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.

PUBLIC HEALTH RELEVANCE: This project seeks to use human induced pluripotent stem cell lines from infants and children with abnormalities of cardiac muscle due to inherited disorders of the RAS/MAPK signaling pathway. These induced pluripotent stem cell lines will be used to derive cardiomyocytes, which will then be characterized thoroughly. If successful, this approach will be important for elaborating the pathogenesis of pediatric heart muscle disorders, which will inform the development of novel therapeutic approaches as well as clinical care pathways.