Human Induced Pluripotent Cell Models of Pediatric Cardiac Disorders

描述(申请人提供)：心血管生物学研究中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。自从2007年证明了将终末分化的人类细胞(如皮肤成纤维细胞)重新编程为多能干细胞的可能性以来，在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性形式：与RAS信号异常相关的肥厚性心肌病(HCM)，以及与HRAS信号增强相关的房肌相关性心动过速。Rasopathies是一种常染色体显性遗传病，由编码RAS/MAPK途径蛋白的基因错义突变引起。HCM在Rasopathies中很常见，多灶性房性心动过速(MAT)专门在一种疾病中观察到，Costello综合征是由功能获得型HRAS突变引起的。对于特定的AIM 1，PI假设与RA相关的HCM是通过信号通路激活而产生的，这在特定的疾病中是不同的。为了测试这一点，他们将使用现有的人类IPSC系来治疗两种表现为心肌细胞肥大的Rasopathy。携带豹和心面皮肤综合征引起突变的分离的心室肌细胞将在信号转导、细胞内钙处理和细胞之间的收缩能力方面具有特征。对于特定的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调节的扰动引起的。为了测试这一点，从Costello综合征IPSC系分化出来的心房心肌细胞将被评估钙瞬变、电生理学和对相关抗心律失常药物的反应。对于特定的AIM 3，PI假设与RA相关的HCM将被解决突变特异性信号扰动的抑制剂逆转。为了研究这一点，IPSC来源的豹和心面部皮肤综合征心肌细胞将被信号通路抑制剂处理以逆转肥厚。将建立剂量响应曲线。联合疗法将在较低剂量下进行疗效测试。治疗对豹综合征心肌细胞自噬缺陷的影响将被确定。广泛地说，这些研究将利用新的IPSC技术来阐明与Rasopathies相关的心肌疾病的发病机制：肥厚性心肌病和房性心律失常。这些研究可能会对这些心肌疾病的新治疗策略的开发产生重要影响，我们目前的方法对这些疾病还不能治愈。

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.