Subtype Switching of T-Type Ca 2+ Channels From Cav3.2 to Cav3.1 During Differentiation of Embryonic Stem Cells to Cardiac Cell Lineage

Abstract

Background The developmental changes of Ni 2+ -sensitivity to automaticity of Nkx2.5-positive cells derived from mouse embryonic stem cell have been identified, suggesting developmental regulation of expressing Ni 2+ -sensitive T-type Ca 2+ channel, although the mechanism of the change has not been fully studied. Methods and Results Transcripts of Cav3.2, Cav3.1 and Cav1.2 genes of beating Nkx2.5-positive cells, which encode the Ni 2+ -sensitive T-type Ca 2+ channel, Ni 2+ -insensitive T-type Ca 2+ channel, and L-type Ca 2+ channel, respectively, were investigated by real-time reverse-transcriptase-polymerase chain reaction, and the current density of each channel was measured by patch-clamp techniques at the early and late stages of differentiation. The expression of the Cav3.2 transcript predominated in the early stage whereas those of Cav3.1 and Cav1.2 transcripts were upregulated in the late stage, which was consistent with the change in each current density, suggesting the expression of channel proteins is largely determined at the transcriptional level. ConclusionThe results indicate that the mechanism of change of Ni 2+ -sensitivity is partly, if not completely, the subtype switch of T-type Ca 2+ channel from Cav3.2 to Cav3.1 at the transcriptional level, and that the expression of the L-type Ca 2+ channel might have an attenuating effect on Ni 2+ -sensitivity to automaticity in the late stage of differentiation. (Circ J 2005; 69: 1284 -1289

Different distribution of Cav3.2 and Cav3.1 transcripts encoding T-type Ca2+ channels in the embryonic heart of mice

“…In Cav3.1 knockout mouse, the ablation of Cav3.1 causes both bradycardia and slowing of atrioventricular conduction, however the ablation of Cav3.2 expression does not affect the beating frequency and heart development (10). We reported subtype switching from Cav3.2 to 3.1 during in vitro differentiation of embryonic stem (ES) cells to cardiomyocytes, which was consistent with the findings in the embryonic heart (11,12), suggesting that role of T-type Ca 2+ channels may vary between the developmental stages. To further explore this issue, it is necessary to clarify the distribution of T-type Ca 2+ channels in the developing heart.…”

“…Nkx2.5 is a common marker of cardiac progenitor cells whereas Tbx5 and Isl-1 are markers of the first and secondary heart field, respectively. All these markers genes are expressed at this stage, (9,11). Thus, we examined the distribution of Cav3.1 and Cav3.2 mRNAs and the marker gene transcripts in E10.5 hearts.…”

“…We analyzed the cardiac progenitor cells derived from mouse ES cell. Nkx2.5/GFP(+) cells were derived from hcgp7 ES cell line carrying GFP reporter gene in one of the Nkx2.5 loci as described elsewhere (11). Their differentiation was induced by formation of embryo bodies as described previously.…”

Maintenance Of Calcium Homeostasis in Embryonic Stem Cell-Derived Cardiomyocytes

“…Again, Ca V 3.2 has been studied in vitro. Its mRNA was detected as early as day 5, but peaked at day 6 and gradually declined and was still detectable up until the last measurement at day 15 post-differentiation in ht7 mESC-derived CMs (Mizuta et al, 2005). In line with this finding, Ca V 3.2 mRNA level was also found to be down-regulated by about 24% from day 9.5 to day 23.5 post-differentiation in EMG7 mESCderived CMs (Yanagi et al, 2007).…”

“…Expression of Ca V 3.1 has also been studied in vitro. Its mRNA could be detected as early as day 5 and generally increased up until its last measurement at day 15 post-differntiation in ht7 mouse ESC-(mESC-) derived CMs (Mizuta et al, 2005). In R1 mESC-derived CMs, however, Ca V 3.1 mRNA could only be detected at two copies per cell by day 12, with its expression reaching the highest level at day 23 and declining by day 34 post-differentiation; the expression pattern reflected the amplitudes of I Ca, T measured at these time-points.…”