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 (Na+ 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 (Ip), 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 alpha1-subunit and alpha3-subunit of the Na+/K+ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast alpha2-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 alpha2-subunit Na+/K+ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.
Otsu
Keishi
K
Department of Cardiovascular Diseases, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
Kuruma
Akinori
A
Yanagida
Eri
E
Shoji
Satoshi
S
Inoue
Takafumi
T
Hirayama
Yoshiyuki
Y
Uematsu
Hiroshi
H
Hara
Yukichi
Y
Kawano
Seiko
S
D000818:Animals; D002316:Cardiotonic Agents / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D002316:Cardiotonic Agents; D019831:Sodium-Calcium Exchanger; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase