Aim: To investigate the effects of the cardiotonic steroid, ouabain, on cardiac differentiation of murine embyronic stem cells (mESCs).
Methods: Cardiac differentiation of murine ESCs was enhanced by standard hanging drop method in the presence of ouabain (20 μmol/L) for 7 d. The dissociated ES derived cardiomyocytes were examined by flow cytometry, RT-PCR and confocal calcium imaging.
Results: Compared with control, mESCs treated with ouabain (20 μmol/L) yielded a significantly higher percentage of cardiomyocytes, and significantly increased expression of a panel of cardiac markers including Nkx 2.5, α-MHC, and β-MHC. The α1 and 2- isoforms Na(+)/K(+)-ATPase, on which ouabain acted, were also increased in mESCs during differentiation. Among the three MAPKs involved in the cardiac hypertrophy pathway, ouabain enhanced ERK1/2 activation. Blockage of the Erk1/2 pathway by U0126 (10 μmol/L) inhibited cardiac differentiation while ouabain (20 μmol/L) rescued the effect. Interestingly, the expression of calcium handling proteins, including ryanodine receptor (RyR2) and sacroplasmic recticulum Ca(2+) ATPase (SERCA2a) was also upregulated in ouabain-treated mESCs. ESC-derived cardiomyocyes (CM) treated with ouabain appeared to have more mature calcium handling. As demonstrated by confocal Ca(2+) imaging, cardiomyocytes isolated from ouabain-treated mESCs exhibited higher maximum upstroke velocity (P<0.01) and maximum decay velocity (P<0.05), as well as a higher amplitude of caffeine induced Ca(2+) transient (P<0.05), suggesting more mature sarcoplasmic reticulum (SR).
Conclusion: Ouabain induces cardiac differentiation and maturation of mESC-derived cardiomyocytes via activation of Erk1/2 and more mature SR for calcium handling.