Ascorbic acid promotes cardiomyogenesis through SMAD1 signaling in differentiating mouse embryonic stem cells

Numerous groups have documented that Ascorbic Acid (AA) promotes cardiomyocyte differentiation from both mouse and human ESCs and iPSCs. AA is now considered indispensable for the routine production of hPSC-cardiomyocytes (CMs) using defined media; however, the mechanisms involved with the inductive process are poorly understood. Using a genetically modified mouse embryonic stem cell (mESC) line containing a dsRED trans-gene driven by the cardiac-restricted portion of the ncx1 promoter, we show that AA promoted differentiation of mESCs to CMs in a dose- and time-dependent manner. Treatment of mPSCs with AA did not modulate total SMAD content; however, the phosphorylated/active forms of SMAD2 and SMAD1/5/8 were significantly elevated. Co-administration of the SMAD2/3 activator Activin A with AA had no significant effect, but the addition of the nodal co-receptor TDGF1 (Cripto) antagonized AA's cardiomyogenic-promoting ability. AA could also reverse some of the inhibitory effects on cardiomyogenesis of ALK/SMAD2 inhibition by SB431542, a TGFß pathway inhibitor. Treatment with BMP2 and AA strongly amplified the positive cardiomyogenic effects of SMAD1/5/8 in a dose-dependent manner. AA could not, however, rescue dorsomorphin-mediated inhibition of ALK/SMAD1 activity. Using an inducible model system, we found that SMAD1, but not SMAD2, was essential for AA to promote the formation of TNNT2⁺-CMs. These data firmly demonstrate that BMP receptor-activated SMADs, preferential to TGFβ receptor-activated SMADs, are necessary to promote AA stimulated cardiomyogenesis. AA-enhanced cardiomyogenesis thus relies on the ability of AA to modulate the ratio of SMAD signaling among the TGFß-superfamily receptor signaling pathways.