Changes in functional expession of voltage-dependent Ca2+ channel and the ryanodine receptor during cardiac myocyte differentiation in P19CL6 cells

P19CL6 cells have been isolated from pluripotent P19 embryonal carcinoma (EC) cells. Following treatment with 1% dimethyl sulfoxide (DMSO), the cells differentiate into mononucleated, spontaneously contracting cardiomyocytes, which are positive for anti-sarcomeric MHC antibody. In the present study, we examined the changes in functional expression of ryanodine receptor and voltage-dependent Ca2+ channel during the differentiation of P19CL6-derived cardiomyocytes. Expression of type-2 ryanodine receptor (RyR2) and voltage-dependent L-type Ca2+ channel (VDCCalC) mRNAs was determined by real-time PCR at the following stages during the differentiation process: before beating (-14 - -1 day), the early stage of beating (1-2 day), the middle stage of beating (3-6 day), and the later stage of beating (7-13 day). Expression of RyR2 mRNA was up-regulated at the early stage of beating, whereas VDCCcclC at middle stage. Spontaneous contractions and Ca2+ transients were almost completely inhibited by 10 uM ryanodine at the early stage. At the middle and later stages, the susceptibility of these spontaneous activities to ryanodine was reduced and further addition of 100 nM nicardipine completely inhibited the activities. Based on the changes in sensitivity to rynanodine in P19CL6 cell-derived cardiomyocytes, it can be suggested that the spontaneous contraction of P19CL6 cells at early stage was mainly regulated by the ryanodine receptors and that the dependence of spontaneous activities to the functional coupling between voltage-dependent Ca2+ channels and ryanodine receptors developed during further differentiation.