基于患者特异性诱导性多能干细胞模型的母系遗传性肥厚型心肌病致病分子机制研究

Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in the young individuals. The maternal transmission of familial HCM in some pedigrees suggests a potential role of mitochondrial DNA (mtDNA) mutations in HCM. However, the underlying molecular mechanisms linking mtDNA mutations to HCM remain poorly understood. We previously identified a novel m.2336T>C homoplasmic mutation in a Chinese maternally inherited HCM family. This mutation disturbs the conserved 2336U-A2438 base-pairing in the stem-loop of the 16S rRNA domain III and causes mitochondrial dysfunctions. The induced pluripotent stem cells (iPSCs)-derived cardiomyocytes from the proband possess the key features of HCM as well as the elevating intracellular Ca2+ concentration and decreasing L-type Ca2+ current. Mitochondria play a fundamental role in regulation of Ca2+ homeostasis. Meanwhile, Ca2+ controls the excitation-contraction coupling and electrophysiological signalling in the heart. So the hypothese are presented out: the altered Ca2+ homeostasis caused by mitochondrial dysfunctions may result in the maternally inherited HCM. In this project we will systematically establish the patient-specific primary cells, iPSCs and derived cardiomyocytes from the maternal members of the HCM family and normal control individuals, then study the mitochondrial content, structures and functions during reprogramming and differentiation. More studies will focus on how mitochondrial dysfunctions induce the imbalances of Ca2+ homeostasis, electrophysiological abnormality and enlarged size, whether mitochondrial drugs rescure the properties of HCM-cardiomyocytes. This project may provide an innovative insight into the molecular pathogenesis of HCM contributed by mtDNA mutations.

肥厚型心肌病（HCM）是青少年心源性猝死的最常见原因。线粒体基因突变是HCM 重要致病因素，机理尚不明确。我们前期发现新的母系遗传性HCM相关m.2336T>C突变，破坏16S rRNA的2336U-A2438配对，影响ATP合成等；初步构建先证者诱导性多能干细胞（iPSCs）分化心肌细胞，具HCM相关特征，且胞内Ca2+浓度增高、Ca2+电流降低。线粒体参与Ca2+稳态，Ca2+调控心肌兴奋-收缩偶联和电生理信号。特提出研究假说：线粒体功能障碍引起的Ca2+稳态失调是母系遗传性HCM的重要致病机制。本项目拟构建上述HCM家系母系成员和正常个体的原代细胞、iPSCs及其心肌细胞模型，研究m.2336T>C突变表型表达的组织特异性和线粒体结构功能变化，阐明线粒体功能障碍对Ca2+稳态、电生理及细胞肥大等影响，线粒体功能拯救对HCM细胞功能修复的可能作用，揭示母系遗传性HCM的致病分子机制。

肥厚型心肌病（HCM）是以左心室及室间隔不对称肥厚为基本特征的原发性心肌病，是青少年心源性猝死的最常见原因之一。线粒体基因（mtDNA）突变是HCM 重要致病因素，机理尚不明确。由于mtDNA突变细胞和动物模型缺乏，严重制约相关线粒体疾病研究。我们前期工作采集到一个确诊的肥厚型心肌病家系，携带新的MT-RNR2 m.2336T>C突变，该位点进化上高度保守，该突变仅见于HCM患者，可能破坏线粒体16S rRNA的2336U-A2438配对，影响线粒体核糖体组装及其蛋白质合成等。本项目成功构建携带MT-RNR2 m.2336T>C突变的肥厚型心肌病患者特异性诱导多能干细胞（iPSCs）及其定向分化心肌细胞等，iPSCs-心肌细胞表达肌钙蛋白（TNNI3）、α-辅肌动蛋白（α-Actin）、心室肌球蛋白-2（MLC2v）和心房肌球蛋白-2（MLC2a）等心肌细胞标志蛋白，可节律跳动；HCM-iPSC-CMs表达利钠肽A（ANF）、利钠肽B（BNP）、活化T细胞的核因子4（NFATC4）等心肌肥大细胞标志蛋白，其细胞体积肥大、细胞自主跳动异常。发现HCM-iPSC-心肌细胞的mtDNA拷贝数增多，MT-RNR2 m.2336T>C突变导致16S rRNA及其结合蛋白的稳态表达水平降低，影响线粒体核糖体的功能，使得线粒体蛋白质合成降低，引起线粒体ATP/ADP比率及膜电势下降等线粒体功能障碍和线粒体超微结构损伤等；HCM-iPSC-心肌细胞的胞内Ca2+浓度增高，Ca2+稳态失调，导致L型Ca2+电流（ICaL）显著降低，引起心肌细胞的动作电位呈现延迟性去极化、动作电位时程延长等电生理异常，以及细胞体积增大等相关表型。本研究创新性地提出mtDNA突变导致心肌细胞ATP合成效率降低是HCM发生的分子基础之一；mtDNA突变引起线粒体膜电势降低、Ca2+稳态平衡失调，导致心肌细胞电生理异常等是母系遗传性HCM的重要致病分子机制。本项目研究为其它线粒体疾病的诊断和防治提供理论和实验基础。