NOS1AP通过离子通道调控心肌梗死后室性心律失常发生机制研究

No breakthrough has been made in the long-term study about the mechanism of ventricular arrhythmia after myocardial infarction. Nitric oxide synthase 1 adaptor protein (NOS1AP) is localized to the sarcoplasmic reticulum and mitochondria, where they exert site specific effects, regulating Ca2+/K+ and many other ion channel functions.NOS1AP redistributes from the SR to the sarcolemmal membrane following injury and may have functional consequences, protecting the heart by inhibiting calcium overload.Our previous study confirmed that NOS1AP polymorphism significantly increased the risk of ventricular tachycardia, and the expression of NOS1AP decreased significantly after myocardial infarction. This subject is proposed in the mouse model of NOS1AP gene knockout acute myocardial infarction: the effect of telemetry on the occurrence of ventricular arrhythmia and sudden death was observed in vivo, and the single-phase action potential technique was used to study the changes of the overall electrophysiological characteristics of isolated ventricular myocardium. Patch-clamp technique studied the changes in the electrophysiological characteristics of ion channels at the cellular level, and molecular biology techniques define the expression and transport mechanisms of NOS1AP-regulated ion channels and NOS1, etc., in order to elucidate the pathophysiological mechanisms of ventricular arrhythmias after myocardial infarction and to provide a theoretical basis for finding the new therapeutic target.

心肌梗死后室性心律失常发生机制虽经长期研究，仍未获得突破。一氧化氮合成酶1衔接蛋白（NOS1AP）是存在于内质网和线粒体中的小分子蛋白，调控K+/Ca2+等多个离子通道，心梗后由细胞质转移到细胞膜发挥重要的生理作用。我们前期研究发现NOS1AP是增加我国汉族人群室速风险的关键基因。急性心梗小鼠的NOS1AP表达水平显著降低，心梗小鼠敲除NOS1AP后室性心律失常发生率显著增加。但NOS1AP如何促进心梗后室性心律失常发生及其具体病理生理机制尚不明确。本课题拟在NOS1AP基因敲除急性心肌梗死小鼠模型中：应用遥测技术在体观察对室性心律失常及猝死发生的影响，单相动作电位技术研究离体心室肌整体电生理特性的改变；膜片钳技术在细胞水平研究离子通道电生理特征的变化；分子生物学技术明确NOS1AP调控离子通道和NOS1等相关蛋白表达及转运机制，为阐明心梗后室性心律失常发生的病理生理机制提供理论依据。

我们前期在中国汉族人群中发现单核苷酸多态性RS12143842与特发性室速密切相关，通过GTEX数据库进行分析发现rs12143842显著改变了NOS1AP的表达，前期研究亦发现了NOS1AP的单核苷酸多态性与心室复极密切相关。因此我们拟在心肌梗死动物模型中研究NOS1AP是否参与了心肌梗死后猝死的发生，为心梗后猝死防控提供靶点。该项目主要研究了NOS1AP在心肌梗死后室性心律失常的发生机制和作用，我们首先制备了NOS1AP基因敲除小鼠模型。基因敲除模型小鼠中我们发现敲除后的老鼠QT间期明显延长，虽然在正常情况下未发生猝死情况，但在使用异丙肾上腺素诱发的情况下，我们发现NOS1AP敲除鼠的室速发生率显著增加，在异丙肾上腺素作用消失后室性心律失常发生减少。我们尝试使用传统左前降支结扎法制备心肌梗死模型，在正常小鼠中成功率在50%左右， 但在NOS1AP基因敲除鼠中，存活率显著下降。我们进一步研究了NOS1AP与离子通道之间的关系，在NOS1AP基因敲除鼠和正常鼠中我们检测了SERCA2/CASQ2 /RYR2/CACNA1C/Kir3.4/ KCNQ1/ KIR2.1等几种离子通道的表达情况，发现SERCA1和Kir3.4基因的表达量在NOS1AP基因敲除后显著下降，其中Kir3.4是由我们团队报道的长QT综合征13型的致病基因。因此我们的研究支持在NOS1AP可能通过钙离子通道和钾离子通道的介导了室性心律失常的发生，为下一步明确治疗靶点、防控长QT综合征和猝死提供了理论基础。我们同时在一个猝死家系中使用外显子测序的方法鉴定了一个新的致病基因，并成功检测到了该基因的突变，为猝死的防控提供了新的靶点目标。