miR-7,26,125在慢性心衰心室晚钠电流异常增强中的作用及恶性心律失常防治新靶点研究

Patients with congestive HF are prone to develop malignant arrhythmias, such as torsade de pointes (TdP),especially under conditions of reduced repolarization reserve that induced by hypokalemia, Ikr inhibitor dofetilide,et al. The mechanisms of TdP involve ion channel remodeling in the failing hearts with prolongation of the action potential. The action potential duration can be prolonged either by a decrease in outward currents or by an increase in inward currents during the plateau phase of the action potential. Downregulation of outward currents such as Ikr and Iks has been well documented in animal models and human HF. The role of late Na+ current (INaL) in action potential prolongation in failure hearts is remain unclear. Recently, some researchers identified that INaL is small in the normal heart but its magnitude is increased in failing ventricular myocytes from human and other animal species. There is a growing body of evidence that SCN5A (Nav1.5) seems to be a major contributor of INaL, increased INaL in failing ventricular myocytes due to downregulation of SCN5A protein expression. Post-transcriptional changes in SCN5A protein expression is remain unclear. MicroRNAs (miRNAs) are endogenous noncoding RNAs, about 22 nucleotides in length that mediate post-transcriptional gene silencing. Evidences has emerged indicating miR-7, 26,125 were significantly up-regulated in failing ventricular myocytes. We used 9 established miRNA target prediction programs (Miranda, PicTar5, PITA, RNA22, DIANA-mT, RNAhybrid, and TargetScan, miRDB, miRWalk) to predict the miRNA targets and confirmed SCN5A is the target gene of miR-7, 26,125. Base on aforementioned studis, we hypothesized that the increased miR-7, 26,125 in failing ventricular myocytes mediate post-transcriptional repressing SCN5A mRNA that related increased INaL.Locked nucleic acid (LNA)-modified multi-target anti-miR chemistries MT-LNA-anti-miR-7, 26,125 can target cardiac expressed miRNAs to therapeutically inhibit miR-7, 26,125 on failing hearts. In this study, we will use miRNA microarray technology miRCURY? LNA Array to analyzed miRNA expression profiles of failing ventricular myocytes. We also confirm increased miR-7, 26,125 in failing hearts by quantitative real-time RT-PCR analysis. Furthermore, dual-luciferase reporter system identified that SCN5A mRNA was the common mRNA target of miR-7, 26,125. Base on canine HF model that created by rapid right ventricular pacing,miR-7, 26,125 transfer technology, q-RT-PCR,western blotting and cell patch-clamp recording, we will investigated the role of the miR-7, 26,125 in increased late Na current of failure ventricular and proarrhythmia. Meanwhile,the effectivity and the safety of MT- LNA-anti-miR-7,26,125 for prevention of malignant arrhythmias will be elaluated.This study will provide the new insights for understanding the mechanisms of malignant arrhythmias of HF and new approach for SCD prevention.

恶性心律失常是慢性心衰患者主要的死亡方式。探讨其发病机制及防治新靶点是目前研究热点。研究表明，衰竭心肌中SCN5A表达水平下调并导致晚钠电流（INaL）异常增强是恶性心律失常的重要发病机制。权威microRNA靶基因预测软件预测内源性的、非编码miR-7,26,125的靶基因是SCN5A。因而提出假说：衰竭心肌中异常增高的miR-7,26,125共同负调控SCN5A并导致INaL异常增强， MT-LNA-anti-miR-7,26,125反义寡核苷酸可以稳定衰竭心肌中SCN5A和INaL于恰当水平，进而防治恶性心律失常。本研究利用犬慢性心衰模型、miR表达谱芯片、双荧光素酶报告系统、实时定量PCR、Western blot及膜片钳技术探讨miR-7,26,125在慢性心衰心室INaL异常增强中的作用，以及MT-LNA-anti-miR-7,26,125防治恶性心律失常的有效性和安全性。

恶性心律失常是慢性心衰患者主要的死亡方式。探讨其发病机制及防治新靶点是目前研究的热点。研究表明，衰竭心肌中SCN5A表达水平下调并导致晚钠电流（INaL）异常增强是恶性心律失常的重要发病机制。权威microRNA靶基因预测软件预测内源性的、非编码miR-7,26,125的靶基因是SCN5A。因而提出假说：衰竭心肌中异常增高的某个或者几个microRNA也许可负调控SCN5A并导致INaL异常增强， MT-LNA-anti-microRNA反义寡核苷酸可以稳定衰竭心肌中SCN5A和INaL于恰当水平，进而防治恶性心律失常。本研究利用犬慢性心衰模型、双荧光素酶报告系统、实时定量PCR、Western blot探讨microRNA在慢性心衰心室INaL异常增强中的作用，以及MT-LNA-anti-microRNA防治恶性心律失常的有效性和安全性。结果发现：慢性心衰伴恶性心律失常患者血浆中差异表达的miR可作为慢性心衰患者发作恶性心律失常的重要生物标记物（biomarker）。同时，在成功建立犬慢性心衰动物模型基础上证实了衰竭心肌中异常上调的cfa-mir-29a在慢性心衰伴恶性心律失常犬心室组织中异常增高，利用靶基因预测软件、荧光素酶报告系统、犬心室肌细胞，我们在表达水平证实了cfa-mir-29a导致慢性心衰促发恶性心律失常的分子机制，即：异常升高的cfa-mir-29a通过多靶点下调SCN5A，SCN2B及COX40离子通道基因及其相应编码蛋白，最终导致晚钠电流（INaL）异常增强及心室肌的折返活动，进而导致慢性心衰促发恶性心律失常的发生发展，这为严重而常见的这一心血管疾病提供了可能的疾病诊断标志物和相应的治疗靶点。