Fibroblast growth factor receptor 1 (Fgfr1) gene knockout impairs cardiomyocyte differentiation in murine embryonic stem cells (mESC). Here, various chemical compounds able to enhance cardiomyocyte differentiation in mESC were tested for their ability to rescue the cardiomyogenic potential of Fgfr1-/- mESC. Among them, only the reduced form of vitC, L-ascorbic acid, was able to recover beating cell differentiation in Fgfr1-/- mESC. The appearance of contracting cells was paralleled by the expression of early and late cardiac gene markers, thus suggesting their identity as cardiomyocytes. In the attempt to elucidate the mechanism of action of vitC on Fgfr1-/- mESC, we analyzed several parameters related to the intracellular redox state, such as reactive oxygen species content, Nox4 expression, and superoxide dismutase activity. The results did not show any relationship between the antioxidant capacity of vitC and cardiomyocyte differentiation in Fgfr1-/- mESC. No correlation was found also for the ability of vitC to modulate the expression of pluripotency genes. Prolyl hydroxylases (PHD) are members of the iron- and 2-oxoglutarate-dependent dioxygenase family, that direct hypoxia inducible factor (HIF)-1α to degradation in normoxic conditions. To test the hypothesis that vitC was acting by maintaining iron ion in the reduced form (Fe2+), we initially evaluated HIF-1α levels in the undifferentiated state of mESC lines. Western blot analysis of isolated nuclei showed a higher HIF-1α content in undifferentiated Fgfr1- /- mESC versus the heterozygous counterpart. Unexpectedly, vitC treatment similarly up regulates HIF- 1α content in both cell lines, thus preventing any conclusion from this result. However, treatment of Fgfr1-/- mESC with both Fe2+ or the HIF inhibitor CAY10585 were able to rescue cardiomyocyte differentiation. In conclusion, our data point to a crucial role for HIF-1α modulation in Fgfr1-dependent cardiomyogenesis.
Ascorbic acid rescues cardiomyocyte development in Fgfr1-/- murineembryonic stem cells
CRESCINI, Elisabetta;GUALANDI, Laura;UBERTI, Daniela Letizia;PRANDELLI, Chiara;PRESTA, Marco;DELL'ERA, Patrizia
2012-01-01
Abstract
Fibroblast growth factor receptor 1 (Fgfr1) gene knockout impairs cardiomyocyte differentiation in murine embryonic stem cells (mESC). Here, various chemical compounds able to enhance cardiomyocyte differentiation in mESC were tested for their ability to rescue the cardiomyogenic potential of Fgfr1-/- mESC. Among them, only the reduced form of vitC, L-ascorbic acid, was able to recover beating cell differentiation in Fgfr1-/- mESC. The appearance of contracting cells was paralleled by the expression of early and late cardiac gene markers, thus suggesting their identity as cardiomyocytes. In the attempt to elucidate the mechanism of action of vitC on Fgfr1-/- mESC, we analyzed several parameters related to the intracellular redox state, such as reactive oxygen species content, Nox4 expression, and superoxide dismutase activity. The results did not show any relationship between the antioxidant capacity of vitC and cardiomyocyte differentiation in Fgfr1-/- mESC. No correlation was found also for the ability of vitC to modulate the expression of pluripotency genes. Prolyl hydroxylases (PHD) are members of the iron- and 2-oxoglutarate-dependent dioxygenase family, that direct hypoxia inducible factor (HIF)-1α to degradation in normoxic conditions. To test the hypothesis that vitC was acting by maintaining iron ion in the reduced form (Fe2+), we initially evaluated HIF-1α levels in the undifferentiated state of mESC lines. Western blot analysis of isolated nuclei showed a higher HIF-1α content in undifferentiated Fgfr1- /- mESC versus the heterozygous counterpart. Unexpectedly, vitC treatment similarly up regulates HIF- 1α content in both cell lines, thus preventing any conclusion from this result. However, treatment of Fgfr1-/- mESC with both Fe2+ or the HIF inhibitor CAY10585 were able to rescue cardiomyocyte differentiation. In conclusion, our data point to a crucial role for HIF-1α modulation in Fgfr1-dependent cardiomyogenesis.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
