Developmental regulation of intracellular calcium transients during cardiomyocyte differentiation of mouse embryonic stem cells

Aim： To investigate the developmental regulation of intracellular Ca^2＋ transients, an essential event in excitation-contraction coupling, during cardiomyocyte differentiation. Methods： Using the embryonic stem （ES） cell in vitro differentiation system and pharmacological intervention, we investigated the molecular and functional regulation of Ca^2＋ handling proteins on the Ca^2＋ transients at early, intermediate and later differentiation stages of ES cell-derived cardiomyocytes （ESCM）. Results： Nifedipine, a selective antagonist of L-type Ca^2＋ channels, totally blocked Ca^2＋ transients even in the condition of field-electric stimulation in ESCM at three differentiation stages. The Ca^2＋ transients of ESCM were also inhibited by both ryanodine [an inhibitor of ryanodine receptors （RyRs）] and 2- aminoethoxydipheylborate [2-APB, an inhibitor of inositol-1,4,5-trisphosphate receptors （IP3Rs）]. The inhibitory effect of ryanodine increased with the time of differentiation, while the effect of 2-APB decreased with the differentiation. Thapsigargin, an inhibitor of SR Ca^2＋-pump ATPase, inhibited Ca^2＋ transients equally at three differentiation stages that matched the expression profile. Na^＋ free solution, which inhibits Na^＋-Ca^2＋ exchanger （NCX） to extrude Ca^2＋ from cytosol, did not affect the amplitude of Ca^2＋ transients of ESCM until the latter differentiation stage, but it significantly enhanced the basal Ca^2＋ concentration. Conclusion： The Ca^2＋ transients in ESCM depend on both the sarcolemmal Ca^2＋ entry via L-type Ca^2＋ channels and the SR Ca^2＋ release from RyRs and IP3Rs even at the early differentiation stage; but NCX seems not to regulate the peak of Ca^2＋ transients until the latter differentiation stage.