老年性窦房结功能减退新机制：miR-125的调控作用及防治新靶点研究

It is known that age-dependent degeneration of sinoatrial node (SAN)function is strong associated with the mortality and severity of cardiovascular diseases,which has been attracted more attentions.Understanding the mechanisms of the age-dependent degeneration of SAN function and find new approaches for bradyarrhythmias therapy has become a hot spot and difficulty.Previous studies indicated that age-dependent degeneration of SAN function may occur as a result of changes of pacemaker genes and the remodeling of ions channels, which provide a novel strategy for prevent this disease.Previous studies and the predictions of bioinformatics tools had been suggested that an endogenous noncodin，single-stranded miR-125 overexpressed and could negatively modulate genes and ion channels that related pacing. Base on aforementioned studis, we hypothesized that the fundmental mechanisms of SAN function decline may be miR-125 mediate post-transcriptional repressing multi-target mRNA that related pacemaker gene and adverse the remodeling of ion channels with SAN aging.A miR-125 inhibitor,2-O-methyl-modified antisense oligoribonucleotides(AMOs) could relieved the adverse remodeling of ion channels,recovery the paceaker currents lineage and improved the SAN function by the multi-targeting. Thus, miR-125 may is a novel antiarrhythmic target for prevented the age-dependent degeneration of SAN.In this study, we will established two experimental groups to investigate the mechanisms of age-dependet degeneration of SAN function,that is:childhood and middle age groups that will be intervented with miR-125,another is old group that will be intervented with AMO-125.The role of miR-125 and the ion channels genes that related miR-125 in age-dependent degneration of SAN function will be investigated by miR-125 and AMO-125 gene transfer,recording the electrophysiologic parameters of the SAN, RT-PCR,western blotting and single-cell patch-clamp recording.Meanwhile,the effectivity and the safety of AMO-125 for treatment of age-dependent degeneration of SAN funcyion will be elaluated.This study will provide the new insights for understanding the mechanisms of the the age-dependent degeneration of SAN function and new approach for bradyarrhythmias therapy.

老年性窦房结功能减退与心血管疾病严重程度和死亡率明显相关而日益受到重视。探索其发病机制并改善窦房结功能是目前缓慢性心律失常领域研究的热点和难点。研究发现，老年性窦房结功能减退的过程是窦房结起搏相关基因及其离子通道重塑的过程。而内源性的、非编码单链miR-125 异常增高并多靶点负向调控起搏相关基因及其离子通道。因而提出假说：miR-125多靶点负向调控起搏相关基因及其离子通道是老年性窦房结功能减退的关键机制，miR-125反义寡核苷酸可多靶标改善离子通道重塑而使窦房结恢复完整起搏电流谱，进而改善窦房结功能。本研究设立幼年组、中年组+miR-125和老年组+AMO-125,通过窦房结电生理参数测定、RT-PCR、western blotting及单细胞膜片钳技术，探索miR125在老年性窦房结功能减退中的作用和涉及到的离子通道途径，以及AMO-125防治老年性窦房结功能减退的有效性和安全性。

老年性窦房结功能减退与心血管疾病严重程度和死亡率明显相关而日益受到重视。探索其发病机制并改善窦房结功能是目前缓慢性心律失常领域研究的热点和难点。研究发现，老年性窦房结功能减退的过程是窦房结起搏相关基因及其离子通道重塑的过程。在几内亚猪窦房结组织中发现，随着动物年龄增加，Cav1.2离子通道基因及其相应编码蛋白表达进行性下调，这种离子通道重塑会导致窦房结自发性电活动下降及对钙离子通道阻断剂敏感性增加。Cav1.3基因敲出小鼠出现钙电流的缺失和显著的心动过缓,提示: Cav1.3离子通道在窦房结起搏活动中扮演重要角色。而内源性的、短链、非编码microRNA可在转录后水平，非特异性的和靶基因(也可以是起搏相关基因及其离子通道) 3’非转录区多靶点结合。本课题分别以病态窦房结综合征患者、模式动物、起搏细胞以及窦房结组织为研究对象，从表达和功能水平阐述异常的microRNA在老年性窦房结功能减退中的作用及其可能分子机制。结果发现：miR-1976在老年性窦房结功能减退患者血浆及老年家兔窦房结组织中异常增高，利用靶基因预测软件、荧光素酶报告系统、人ips细胞来源的起搏细胞及miR-1976过表达小鼠，我们在表达和功能水平证实了hsa-miR-1976导致年龄相关性病态窦房结综合征的分子机制，即：异常升高的hsa-miR-1976通过多靶点下调Cav1.2 及 Cav1.3离子通道基因及其相应编码蛋白，最终导致窦房结功能减退，进而导致老年相关性疾病-病态窦房结综合征的发生发展，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。