Na⁺/K⁺ ATPase and its functional coupling with Na⁺/Ca²⁺ exchanger in mouse embryonic stem cells during differentiation into cardiomyocytes

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na⁺/K⁺ ATPase⁺ pump) or Na⁺/Ca²⁺ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na⁺/K⁺ ATPase and Na⁺/Ca²⁺ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na⁺]_i and Na⁺ pump current (I_p), it was shown that an ouabain-high sensitive Na⁺/K⁺ ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for α1-subunit and α3-subunit of the Na⁺/K⁺ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast α2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na⁺/Ca²⁺ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca²⁺ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca²⁺]_i oscillations by blocking of Na⁺/K⁺ ATPase, suggesting the functional coupling with Na⁺/Ca²⁺ exchanger. From these results and RT-PCR analysis, we conclude that α2-subunit Na⁺/K⁺ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.