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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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Related in: MedlinePlus

Nifedipine repressed the expression level of most specific cardiac and mesoderm genes.(A) RT-PCR for L-type Ca2+ channel α-subunit isoforms in undifferentiated ES cells and CMs at day 12 of differentiation, demonstrating (i) presence of L-type Ca2+ channel mRNA even in undifferentiated ES cells and (ii) an increase in mRNA encoding the CaV1.2 subunit of the channel. (B) RT-PCR analyses of cardiac-specific and transcription factors α-MHC/Myh6, ANF/Nppa, MLC2v/Myl2, NKx2.5 and GATA4 in beating cluster at day 12 of both ES and iPS differentiation. (C) RT-PCR analyses of representative markers, PECAM-1 (mesoderm, endothelial cell), AFP (endoderm, early hepatocyte progenitor), Nestin and NF-H (ectoderm, neural progenitor) and α-SMA (mesoderm) in ES and iPS beating cluster at day 12 of differentiation. (D) Quantitative real-time PCR analyses of mesoderm and cardiac markers and specific transcript (Brachyury T, Mesp1, NKx2.5, Tbx5, Mest, Myocd, Myh6/α-MHC, Myl2/MLC2v, and Nppa/ANF), as well as cardiac ionic channels (CACNA1c, KCNH2 and SCN5A) expression at different stage of ES cell differentiation. GAPDH was used as housekeeping gene and served to normalize the result. Results are reported as the means±SEM (n = 3). *P<0.05 vs. control CMs.
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pone-0053407-g003: Nifedipine repressed the expression level of most specific cardiac and mesoderm genes.(A) RT-PCR for L-type Ca2+ channel α-subunit isoforms in undifferentiated ES cells and CMs at day 12 of differentiation, demonstrating (i) presence of L-type Ca2+ channel mRNA even in undifferentiated ES cells and (ii) an increase in mRNA encoding the CaV1.2 subunit of the channel. (B) RT-PCR analyses of cardiac-specific and transcription factors α-MHC/Myh6, ANF/Nppa, MLC2v/Myl2, NKx2.5 and GATA4 in beating cluster at day 12 of both ES and iPS differentiation. (C) RT-PCR analyses of representative markers, PECAM-1 (mesoderm, endothelial cell), AFP (endoderm, early hepatocyte progenitor), Nestin and NF-H (ectoderm, neural progenitor) and α-SMA (mesoderm) in ES and iPS beating cluster at day 12 of differentiation. (D) Quantitative real-time PCR analyses of mesoderm and cardiac markers and specific transcript (Brachyury T, Mesp1, NKx2.5, Tbx5, Mest, Myocd, Myh6/α-MHC, Myl2/MLC2v, and Nppa/ANF), as well as cardiac ionic channels (CACNA1c, KCNH2 and SCN5A) expression at different stage of ES cell differentiation. GAPDH was used as housekeeping gene and served to normalize the result. Results are reported as the means±SEM (n = 3). *P<0.05 vs. control CMs.

Mentions: To gain more insight on the effect of nifedipine, we afterwards performed RT-PCR to confirm our microarrays results. As shown in Figure 3A, LTCCs subunit genes CACNA1C (α1c), CACNAB2 (β2) and CACNA2D2 (α2δ2) were present already in undifferentiated ES cells (conditioned medium, feeder-free) with higher expression at day 12. We also found that the cardiac markers α-MHC or Myh6, ANF or Nppa, MLC2v or Myl2, GATA4 and Nkx2.5 were lower expressed in cells generated under nifedipine-treated cultures at day 12 of ES and iPS differentiation (Figure 3B). In addition, we examined the mRNA expression level of AFP (endoderm, early hepatocyte progenitor), Nestin and NF-H (ectoderm, neural progenitor) of cell generated under nifedipine. For both ES and iPS cells, only a weak decrease of NF-H expression level was observed in nifedipine-treated cells (Figure 3C) whereas Nestin and AFP expression levels were unchanged. Moreover, in both ES and iPS cells, significant decrease of PECAM-1 (mesoderm, endothelial cell) expression level was observed in nifedipine-treated condition as compare to control. The analysis of α-smooth muscle actins (α-SMA), another mesodermal sub lineage, revealed no significant effect on the mRNA expression level of both cell lines. These results indicates the presence of other differentiated cell types along with cardiomyocytes and confirming, at least in part, the specific action of nifedipine on cardiac precursor cells.


