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The L-type Ca2+ channels blocker nifedipine represses mesodermal fate determination in murine embryonic stem cells.

Nguemo F, Fleischmann BK, Gupta MK, Sarić T, Malan D, Liang H, Pfannkuche K, Bloch W, Schunkert H, Hescheler J, Reppel M - PLoS ONE (2013)

Bottom Line: 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.This was accompanied by the inhibition of spontaneously occurring Ca(2+) 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 Ca(2+) channel α(1C) (Ca(V)1.2) 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurophysiology, University of Cologne, Cologne, Germany.

ABSTRACT
Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca(2+) channels of different cell types. Moreover, influx of Ca(2+) through L-type Ca(2+) channels (LTCCs) activates Ca(2+) 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 Ca(2+) 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 Ca(2+) channel α(1C) (Ca(V)1.2) 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 Ca(2+) concentration and pathways are essentially required for cardiac gene expression, differentiation and function.

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The presence of nifedipine in culture medium inhibits differentiation of ES cell-derived CMs.(A) Protocol used to determine the effect of nifedipine application during cell differentiation. Note that for beating EBs and single cell electrophysiology and Ca2+ imaging experiments, analysis was performed at least 24 hours after washout to reflect long-term changes instead of acute drugs effects. (B–C) Representative experiments showing EBs with GFP expressing areas cultured under control conditions (B) and after nifedipine-treatment (10 µM) (C) of ES cells. (D–F) Representative EBs showing EGFP+ CMs differentiated of ES cells under control conditions (D), after nifedipine- (E) and BayK8644- (F) treatment. (G) Representative FACS analysis of ES cell-derived CMs generated under control, nifedipine- and BayK8644-treated conditions. (Scale bars 50 µm (B, C) and 20 µm (D–F).
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pone-0053407-g001: The presence of nifedipine in culture medium inhibits differentiation of ES cell-derived CMs.(A) Protocol used to determine the effect of nifedipine application during cell differentiation. Note that for beating EBs and single cell electrophysiology and Ca2+ imaging experiments, analysis was performed at least 24 hours after washout to reflect long-term changes instead of acute drugs effects. (B–C) Representative experiments showing EBs with GFP expressing areas cultured under control conditions (B) and after nifedipine-treatment (10 µM) (C) of ES cells. (D–F) Representative EBs showing EGFP+ CMs differentiated of ES cells under control conditions (D), after nifedipine- (E) and BayK8644- (F) treatment. (G) Representative FACS analysis of ES cell-derived CMs generated under control, nifedipine- and BayK8644-treated conditions. (Scale bars 50 µm (B, C) and 20 µm (D–F).

Mentions: We have used the conventional murine ES cell line D3 (clone α-pig44) [26], engineered to express eGFP under control of α-MHC promoter, to specifically detect cardiomyocytes during the developmental process. Cells were maintained in an undifferentiated state by culturing on a monolayer of mitotically inactivated embryonic fibroblast cells feeders in DMEM supplemented with nonessential amino acids (0.1 mM), L-glutamine (2 mM), penicillin, streptomycin (50 µg/ml each), β-mercaptoethanol (0.1 mM), leukemia inhibitory factor (LIF) (500 U/ml), and 15% fetal calf serum (FCS). The ES cell D3 α-pig44 [26] were differentiated into spontaneously beating CMs as reported [27]. Induced pluripotent stem (iPS) cell clone 09, generated from murine embryonic fibroblasts (MEFs) by Wernig and coworkers [28] and as previously described [6], [29], was also used to confirm the initial results obtained from native murine ES cells. The principle of differentiation is schematically illustrated in Figure 1A and described in Methods S1.


The L-type Ca2+ channels blocker nifedipine represses mesodermal fate determination in murine embryonic stem cells.

Nguemo F, Fleischmann BK, Gupta MK, Sarić T, Malan D, Liang H, Pfannkuche K, Bloch W, Schunkert H, Hescheler J, Reppel M - PLoS ONE (2013)

The presence of nifedipine in culture medium inhibits differentiation of ES cell-derived CMs.(A) Protocol used to determine the effect of nifedipine application during cell differentiation. Note that for beating EBs and single cell electrophysiology and Ca2+ imaging experiments, analysis was performed at least 24 hours after washout to reflect long-term changes instead of acute drugs effects. (B–C) Representative experiments showing EBs with GFP expressing areas cultured under control conditions (B) and after nifedipine-treatment (10 µM) (C) of ES cells. (D–F) Representative EBs showing EGFP+ CMs differentiated of ES cells under control conditions (D), after nifedipine- (E) and BayK8644- (F) treatment. (G) Representative FACS analysis of ES cell-derived CMs generated under control, nifedipine- and BayK8644-treated conditions. (Scale bars 50 µm (B, C) and 20 µm (D–F).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3539992&req=5

pone-0053407-g001: The presence of nifedipine in culture medium inhibits differentiation of ES cell-derived CMs.(A) Protocol used to determine the effect of nifedipine application during cell differentiation. Note that for beating EBs and single cell electrophysiology and Ca2+ imaging experiments, analysis was performed at least 24 hours after washout to reflect long-term changes instead of acute drugs effects. (B–C) Representative experiments showing EBs with GFP expressing areas cultured under control conditions (B) and after nifedipine-treatment (10 µM) (C) of ES cells. (D–F) Representative EBs showing EGFP+ CMs differentiated of ES cells under control conditions (D), after nifedipine- (E) and BayK8644- (F) treatment. (G) Representative FACS analysis of ES cell-derived CMs generated under control, nifedipine- and BayK8644-treated conditions. (Scale bars 50 µm (B, C) and 20 µm (D–F).
Mentions: We have used the conventional murine ES cell line D3 (clone α-pig44) [26], engineered to express eGFP under control of α-MHC promoter, to specifically detect cardiomyocytes during the developmental process. Cells were maintained in an undifferentiated state by culturing on a monolayer of mitotically inactivated embryonic fibroblast cells feeders in DMEM supplemented with nonessential amino acids (0.1 mM), L-glutamine (2 mM), penicillin, streptomycin (50 µg/ml each), β-mercaptoethanol (0.1 mM), leukemia inhibitory factor (LIF) (500 U/ml), and 15% fetal calf serum (FCS). The ES cell D3 α-pig44 [26] were differentiated into spontaneously beating CMs as reported [27]. Induced pluripotent stem (iPS) cell clone 09, generated from murine embryonic fibroblasts (MEFs) by Wernig and coworkers [28] and as previously described [6], [29], was also used to confirm the initial results obtained from native murine ES cells. The principle of differentiation is schematically illustrated in Figure 1A and described in Methods S1.

Bottom Line: 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.This was accompanied by the inhibition of spontaneously occurring Ca(2+) 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 Ca(2+) channel α(1C) (Ca(V)1.2) 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurophysiology, University of Cologne, Cologne, Germany.

ABSTRACT
Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca(2+) channels of different cell types. Moreover, influx of Ca(2+) through L-type Ca(2+) channels (LTCCs) activates Ca(2+) 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 Ca(2+) 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 Ca(2+) channel α(1C) (Ca(V)1.2) 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 Ca(2+) concentration and pathways are essentially required for cardiac gene expression, differentiation and function.

Show MeSH
Related in: MedlinePlus