Human Induced Pluripotent Cell Models of Pediatric Cardiac Disorders

描述（由申请人提供）：心血管生物学研究的一个长期障碍是无法在长期细胞培养中维持人类心肌细胞。自2007年以来，人们证实了将皮肤成纤维细胞等终末分化的人类细胞重编程为多能干细胞的可能性，从而使体外生成人类心肌细胞成为可能，从而使人类原发性心肌疾病的研究成为可能。在这个项目中，我们打算研究儿童心肌疾病的遗传形式：与RAS信号异常相关的肥厚性心肌病（HCM）和与HRAS信号增加相关的心房肌相关性心动过速。“RASopathies”是由编码RAS/MAPK通路蛋白的基因错义突变引起的常染色体显性疾病家族。HCM在ras病中很常见，多灶性房性心动过速（MAT）在一种疾病Costello综合征中特别观察到，这是由HRAS功能获得突变引起的。对于SPECIFIC AIM 1， pi假设RASopathy相关的HCM是通过信号通路激活产生的，而信号通路在特定疾病中是不同的。为了验证这一点，他们将使用现有的人类iPSC细胞系治疗两种表现心肌细胞肥大的ras病变。分离的心室心肌细胞携带LEOPARD和心表皮综合征引起的突变，将在信号转导、细胞内钙处理和细胞间的收缩性方面进行表征。对于SPECIFIC AIM 2， pi假设Costello综合征中的MAT是由HRAS信号改变引起的细胞内钙处理扰动引起的。为了验证这一点，将评估从Costello综合征iPSC系分化出来的心房心肌细胞的Ca2+瞬态、电生理和对相关抗心律失常药物的反应。对于SPECIFIC AIM 3， pi假设rasopathy相关的HCM可以通过处理突变特异性信号扰动的抑制剂逆转。为了研究这一点，ipsc衍生的LEOPARD和心表皮综合征心肌细胞将被信号通路抑制剂治疗以逆转肥大。建立剂量响应曲线。将在较低剂量下测试联合疗法的疗效。治疗对LEOPARD综合征心肌细胞自噬缺陷的影响将被确定。总的来说，这些研究将利用新的iPSC技术的力量来阐明与rasopathy相关的心肌疾病的发病机制：HCM和心房心律失常。这些研究可能对这些心肌疾病的新治疗策略的发展产生重要影响，我们目前的方法无法治愈这些疾病。

DESCRIPTION (provided by applicant): A long-standing barrier in cardiovascular biology research has been the inability to maintain human cardiomyocytes in long-term cell culture. Since 2007 when the possibility of reprogramming terminally differentiated human cells like skin fibroblasts into pluripotent stem cells was demonstrated, it became possible to generate human cardiomyocytes in vitro, enabling the study of human primary myocardial diseases. For this project, we intend to study inherited forms of childhood myocardial disease: hypertrophic cardiomyopathy (HCM) associated with RAS signaling abnormalities and atrial muscle-related tachycardia associated with increased HRAS signaling. The "RASopathies" are a family of autosomal dominant disorders caused by missense mutations in genes encoding RAS/MAPK pathway proteins. HCM is common in the RASopathies and multifocal atrial tachycardia (MAT) is specifically observed in one disorder, Costello syndrome, which is caused by gain-of-function HRAS mutations. For SPECIFIC AIM 1, the PIs hypothesize that RASopathy- associated HCM arises through signaling pathway activation that differs among the specific disorders. To test this, they will use existing human iPSC lines for two RASopathies that exhibit cardiomyocyte hypertrophy. Isolated ventricular cardiomyocytes harboring LEOPARD and cardiofaciocutaneous syndrome-causing mutations will be characterized with respect to signal transduction, intracellular calcium handling and contractility among cells. For SPECIFIC AIM 2, the PIs hypothesize that MAT in Costello syndrome is caused by perturbations in intracellular calcium handling induced by altered signaling from HRAS. To test this, atrial cardiomyocytes, differentiated from Costello syndrome iPSC lines, will be assessed for Ca2+ transients, electrophysiology and response to relevant anti-arrhythmic drugs. For SPECIFIC AIM 3, the PIs hypothesize that RASopathy-associated HCM will be reversed by inhibitors that address mutation-specific signaling perturbations. To study this, iPSC-derived LEOPARD and cardiofaciocutaneous syndrome cardiomyocytes will be treated with signaling pathway inhibitors to reverse hypertrophy. Dose response curves will be established. Combination therapies will be tested for efficacy at lower doses. Effects of therapies on the autophagy defect in LEOPARD syndrome cardiomyocytes will be determined. Broadly, these studies will harness the power of the new iPSC technology to elucidate the pathogenesis of myocardial disease associated with RASopathies: HCM and atrial arrhythmias. These studies could have important impact on the development of novel therapeutic strategies for these myocardial diseases, for which our current approaches are not curative.