Modeling RASopathy-associated Cardiac Disease using Patient-Specific Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

The RASopathies are a family of autosomal dominant developmental disorders due to germline mutations in the RAS/MAPK signaling pathway. Among the pleomorphic defects associated with these disorders, about 80% of RASopathy patients display cardiovascular abnormalities, with hypertrophic cardiomyopathy (HCM) being one of the most common manifestations. For one RASopathy, called cardio-facio-cutaneous syndrome (CFCS), caused primarily by activating BRAF mutations, 40% of patients develop HCM. While the RAS/MAPK pathway has been implicated in cardiac hypertrophy, the role of this pathway in HCM is still unclear. In order to better understand the role of RAS/MAPK activation in HCM, we generated a human induced pluripotent stem cell model for CFCS from three patients with activating BRAF mutations. Using cell sorting, we have developed a method to examine hiPSC-derived cell-type specific phenotypes and cellular interactions underpinning HCM. We show that purified BRAF-mutant cardiomyocytes display cellular hypertrophy with prohypertrophic gene expression and intrinsic calcium handling defects. In addition, purified BRAF-mutant fibroblast-like cells exhibit a pro-fibrotic phenotype and critically modulate cardiomyocyte hypertrophy through TGFbeta paracrine signaling. Inhibition of TGFbeta or RAS/MAPK signaling is capable of rescuing the hypertrophic phenotype. Thus, we show for the first time that cell autonomous and non-autonomous defects underlie RASopathy-associated HCM, as has been demonstrated in non-syndromic HCM models. TGFbeta inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies.;Secondarily, we have developed a method for the purification of hiPSC-derived atrial-like cardiomyocytes, as there is currently no reliable method for stem cell-derived atrial cell labeling and purification. We have created a bacterial artificial chromosome (BAC) reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN). When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find utility in the study of atrial-specific pathologies and chamber-specific lineage development.