Induced pluripotent stem cell-derived cardiomyocytes as a model to study cardiac defects in Noonan syndrome and related disorders
Rebecca Josowitz1, Sonia Mulero-Navarro1, Ilan Riess1, Sherly Pardo3 Sunita D’Souza2, Xonia Carvajal-Vergara2, Marco Tartaglia4, Ihor Lemischka2, Bruce D. Gelb1
1Child Health and Development and 2Black Family Stem Cell Institutes, Mount Sinai School of Medicine, New York, NY, USA, 10029, 3Recinto de Ciencias Medicas, Universidad de Puerto Rico, San Juan, Puerto Rico, 00936-5067, 4Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy, 00161.
Mutations in PTPN11 and BRAF, genes involved in the RAS/MAPK pathway, are implicated in a variety of clinical disorders, such as Noonan syndrome (NS), LEOPARD syndrome (LS), and cardio-facial-cutaneous syndrome (CFCS), members of a family of disorders termed the “RASopathies”. These disorders are characterized by skeletal and neurological defects, as well as a high prevalence of cardiovascular abnormalities. Hypertrophic cardiomyopathy (HCM) is observed in 90% of patients with PTPN11 mutations causing LS and 40% of patients with CFCS, yet PTPN11 mutations causing NS are negatively associated with HCM. In order to further elucidate the molecular mechanisms of RASopathy-associated HCM, we have generated human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes as representative models of these syndromes. We have previously demonstrated that hiPSCs derived from patients with LS exhibit altered RAS/MAPK signaling and their derived cardiomyocytes display a molecular phenotype consistent with the cardiac hypertrophy observed clinically. We have now introduced lentiviral selection cassettes driven by the cardiomyocyte-specific gene α-myosin heavy chain (αMHC) (gift from Mark Mercola), in order to purify cardiomyocytes from a heterogeneous population of hiPSC-derived cells. Additionally, the use of bacterial artificial chromosomes (BACs) containing selection markers driven by the ventricular cardiomyocyte specific gene myosin light chain 2v (MLC2V), will allow specific isolation and characterization of ventricular cells, and thus more thorough investigation of the mechanism underlying RASopathy-associated HCM. We hypothesize that purified hiPSC-derived cardiomyocytes from patients harboring LS PTPN11 mutations and CFCS BRAF mutations will display increased cellular area and altered signaling pathway activation compared to hiPSC-derived cardiomyocytes from patients harboring NS PTPN11 mutations and control cardiomyocytes. Using a directed cardiac differentiation protocol, we have obtained differentiated beating embryoid bodies (EBs) in all samples. After selection with the αMHC selection cassette, counting cardiac troponin T+ (cTNT) cells revealed an enrichment of up to 90% cardiomyocytes. ImageJ analysis of cTNT+ cells revealed a similar average cellular area between CFC-BRAF and LS-PTPN11 samples, double in size compared to the average cellular area in the NS-PTPN11 sample. Our data indicate that the phenotype of iPSC-derived cardiomyocytes from patients with LS, CFCS, and NS correlates with the clinically observed cardiac phenotype of their respective disorders. Using purified cardiomyocytes from these lines, we will be able to study the molecular mechanisms underlying the cardiovascular manifestations characteristic of their corresponding disorders, with the goal of further understanding the role of RAS/MAPK signaling in normal and abnormal cardiac function.