Aims: 

Toinvestigate whether targeted activation of calcium-activated potassium (K_Ca) channels by zoxazolamine (ZOX) has an impact on cardiomyocyte differentiation of embryonic stem (ES) cells.

Methods and Results: 

ES cells were treated with 100 mM and 200 mM ZOX, respectively, between day 5 and day 15 of cell culture. ZOX increased the diameter of cardiac foci and frequency of contractions compared with non-treated cells. Moreover, enhancement in mRNA expression levels of cTnI, a-MHC and HCN4 suggested an increase in the number of sinus node-like cells. Physiological measurements of membrane potential as well as Ca²⁺ and NO concentrations upon exposure to ZOX using fluorescence indicators revealed that ZOX induced a hyperpolarization of the cell membrane, presumably due to activation of IK_Ca, transiently raised [Ca²⁺]_i and increased NO generation. The intracellular Ca²⁺ chelator BAPTA-AM completely abolished the Ca²⁺ signal triggered by ZOX, indicating that an intracellular Ca²⁺ transient precedes subsequent Ca²⁺ entry from the extracellular space. Cytosolic Ca²⁺ mobilization was abolished in presence of MRS-2179, a specific P2Y₁ receptor antagonist. The involvement of PLC in the signalling cascade was confirmed using the specific inhibitor U73122 which suppressed the ZOX-mediated [Ca²⁺]_i response. In the absence of intra- and extracellular Ca²⁺ ZOX failed to stimulate NO generation, demonstrating that ZOX-evoked NO synthesis is strongly dependent on Ca²⁺.

Conclusions: 

ZOX hyperpolarizes the cell membrane potential of ES cells, evokes a transient [Ca²⁺]_i response as well as a consistent increase in NO. Both signalling molecules may interfere with transcriptional signalling cascades leading to enhanced cardiomyocyte differentiation.