Electrophysiological determination of P19CL6-derived cardiomyocytes and their modification by the transcription factor Csx/Nkx2-5

同源异型盒基因CSX/Nkx2-5是一种心脏富集型转录因子，在早期心脏发生中起着关键作用。我们研究了CSX/Nkx2-5对P19CL6来源的心肌细胞心肌离子通道表达的影响。二甲基亚砜可使P19CL6细胞和CSX/Nkx2-5高表达的P19CL6细胞(P19CL6-CSX细胞)有效地分化为心肌细胞。在不同分化阶段：早期(搏动后1~5d)和晚期(搏动后10~15d)，用全细胞膜片钳技术测定动作电位和膜电流。在P19CL6和P19CL6-CSX细胞系中，自发搏动频率随着分化的推进而增加。随着细胞分化，P19CL6-CSX细胞的最大舒张期电位改变为去极化。在分化过程中，两种细胞系的APD50均缩短。P19CL6细胞体积增大。在P19CL6细胞中，I_&lt；to&gt；，I_&lt；Kr&gt；，I_&lt；Ks&gt；，I_&lt；K1&gt；，I_&lt；K，ACh&gt；，其中I_&lt；K，ATP&gt；；通过CSX/Nkx2-5的过度表达，Itt；to Gt；的发育性降低一直受到抑制。同源异型盒基因CSX/Nkx2-5在分化期改变心脏离子通道的表达，主要发生在It1~Gt；。

The homeobox-containing gene Csx/Nkx2-5 is one of cardiac-enriched transcription factors and it plays a critical role for early cardiogenesis. We investigated the effect of Csx/Nkx2-5 on the expression of cardiac ion channels with P19CL6-derived cardiomyocytes. P19CL6 cells and P19CL6 cells with Csx/Nkx2-5 overexpression(P19CL6-Csx cells) were efficiently differentiated into cardiomyocytes by a treatment with dimethyl sulfoxide. Action potentials and membrane currents were measured by whole cell patch clamp at distinct differentiation stage : the early stage(1-5 days after beating) and the late stage(10-15 days after beating). Spontaneous beating rate increased as differentiation advanced both in P19CL6 and P19CL6-Csx cell lines. Maximal diastolic potential of P19CL6-Csx cells was changed to more depolarized as differentiated. APD_<50> was shortened as differentiated in both cell lines. Cell size increased in P19CL6 cells. In P19CL6 cells, I_<to>,I_<Kr>,I_<Ks>,I_<K1>,I_<K,ACh>, where I_<K,ATP> were expressed from the early stage, and I_<to> was predominant in the all K^+ currents. By overexpression of Csx/Nkx2-5, developmental decrease of I_<to> was suppressed invariantly. Homeobox-containing gene Csx/Nkx2-5 modified the expression of cardiac ionic channels during differentiation period, dominantly in I_<to>.