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)

Nifedipine repressed the expression level of most specific cardiac and mesoderm genes.(A) RT-PCR for L-type Ca2+ channel α-subunit isoforms in undifferentiated ES cells and CMs at day 12 of differentiation, demonstrating (i) presence of L-type Ca2+ channel mRNA even in undifferentiated ES cells and (ii) an increase in mRNA encoding the CaV1.2 subunit of the channel. (B) RT-PCR analyses of cardiac-specific and transcription factors α-MHC/Myh6, ANF/Nppa, MLC2v/Myl2, NKx2.5 and GATA4 in beating cluster at day 12 of both ES and iPS differentiation. (C) RT-PCR analyses of representative markers, PECAM-1 (mesoderm, endothelial cell), AFP (endoderm, early hepatocyte progenitor), Nestin and NF-H (ectoderm, neural progenitor) and α-SMA (mesoderm) in ES and iPS beating cluster at day 12 of differentiation. (D) Quantitative real-time PCR analyses of mesoderm and cardiac markers and specific transcript (Brachyury T, Mesp1, NKx2.5, Tbx5, Mest, Myocd, Myh6/α-MHC, Myl2/MLC2v, and Nppa/ANF), as well as cardiac ionic channels (CACNA1c, KCNH2 and SCN5A) expression at different stage of ES cell differentiation. GAPDH was used as housekeeping gene and served to normalize the result. Results are reported as the means±SEM (n = 3). *P<0.05 vs. control CMs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539992&req=5

pone-0053407-g003: Nifedipine repressed the expression level of most specific cardiac and mesoderm genes.(A) RT-PCR for L-type Ca2+ channel α-subunit isoforms in undifferentiated ES cells and CMs at day 12 of differentiation, demonstrating (i) presence of L-type Ca2+ channel mRNA even in undifferentiated ES cells and (ii) an increase in mRNA encoding the CaV1.2 subunit of the channel. (B) RT-PCR analyses of cardiac-specific and transcription factors α-MHC/Myh6, ANF/Nppa, MLC2v/Myl2, NKx2.5 and GATA4 in beating cluster at day 12 of both ES and iPS differentiation. (C) RT-PCR analyses of representative markers, PECAM-1 (mesoderm, endothelial cell), AFP (endoderm, early hepatocyte progenitor), Nestin and NF-H (ectoderm, neural progenitor) and α-SMA (mesoderm) in ES and iPS beating cluster at day 12 of differentiation. (D) Quantitative real-time PCR analyses of mesoderm and cardiac markers and specific transcript (Brachyury T, Mesp1, NKx2.5, Tbx5, Mest, Myocd, Myh6/α-MHC, Myl2/MLC2v, and Nppa/ANF), as well as cardiac ionic channels (CACNA1c, KCNH2 and SCN5A) expression at different stage of ES cell differentiation. GAPDH was used as housekeeping gene and served to normalize the result. Results are reported as the means±SEM (n = 3). *P<0.05 vs. control CMs.
Mentions: To gain more insight on the effect of nifedipine, we afterwards performed RT-PCR to confirm our microarrays results. As shown in Figure 3A, LTCCs subunit genes CACNA1C (α1c), CACNAB2 (β2) and CACNA2D2 (α2δ2) were present already in undifferentiated ES cells (conditioned medium, feeder-free) with higher expression at day 12. We also found that the cardiac markers α-MHC or Myh6, ANF or Nppa, MLC2v or Myl2, GATA4 and Nkx2.5 were lower expressed in cells generated under nifedipine-treated cultures at day 12 of ES and iPS differentiation (Figure 3B). In addition, we examined the mRNA expression level of AFP (endoderm, early hepatocyte progenitor), Nestin and NF-H (ectoderm, neural progenitor) of cell generated under nifedipine. For both ES and iPS cells, only a weak decrease of NF-H expression level was observed in nifedipine-treated cells (Figure 3C) whereas Nestin and AFP expression levels were unchanged. Moreover, in both ES and iPS cells, significant decrease of PECAM-1 (mesoderm, endothelial cell) expression level was observed in nifedipine-treated condition as compare to control. The analysis of α-smooth muscle actins (α-SMA), another mesodermal sub lineage, revealed no significant effect on the mRNA expression level of both cell lines. These results indicates the presence of other differentiated cell types along with cardiomyocytes and confirming, at least in part, the specific action of nifedipine on cardiac precursor cells.

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