PRMT1-ADMA-DDAH2通路促糖尿病心肌病形成及机制

The diabetic patients are easy to suffer from cardiovascular disease, especially diabetic cardiomyopathy (DCM), which is the leading cause of morbidity and mortality in diabetic patients. However, the underlying mechanisms were remained obscure. Previous studies had indicated that the endogenous inhibitor of nitric oxide synthase asymmetric dimethylarginin (ADMA) was elevated in cardiovascular diseases and daibetes mellitus and closely related to the vascular endothelial dysfunction. Our current preliminary experiments of cardiomyocytes incubated with ADMA have demonstrated that exogenous ADMA directly inhibits insulin signal transduction and disturbs intracellular calcium homeostasis as well as upregulates the transcription and expression of promoting cardiac hypertrophy, fibrosis and apoptosis related genes. All above chagnes in cardiomyocytes induced by ADMA are very similar to the major pathological features of DCM. Accordingly, the speculation is made that the elevated endogenous ADMA may be the link between daibetes mellitus and cardiovascular diseases and plays a important role in the development of DCM. The present study is to determine the relationship between ADMA and DCM by investigation of the dynamic changes of ADMA pathway, which includes ADMA generating enzyme protein arginine methyltransferase1 (PRMT1) and ADMA degradating enzyme dimethylarginine-dimethylamine hydrolase 2 (DDAH2), in the development of DCM in various diabetic animal models with defferent duration of diabetes, and then to reveal the important role of ADMA in development of DCM by observation the impact of ADMA on cardiac function, structure and remodeling in endogenous and exogenous high ADMA animal models with different courses. Furthermore, the potential mechanisms underlying ADMA leading to DCM will be explored in multiple animal models with diabetes mellitus and high ADMA as well as cadiac cell models of high glucose and ADMA incubation. Finally, the gene therapy and drug treatment were performed in diabetic rat models and cardiac cell model against the pathway of PRMT1-ADMA-DDAH2 in order to explore new target for clinical diagnosis, prevention and treatment of DCM as well as the development of new drug.

糖尿病(DM)易并发心血管疾病，糖尿病心肌病(DCM)是导致DM死亡的主要原因，但发病机制不清楚。研究表明，在心血管疾病和糖尿病均升高的内源性一氧化氮合酶抑制物ADMA与其血管内皮功能不全密切相关；本项目预实验发现，外源性ADMA可抑制心肌细胞胰岛素信号转导，干扰细胞内钙稳态，上调促心肌肥大、纤维化及凋亡相关因子表达；这些都是DCM的主要病理改变，据此申请人假设：内源性ADMA升高是联系DM与心血管疾病的“枢纽分子"并在DCM发生发展中起重要作用。本项目先在不同病程DM大鼠心脏检测ADMA及其生成酶PRMT1和代谢酶DDAH2通路动态变化，明确ADMA与DCM的关系；再在不同时程高ADMA大鼠和心肌细胞模型，观测ADMA对心脏功能和形态结构的影响，阐明ADMA促DCM形成的重要作用及机制；并针对ADMA通路给予糖尿病大鼠和心肌细胞模型基因和药物治疗，为临床DCM防治及新药研发揭示新靶点。

本项目在多种糖尿病大鼠揭示PRMT1-ADMA-DDAH通路紊乱所致内源性NOS抑制物ADMA蓄积与糖尿病心肌病（DCM）密切相关；并发现外源性NOS抑制物诱导4~16w大鼠的心脏损害与DCM大鼠心脏功能和形态结构变化非常相似，从而证明内源性ADMA蓄积是促DCM形成的重要因素。采用N-乙酰半胱氨酸（NAC）和白藜芦醇或二甲双胍慢性治疗1型和2型糖尿病大鼠、用PRMT1抑制剂TC-E或PRMT1干扰慢病毒急性治疗Zucker糖尿病大鼠以及用L-精氨酸慢性治疗外源性高NOS抑制物大鼠，不仅可降低心肌PRMT1表达、减少内源性ADMA蓄积或上调DDAH和eNOS表达、增加NO含量，又可改善大鼠心功能障碍和形态结构的病理变化，从而确认PRMT1-ADMA-DDAH通路是防治DCM的潜在靶点。在原代培养的大鼠心肌细胞，ADMA孵育呈剂量和时间依赖性地抑制胰岛素信号转导、线粒体生物合成与ATP含量，引起钙调节紊乱，上调促细胞肥大和纤维化相关基因转录并诱导心肌细胞凋亡，这些都是ADMA促DCM形成的分子机制。用二甲双胍或白芦藜醇预处理可对抗ADMA和高糖引起的胰岛素抵抗，用L-精氨酸或NAC处理可逆转ADMA和高糖引起的钙调节相关蛋白表达下调，用NAC或罗格列酮处理可对抗ADMA和高糖引起的线粒体功能和生物合成障碍及其调节紊乱，提示这些药物具有防治ADMA促DCM形成作用。在稳定过表达神经酰胺合成酶1（Cers1）和转空载体的H9C2大鼠心肌细胞系，发现ADMA和神经酰胺（C18）不仅各自可分别导致心肌细胞胰岛素抵抗、糖脂代谢和钙调节紊乱，还可相互促进对方信号通路紊乱，Cers1过表达对ADMA和C18的作用有正反馈效应，导致恶性循环。本项目为阐明DCM发生机制及其防治提供了重要的实验数据，为深入研究DCM开拓了新思路，并为防治DCM新药研发提供了新靶点。. 该项目已发表论文10篇，其中SCI论文3篇，在北大核心期刊发表论文3篇，科技核心期刊发表论文4篇；课题负责人连续参加美国糖尿病协会（ADA）的科学年会5次，并在《Diabetes》期刊发表会议摘要5篇，参加全国心血管药理学术会议3人次；培养硕、博士研究生及博士后9人；此外，尚有2篇SCI论文在投稿中，还有3篇SCI论文正在整理、完善及写作中。

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