Fibroblast Growth Factor Receptor-1 Is Essential for In Vitro Cardiomyocyte Development

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Abstract
Fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling plays a crucial role in mesoderm formation and patterning. Heartless mutant studies in Drosophila suggest that FGFR1, among the different FGFRs, may play a role in cardiogenesis. However, fgfr1 −/− mice die during gastrulation before heart formation. To establish the contribution of FGFR1 in cardiac development, we investigated the capacity of murine fgfr1 +/− and fgfr1 −/− embryonic stem (ES) cells to differentiate to cardiomyocytes in vitro. Clusters of pulsating cardiomyocytes were observed in >90% of 3-dimensional embryoid bodies (EBs) originated from fgfr1 +/− ES cells at day 9 to 10 of differentiation. In contrast, 10% or less of fgfr1 −/− EBs showed beating foci at day 16. Accordingly, fgfr1 −/− EBs were characterized by impaired expression of early cardiac transcription factors Nkx2.5 and d-Hand and of late structural cardiac genes myosin heavy chain ( MHC )-α, MHC -β, and ventricular myosin light chain . Homozygous fgfr1 mutation resulted also in alterations of the expression of mesoderm-related early genes, including nodal , BMP2, BMP4 , T ( bra ), and sonic hedgehog . Nevertheless, fgfr1 +/− and fgfr1 −/− EBs similarly express cardiogenic precursor, endothelial, hematopoietic, and skeletal muscle markers, indicating that fgfr1 -null mutation exerts a selective effect on cardiomyocyte development in differentiating ES cells. Accordingly, inhibitors of FGFR signaling, including the FGFR1 tyrosine kinase inhibitor SU 5402, the MEK1/2 inhibitor U0126, and the protein kinase C inhibitor GF109 all prevented cardiomyocyte differentiation in fgfr1 +/− EBs without affecting the expression of the hematopoietic/endothelial marker flk-1 . In conclusion, the data point to a nonredundant role for FGFR1-mediated signaling in cardiomyocyte development.

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