MYL4功能异常致心房纤维化性心肌病的机制研究

Atrial fibrosis is one of the most important mechanism leading to atrial structural abnormalities and electrical conduction disorders. We found a Chinese family with atrial standstill, which is the failure of the atria to contract, characterized electrocardiographically by serious bradycardia or junctional escape rhythm, and the absence of P waves. After exome sequencing, we identified a rare missense variant of MYL4 (p.E11K) which had perfect co-segregation in this large multiplex atrial standstill pedigree. Then we built a knock-in rat model by the CRISPR/Cas-mediated genome editing. Results show that rats exhibit the phenotypes consistent with human patients, with atrial electrical activity gradual disappearance and significant atrial fibrosis. However, the mechanism of MYL4 in atrial fibrosis is still unclear. Atrial fibrosis was always in parallel with myocyte apoptosis and resulted from a variety of cardiac insults that share common fibro-proliferative signaling pathways. We found that TUNEL positive nuclei in atrial tissue significantly increased in either heterozygous or homozygous MYL4 p.E11K rats, and apoptosis was earlier than fibroblast proliferation. So we hypothesized that MYL4 p.E11K mutation leads to atrial fibrosis through MYL4 dysfunction induced atrial myocyte calcium overload/endoplasmic reticulum stress/apoptosis, and the apoptotic cells release fibrotic signaling protein TGFβ1 leading to reactive atrial fibrosis; correct the MYL4 dysfunction may slow down or reverse the disease process. Specifically, we will pursue following research objectives to test this hypothesis: firstly, atrial myocyte apoptosis, pro-fibrotic signaling pathways and fibroblasts proliferation and differentiation were evaluated in different age of MYL4 p.E11K knock-in rat model to determine the relevance of apoptosis and fibrosis. In vitro experiments were performed to validate the results of in vivo data. Then it will be explored whether over-expression normal MYL4 protein by adenovirus would slow down or reverse the disease process. This study will clarify the role and mechanism of MYL4 in atrial fibrosis, which may facilitated the novel insights into how MYL4 influence atrial fibrosis and explore the therapeutic potential of over-expression normal MYL4 protein as a treatment for atrial fibrosis.

心房纤维化是导致心房结构异常和电传导障碍的重要机制。申请人前期发现一心房静止家系，定位了一个新的致病位点MYL4 c.31G>A (p.E11K)，并建立点突变大鼠模型。该大鼠表现为心房电活动逐渐消失和明显心房纤维化，但机制未明。我们预实验发现p.E11K可引起大鼠心房肌细胞凋亡且早于成纤维细胞的增殖。故提出假设：MYL4功能异常可引起心房肌细胞钙超载/内质网应激/凋亡，凋亡细胞释放纤维化信号TGFβ1导致反应性心房肌纤维化；纠正MYL4功能异常可以减慢/逆转疾病过程。本课题拟通过对不同年龄的MYL4 p.E11K大鼠连续评估其心房肌细胞凋亡、TGFβ1-Smad信号通路激活及成纤维细胞增殖分化，确定凋亡与纤维化的相关性，并通过体外实验进行验证。然后进一步探索过表达正常MYL4蛋白是否可以减慢/逆转疾病过程。本研究将阐明MYL4在心房纤维化中的作用机制，为心房纤维化的防治提供新的分子靶点。

心房纤维化是导致心房结构异常和电传导障碍的重要机制。申请人前期发现一心房静止家系，定位了一个新的致病位点MYL4 c.31G>A (p.E11K)，并建立点突变大鼠模型。该大鼠表现为心房电活动逐渐消失和明显心房纤维化，但机制未明。我们在不同年龄点连续评价MYL4 p.E11K对心房肌细胞凋亡、心房纤维化及心房电生理的影响，研究发现，MYL4 p.E11K突变后是先导致心房结构异常，然后导致电生理异常；MYL4 p.E11K是先导致心房肌细胞凋亡进而激活纤维化信号通路及心肌成纤维细胞。我们构建携带正常MYL4和MYL4 p.E11K的质粒载体，表达和纯化正常MYL4和MYL4 p.E11K蛋白，在分子水平检测MYL4 p.E11K对肌球蛋白ATPase活性的影响，结果发现该蛋白突变后导致MYL4功能异常。通过CRISPR/Cas9基因修饰技术治疗心房心肌病，在使用CRISPR/Cas9基因修饰之前，我们使用腺相关病毒在点突变大鼠过表达正常MYL4编码的蛋白，发现过表达正常MYL4后可以得到一定程度的缓解。这也提示基因修饰MYL4基因表达正常MYL4蛋白课题对疾病起到治疗作用。通过MYL4 p.E11K大鼠心房肌组织行电镜检查，发现点突变大鼠与WT大鼠相比，表现为大量的溶酶体聚集，提示溶酶体在MYL4 p.E11K大鼠心房纤维化及心房凋亡过程中发挥重要作用。进一步研究发现突变大鼠自噬流障碍，结构蛋白被破坏。此外，临床研究发现与肌球蛋白轻链4（MYL4）相关的Rs4968309可以预测无结构性心脏病的房颤患者的疾病发作并预测房颤患者导管消融的预后。综上，我们的课题围绕MYL4基因，研究了MYL4异常与房颤及心房心肌病发病的机制，以及MYL4基因在临床诊断及治疗的应用，为阐明MYL4与心房疾病的相关性提供了实验数据和理论基础。