a 敲除小鼠心脏不同部位心肌细胞，分别对心外膜和心内膜心肌细胞进行缺氧诱导；结扎新西兰大白兔冠状动脉建立MI模型，向术后存活心梗模型梗死边缘区微注射miR-1模拟物或miR-1敲除慢病毒。MI后1周，行心电图、心超检查；体内程序性电刺激评估心律失常诱发；电生理检查结束后整体心脏取材， Masson染色检测梗死面积，免疫组化染色检测GAP43、TH表达，ELISA 检测心肌ET-1水平，Real-Time PCR、Western Blot检测ET-1、NGF、miR-1等mRNA和蛋白表达。结果：1）心外膜（ET-1、NGF表达为主）、心内膜（Sema3a表达为主）表达具统计学差异，提示ET-1/NGF及Sema3A平衡在心肌梗死后心脏交感神经重构中表起到重要作用。2）心肌ET-1的表达存在由心外膜到心内膜由高至低的浓度梯度分布；miR-1 对靶基因ET-1有明确的调节作用，心肌细胞miR-1升高和降低通过转录后调节分别使得对内皮素-1合成减少和增多。3）缺氧诱导下，Sema3a 敲除/野生型小鼠心肌ET-1、NGF的表达存在由心外膜到心内膜由高至低的浓度梯度分布；ET-1 对NGF有明确的调节作用，促进NGF蛋白合成。4）MI后，整体心脏GAP43、TH阳性神经纤维密度明显增加，梗死边缘区变化最明显； miR-1过表达组， ET-1、NGF蛋白表达下调，梗死边缘区和远隔区的心肌GAP43、TH阳性神经纤维密度下降，心律失常评分结果明显降低，有统计学意义。结论：MI后，梗死边缘区miR-1高水平表达，负向调节ET-1表达，NGF蛋白合成下降，能抑制整体心脏的神经出芽及交感神经过度增生，并降低室性心律失常的发生。miR-1的差异性表达通过调节ET-1，与sema3a 共同导致心脏交感重构。