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

使用患者来源的诱导多能干细胞模拟家族性扩张型心肌病

• 批准号: 9325330
• 负责人: Cassandra Lynn Happe
• 金额: $ 5.61万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-06-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9325330
• 关键词: Actins; Address; Adult; Affect; Atomic Force Microscopy; Biological Assay; Calcium; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Pathology; Cell Line; Cell physiology; Cells; Cellular biology; Charge; Code; Codon Nucleotides; Complex; Coupling; Cytoskeletal Proteins; Development; Diagnosis; Dilated Cardiomyopathy; Disease; Disease model; Dyes; Exhibits; Extracellular Matrix; Family; Family member; Fluo 4; Functional disorder; Future; Gene Mutation; Genetic; Genome engineering; Genotype; Glutamic Acid; Goals; Heart; Heart Diseases; Heart Transplantation; Heart failure; Human; Image; Immunofluorescence Immunologic; Impairment; In Vitro; Incidence; Individual; Intercalated disc; Intercellular Junctions; Lead; Left; Link; Lysine; Mediating; Modeling; Morbidity - disease rate; Mutate; Mutation; Myosin ATPase; Nonsense Codon; Patients; Play; Positioning Attribute; Proteins; Protocols documentation; Public Health; Reading Frames; Role; Sagittaria; Sarcomeres; Societies; Speed; Structural defect; Structure; Therapeutic; Thin Filament; Traction; Variant; Ventricular; Vinculin; Work; alpha Tropomyosin; cellular pathology; cohort; disease heterogeneity; disease mechanisms study; effective therapy; electrical property; familial dilated cardiomyopathy; genetic pedigree; genome editing; genome wide association study; improved; in vitro Model; in vivo; induced pluripotent stem cell; insight; interest; mortality; multi-electrode arrays; mutant; protein E; public health relevance; repaired; small molecule; voltage; voltage sensitive dye

 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.

 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 人中就有 1 人受影响，并且由于其高死亡率和发病率而成为重大的公共卫生问题。该疾病的特点是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。由于该疾病的异质性，DCM 的病理生理学仍然难以捉摸，其中细胞骨架蛋白存在大量突变，例如纽蛋白、VCL 和肌节蛋白，例如α-原肌球蛋白，TPM1，已被牵连；由于成人心脏的心肌细胞 (CM) 难以分离和培养，使得体外分析变得困难，这一事实加剧了这些多样性。 DCM 给受影响的家庭和社会带来了巨大的负担，因此迫切需要开发准确的疾病模型，以阐明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类诱导多能干细胞（hiPSC）产生的 CM 开发 DCM 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 源自携带 V​​CL 和 TPM1 突变的家庭队列，其中多个具有两种突变杂合子的家庭成员已被诊断患有 DCM。在目标 1 中，我们将从患者来源的 hiPSC 和家族对照中生成 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目标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，例如肌节或闰盘紊乱，并与家族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 的机电耦合。该检测将确定家族和等基因系内 VCL/TPM1 突变引起的任何功能畸变。这些研究的结果将有助于更好地理解 VCL/TPM1 突变在结构和功能异常发展中的作用，这些异常可能共同导致患者 DCM，并有可能为未来的治疗提供见解。