| Co-Investigator(Kenkyū-buntansha) |
YAMAGATA Kazuo Division of Life Science, Graduated School of Integrated Science and Art, University of East Asia, 工学部, 助教授 (10299323)
ICHINOSE Shizuko Instrumental Analysis Research Center, Tokyo Medical and Dental University, Assistant Professor, 機器分析センター, 助手 (60014156)
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| Research Abstract |
Mouse embryonic stem (ES) cells differentiate to all cell lineages including cardiomyocytes. In this study, we investigated the functional development of excitation-contraction coupling during cardiomyogenesis in mouse embryonic stem cells. During the differentiation to cardiomyocytes from ES cells (Cell line D3), the Ca^<2+> signaling pathway in each stage was examined using Ca^<2+> or Na^+ imaging, patch clamp techniques and RT-PCR. Our results showed that in undifferentiated ES cells Ca^<2+> was released from ER by the stimulation of G-protein coupled receptors. Using RT-PCR, mRNA for IP3 type I, II and III were detected, confirming IP3-induced Ca^<2+> release (IICR). On the other hand, function of ryanodine receptors or voltage-operated Ca^<2+> channels (VOCC) could not be detected, indicating no function of Ca^<2+>-induced Ca^<2+> release (CICR). For the Ca^<2+> entry pathway, store-operated Ca^<2+> entry (ISOC) was observed. From these results we conclude that Ca^<2+> entry through plasma membrane is mainly mediated by ISOC with little contribution of VOCCs. In this study, we also found that Na^+-Ca^<2+> exchangers and plasma membrane pumps functioned as Ca^<2+> extrusion system. The function of Na^+-K^+ pump examined using ouabain sensitivity could be detected in ES cells. During the differentiation to cardiomyocytes, IICR function was observed in all stages. In the 7-day differentiated cells, CICR could be observed. Both T-type and L-type Ca^<2+> channel were recorded by patch clamp experiments. Ouabain-sensitive Na^+-K^+ pump functions were increased with the differentiation. In whole cell patch clamp, Na^+-K^+ pump currents could be recorded in cells from at 8-day-differentiated stage but not earlier stages. Thus, we clarified the Ca^<2+> signaling pathways in undifferentiated mouse ES cells for the first time in this study. However, the further studies are needed to explore the mechanisms how these signaling pathways differentiate or develop.
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