Modeling Familial Dilated Cardiomyopathy Using Patient-derived Induced Pluripotent Stem Cells

 描述（由申请人提供）：扩张型心肌病（DCM）是最常见的心肌病类型，每250例患者中就有1例受影响，由于其高死亡率和发病率，因此是一个重大的公共卫生问题。该疾病的特征在于收缩功能障碍和左心室扩张，是心力衰竭的主要原因，也是心脏移植的普遍适应症。DCM的病理生理学仍然难以捉摸，因为疾病的异质性，其中涉及细胞骨架蛋白（例如黏着斑蛋白、VCL）和肌节蛋白（例如α-原肌球蛋白、TPM 1）的许多突变;这些疾病由于来自成人心脏的心肌细胞（CM）难以分离和培养而复杂化，使得体外分析困难。由于DCM给受影响的家庭和社会造成了巨大的负担，因此迫切需要开发准确的疾病模型，以揭示复杂的病理生理学并开发有效的治疗方法。为了满足这一需求，我们建议使用源自患者的人诱导多能干细胞（hiPSC）产生的CM开发DCM的体外模型，并研究与该疾病相关的细胞病理学。这些研究中使用的hiPSC-CM来源于携带VCL和TPM 1突变的家族队列，其中多个两种突变杂合的家族成员已被诊断患有DCM。在目标1中，我们将从患者来源的hiPSC和家族对照产生CM。此外，我们将使用基因组工程创建等基因系，以突变家族对照系中的感兴趣的基因座，并修复患者系中的突变。将评估患者hiPSC-CM的结构异常，如肌节或闰盘解体，并与家族性和同基因对照进行比较。在目标2中，我们将利用功能分析-即钙成像、电压成像、牵引力显微镜、多电极阵列记录-来表征患者和对照hiPSC-CM中的机电耦合。该试验将确定家族和等基因系内VCL/TPM 1突变导致的任何功能畸变。这些研究的结果将更好地理解VCL/TPM 1突变在结构和功能异常发展中的作用，这些结构和功能异常可能联合收割机导致患者的DCM，并可能为未来的治疗提供见解。

 DESCRIPTION (provided by applicant): Dilated cardiomyopathy (DCM) is the most common type of cardiomyopathy, affecting as many as 1 in every 250 individuals, and represents a significant public health concern due to its high mortality and morbidity rates. The disease, characterized by systolic dysfunction and left ventricular dilation, is a leading cause of heart failure and a prevalent indication for heart transplant. The pathophysiology of DCM has remained elusive because of the heterogeneity of the disease, in which numerous mutations to cytoskeletal proteins, e.g. vinculin, VCL, and sarcomeric proteins, e.g. α-tropomyosin, TPM1, have been implicated; these diversities are compounded by the fact that cardiomyocytes (CMs) from adult hearts are difficult to isolate and culture, making in vitro analysis difficult. With th huge burden DCM creates for affected families and society, there is a significant need to develop accurate disease models that unravel complex pathophysiology and enable the development of effective treatments. To address this need, we propose to develop an in vitro model of DCM using CMs generated from patient-derived human induced pluripotent stem cells (hiPSCs) and investigate the cellular pathology associated with the disease. hiPSC-CMs used in these studies originate from a family cohort carrying mutations in VCL and TPM1, where multiple family members heterozygous for both mutations have been diagnosed with DCM. In Aim 1, we will generate CMs from the patient-derived hiPSCs and familial controls. Additionally, we will create isogenic lines using genome engineering to mutate the loci of interest in familial control lines and to repair mutations in patient lines. Patient hiPSC-CMs will be assessed for structural abnormalities, such as sarcomere or intercalated disc disorganization, and compared to familial and isogenic controls. In Aim 2, we will utilize functional analyses - i.e. calcium imaging, voltage imaging, traction force microscopy, multielectrode array recording - to characterize the electro-mechanical coupling in patient and control hiPSC-CMs. The assays will determine any functional aberrations resulting from VCL/TPM1 mutations within the familial and isogenic lines. Results from these studies will create a better understanding of the role of VCL/TPM1 mutations in the development of structural and functional abnormalities that may combine to cause DCM in patients, and potentially provide insight into future therapeutics.