5-Azacytidin Induces Multiple Ion Channel Current Changes In Rat Bone Marrow Mesenchymal Stem Cells

Objective: To study ion channel current changes in the process of the rat bone marrow mesenchymal stem cells (MSCs) differentiating into cardiomyocytes after being induced by 5-azacytidine (5-Aza) in vitro and determine the underlying mechanism.Methods : The MSCs were obtained from bone marrow of Sprague-Dawley rats as described previously and cultured for 2 weeks. Cells were randomly divided tested group (induced with 10μM 5-Aza) or control group (non-induced cells) for additional 4 weeks. The presence and intensity of membrane current were tested by whole-cell patch clamp and the inhibited drugs were applied to identify the ion channels. Cells were randomly tested with Na+-free solution (20 samples) or Na+-contained solution (10 samples) weekly. The cardiac troponin T (cTnT) and gap junction protein43(Cx43) expression were identified using immunohistochemistry fluorescence method.Results:⑴Membrane Current:①Three types of K+ currents were observed single or multi-expression in each test week and had aniso-expression. Delayed rectifier K+ current (IkDR),inward rectifier K+ current(Ikir) in majorities MSCs and transient outward K+ current (Ito) in few cells. The detection rate of the same type of K+ current weekly tested had no significant deviation.②Tetrodotoxin-sensitive sodium current (INa) and inward Ca2+ current(ICa) were detected after being induced for two weeks. The uninduced and one-week induced cells had no expression.③The three types of K+ current intensity were gradually augmented after being inducing cultured for 1﹑2﹑3﹑4weeks(p<0.05).④When compared to the uninduced cells, the same type of K+ current intensity of one-week-induced cells had no significant deviation. The current intensity was augmented begin at two-weeks-induced cells(p<0.05)and significant augmented at four-weeks-induced cell(sp<0.01).⑵Immunohistochemistry fluorescence results:Positive immunohistochemistry fluorescence presence of cTnT and Cx43 were observed in four-weeks induced MSCs, however, results in uninduced cells were negative.Conclusion:⑴5-Aza induction can make some of the MSCs differentiating into cardiomyocytes with Multiple-expression of ion currents (IkDR,Ito,Ikir,INa and ICa).⑵5-Aza induction promotes the maturity of K+,Na+ and Ca2+ channels and augmented K+ current density in MSCs in the early stage.