Human Induced Pluripotent Cell Models of Pediatric Cardiac Disorders

描述（由申请人提供）：心血管生物学研究中的一个长期障碍是无法在长期细胞培养中维持人心肌细胞。自2007年证明了将终末分化的人类细胞如皮肤成纤维细胞重编程为多能干细胞的可能性以来，体外产生人类心肌细胞成为可能，从而能够研究人类原发性心肌疾病。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型心肌病（HCM）和与HRAS信号增加相关的心房肌相关性心动过速。“RAS病”是由编码RAS/MAPK途径蛋白的基因中的错义突变引起的常染色体显性疾病家族。HCM在RASopathies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，这是由功能获得性HRAS突变引起的。对于特定AIM 1，PI假设RAS病相关HCM通过信号通路激活而产生，该信号通路激活在特定疾病中不同。为了测试这一点，他们将使用现有的人类iPSC细胞系用于两种表现出心肌细胞肥大的RASopathies。携带LEOPARD和心面皮肤综合征引起突变的分离的心室心肌细胞将在信号转导、细胞内钙处理和细胞间收缩性方面进行表征。对于特定目的2，PI假设Costello综合征中的MAT是由HRAS信号改变诱导的细胞内钙处理扰动引起的。为了测试这一点，将评估从Costello综合征iPSC系分化的心房心肌细胞的Ca 2+瞬变、电生理学和对相关抗心律失常药物的反应。对于特异性AIM 3，PI假设RASopathy相关HCM将被解决突变特异性信号转导干扰的抑制剂逆转。为了研究这一点，iPSC衍生的LEOPARD和心面皮肤综合征心肌细胞将用信号传导通路抑制剂处理以逆转肥大。将建立剂量反应曲线。将在较低剂量下测试组合疗法的功效。将确定治疗对LEOPARD综合征心肌细胞中自噬缺陷的影响。概括地说，这些研究将利用新的iPSC技术的力量来阐明与RASopathies相关的心肌疾病的发病机制：HCM和房性心律失常。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.