Autonomous and Non-autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes.

Germline mutations in BRAF cause cardio-facio-cutaneous syndrome (CFCS), whereby 40% of patients develop hypertrophic cardiomyopathy (HCM). As the role of the RAS/MAPK pathway in HCM pathogenesis is unclear, we generated a human induced pluripotent stem cell (hiPSC) model for CFCS from three patients with activating BRAF mutations. By cell sorting for SIRPα and CD90, we generated a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM. BRAF-mutant SIRPα(+)/CD90(-) cardiomyocytes displayed cellular hypertrophy, pro-hypertrophic gene expression, and intrinsic calcium-handling defects. BRAF-mutant SIRPα(-)/CD90(+) cells, which were fibroblast-like, exhibited a pro-fibrotic phenotype and partially modulated cardiomyocyte hypertrophy through transforming growth factor ß (TGFß) paracrine signaling. Inhibition of TGFß or RAS/MAPK signaling rescued the hypertrophic phenotype. Thus, cell autonomous and non-autonomous defects underlie HCM due to BRAF mutations. TGFß inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies.

Multifaceted roles of miR-1s in repressing the fetal gene program in the heart.

miRNAs are an important class of regulators that play roles in cellular homeostasis and disease. Muscle-specific miRNAs, miR-1-1 and miR-1-2, have been found to play important roles in regulating cell proliferation and cardiac function. Redundancy between miR-1-1 and miR-1-2 has previously impeded a full understanding of their roles in vivo. To determine how miR-1s regulate cardiac function in vivo, we generated mice lacking miR-1-1 and miR-1-2 without affecting nearby genes. miR-1 double knockout (miR-1 dKO) mice were viable and not significantly different from wild-type controls at postnatal day 2.5. Thereafter, all miR-1 dKO mice developed dilated cardiomyopathy (DCM) and died before P17. Massively parallel sequencing showed that a large portion of upregulated genes after deletion of miR-1s is associated with the cardiac fetal gene program including cell proliferation, glycolysis, glycogenesis, and fetal sarcomere-associated genes. Consistent with gene profiling, glycogen content and glycolytic rates were significantly increased in miR-1 dKO mice. Estrogen-related Receptor ß (Errß) was identified as a direct target of miR-1, which can regulate glycolysis, glycogenesis, and the expression of sarcomeric proteins. Cardiac-specific overexpression of Errß led to glycogen storage, cardiac dilation, and sudden cardiac death around 3-4 weeks of age. We conclude that miR-1 and its primary target Errß act together to regulate the transition from prenatal to neonatal stages by repressing the cardiac fetal gene program. Loss of this regulation leads to a neonatal DCM.

Identification and purification of human induced pluripotent stem cell-derived atrial-like cardiomyocytes based on sarcolipin expression.

The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. 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 many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.