Acta Pharmacologica Sinica 2006 July; 27 (7): 901-910; doi: 10.1111/j.1745-7254.2006.00380.x

Aim: To investigate the developmental regulation of intracellular Ca²⁺ 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 Ca²⁺ handling proteins on the Ca²⁺ transients at early, intermediate and later differentiation stages of ES cell-derived cardiomyocytes (ESCM).

Results: Nifedipine, a selective antagonist of L-type Ca²⁺ channels, totally blocked Ca²⁺ transients even in the condition of field-electric stimulation in ESCM at three differentiation stages. The Ca²⁺ 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 Ca²⁺-pump ATPase, inhibited Ca²⁺ transients equally at three differentiation stages that matched the expression profile. Na⁺ free solution, which inhibits Na⁺-Ca²⁺ exchanger (NCX) to extrude Ca²⁺ from cytosol, did not affect the amplitude of Ca²⁺ transients of ESCM until the latter differentiation stage, but it significantly enhanced the basal Ca²⁺ concentration.

Conclusion: The Ca²⁺ transients in ESCM depend on both the sarcolemmal Ca²⁺ entry via L-type Ca²⁺ channels and the SR Ca²⁺ release from RyRs and IP₃Rs even at the early differentiation stage; but NCX seems not to regulate the peak of Ca²⁺ transients until the latter differentiation stage.

Keywords: Ca²⁺ transients; cardiac differentiation; embryonic stem cells; Ca²⁺ handing proteins