The L-type Ca2+ Channels Blocker Nifedipine Represses Mesodermal Fate Determination in Murine Embryonic Stem Cells

Abstract

Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca2+ channels of different cell types. Moreover, influx of Ca2+ through L-type Ca2+ channels (LTCCs) activates Ca2+ signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca2+ transient and reduction of LTCCs current density (I CaL) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca2+ channel α1C (CaV1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca2+ concentration and pathways are essentially required for cardiac gene expression, differentiation and function.

L-Type Ca2+ Channels and SK Channels in Mouse Embryonic Stem Cells and Their Contribution to Cell Proliferation

“…-channel was detected in about 25 % of mESCs. This current is strikingly similar to that described previously in mesenchymal stem cells (Kawano et al 2002;Heubach et al 2004;Nguemo et al 2013). Our study also demonstrates that SK channels are expressed in all mESCs.…”

Functional Expression and Regulation of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels (HCN) in Mouse iPS Cell-derived Cardiomyocytes afterUTF1-Neo Selection

“…6). In fact, the efflux of Ca 2+ from intracellular storage via Inositoltriphosphate (IP3), the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) or the ryanodine receptor (RyR) may contribute to the beating activity of CMs that do not possess functional and measurable I f currents [4,44] because a block of L-type Ca 2+ channels has been shown to abolish the beating activity in iPS-CMs [2,22]. Most CMs recorded in the late stage of differentiation possess detectable I f currents and beat faster with regular rhythm.…”

“…Early developmental stage (EDS) was defined according to the day of differentiation when the majority of EBs started beating (day 10-11), intermediate developmental stage (IDS) two to three days later (day 13) and late developmental stage (LDS) as differentiation on day 15. For immunostaining and whole-cell patch-clamp experiments, single CMs were prepared as previously described [22]. …”

Modulation of L-type Calcium Current by Intracellular Magnesium in Differentiating Cardiomyocytes Derived from Induced Pluripotent Stem Cells

“…We also previously revealed the expression of all major LTCCs subunit genes in embryonic stem (ES) and iPS cell-derived CMs during the differentiation process [24], suggesting that different response of LTCCs to similar [Mg 2 + ] i observed is not due to the subunit composition of the Ca 2 + channel. Moreover, since the T-tubule is absent at EDS and LDS of CMs even at fetal developmental stages [7,54], we assume that Mg 2 + may at least, in part, modulate the Ca 2 + channel located in the sarcolemma membrane of EDS and LDS cells.…”

“…Using the whole-cell patch-clamp technique to record I CaL , we examined the [Mg 2 + ] i effects on I CaL under different phosphorylation conditions at different stages of induced pluripotent stem (iPS) cell-derived CMs that have been previously well characterized [22][23][24]. Our data demonstrate that LTCCs was markedly regulated by [Mg 2 + ] i under conditions that activate channel phosphorylation in late differentiation stage (LDS) compared with early differentiation stage (EDS), suggesting important developmental changes in terms of receptors (ie, sensitivity to agonists and antagonists), signaling pathways, and interactions.…”