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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 cardiac differentiation profile.(A) Time course of the incidence of ES cell-derived contracting EBs generated in the absence (control) or presence of nifedipine or BayK8644. The mean±SEM of the percentage of EBs with contracting areas during differentiation is depicted. (B) Percentage of beatings EBs at day 12 of differentiation derived from ES cells following nifedipine and BayK8644 administration at different times. Note that addition of nifedipine before day 4 of differentiation reduced the percentage of EBs containing beating CMs significantly below both control and BayK8644 levels. (C) Percentage of CMs changes from control condition obtained by FACS analysis after 12 days of cell differentiation under nifedipine and BayK8644. Nifedipine and Bayk8644 were applied on days 0 (D0), 2 (D2) and 4 (D4) of differentiation. (D–E) Immunofluorescence detection of CMs (green) in EBs after 12 days of differentiation. (F–G) Immunostaining of representative dispersed cells from beating clusters of EBs derived under control and nifedipine-treated conditions with anti-sarcomeric α-actinin (red) (1∶800). (H–I) Cardiomyocytes isolated from beating areas of EBs at day 12 of differentiation in both control and nifedipine-treated conditions show sarcomeric striations when stained for α-sarcomeric actinin. Insets show the fluorescence intensity of EGFP (red). Hoechst 33342 was used to stain nuclei (blue or magenta). Scale bars: 20 µm.
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pone-0053407-g002: Nifedipine repressed the cardiac differentiation profile.(A) Time course of the incidence of ES cell-derived contracting EBs generated in the absence (control) or presence of nifedipine or BayK8644. The mean±SEM of the percentage of EBs with contracting areas during differentiation is depicted. (B) Percentage of beatings EBs at day 12 of differentiation derived from ES cells following nifedipine and BayK8644 administration at different times. Note that addition of nifedipine before day 4 of differentiation reduced the percentage of EBs containing beating CMs significantly below both control and BayK8644 levels. (C) Percentage of CMs changes from control condition obtained by FACS analysis after 12 days of cell differentiation under nifedipine and BayK8644. Nifedipine and Bayk8644 were applied on days 0 (D0), 2 (D2) and 4 (D4) of differentiation. (D–E) Immunofluorescence detection of CMs (green) in EBs after 12 days of differentiation. (F–G) Immunostaining of representative dispersed cells from beating clusters of EBs derived under control and nifedipine-treated conditions with anti-sarcomeric α-actinin (red) (1∶800). (H–I) Cardiomyocytes isolated from beating areas of EBs at day 12 of differentiation in both control and nifedipine-treated conditions show sarcomeric striations when stained for α-sarcomeric actinin. Insets show the fluorescence intensity of EGFP (red). Hoechst 33342 was used to stain nuclei (blue or magenta). Scale bars: 20 µm.

Mentions: To determine the development of contracting EBs over time in control and nifedipine-treated cultures, plated EBs were examined daily from day 7 to day 20 of differentiation for spontaneously CAs (Figure 2A). On day 7 of differentiation (2 days after plating), less than 2% of the plated EBs show spontaneously CAs in control cultures. The percentage of EBs contained such CAs continued to rise until 64% at day 11 of differentiation, whereas very few EBs (5, 7%) with spontaneously CAs were observe on day 10 of differentiation in nifedipine-treated cultures. The percentage of EBs with CAs increases slightly and remains low until day 12 of differentiation. After day 12, the percentage continued to increase until 41, 7% of the EBs show CAs on day 14 of differentiation. However, the maximum percentage of EBs with CAs were seen on day 10 (one day before as compared to control cultures) in BayK8644-treated cultures and were remain unchanged and continued to beat vigorously until day 20 (the longest period observed) of differentiation (Figure 2A). EGFP-positive cells were seen to have more synchronized contracting in BayK8644 and control treatment group through real-time imaging of the beating Ebs. This opposite and positive effect of BayK8644 suggests that it may increases Ca2+ current in a way that should allow extra Ca2+ influx to be used efficiently during cardiomyogenesis.


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 cardiac differentiation profile.(A) Time course of the incidence of ES cell-derived contracting EBs generated in the absence (control) or presence of nifedipine or BayK8644. The mean±SEM of the percentage of EBs with contracting areas during differentiation is depicted. (B) Percentage of beatings EBs at day 12 of differentiation derived from ES cells following nifedipine and BayK8644 administration at different times. Note that addition of nifedipine before day 4 of differentiation reduced the percentage of EBs containing beating CMs significantly below both control and BayK8644 levels. (C) Percentage of CMs changes from control condition obtained by FACS analysis after 12 days of cell differentiation under nifedipine and BayK8644. Nifedipine and Bayk8644 were applied on days 0 (D0), 2 (D2) and 4 (D4) of differentiation. (D–E) Immunofluorescence detection of CMs (green) in EBs after 12 days of differentiation. (F–G) Immunostaining of representative dispersed cells from beating clusters of EBs derived under control and nifedipine-treated conditions with anti-sarcomeric α-actinin (red) (1∶800). (H–I) Cardiomyocytes isolated from beating areas of EBs at day 12 of differentiation in both control and nifedipine-treated conditions show sarcomeric striations when stained for α-sarcomeric actinin. Insets show the fluorescence intensity of EGFP (red). Hoechst 33342 was used to stain nuclei (blue or magenta). Scale bars: 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539992&req=5

pone-0053407-g002: Nifedipine repressed the cardiac differentiation profile.(A) Time course of the incidence of ES cell-derived contracting EBs generated in the absence (control) or presence of nifedipine or BayK8644. The mean±SEM of the percentage of EBs with contracting areas during differentiation is depicted. (B) Percentage of beatings EBs at day 12 of differentiation derived from ES cells following nifedipine and BayK8644 administration at different times. Note that addition of nifedipine before day 4 of differentiation reduced the percentage of EBs containing beating CMs significantly below both control and BayK8644 levels. (C) Percentage of CMs changes from control condition obtained by FACS analysis after 12 days of cell differentiation under nifedipine and BayK8644. Nifedipine and Bayk8644 were applied on days 0 (D0), 2 (D2) and 4 (D4) of differentiation. (D–E) Immunofluorescence detection of CMs (green) in EBs after 12 days of differentiation. (F–G) Immunostaining of representative dispersed cells from beating clusters of EBs derived under control and nifedipine-treated conditions with anti-sarcomeric α-actinin (red) (1∶800). (H–I) Cardiomyocytes isolated from beating areas of EBs at day 12 of differentiation in both control and nifedipine-treated conditions show sarcomeric striations when stained for α-sarcomeric actinin. Insets show the fluorescence intensity of EGFP (red). Hoechst 33342 was used to stain nuclei (blue or magenta). Scale bars: 20 µm.
Mentions: To determine the development of contracting EBs over time in control and nifedipine-treated cultures, plated EBs were examined daily from day 7 to day 20 of differentiation for spontaneously CAs (Figure 2A). On day 7 of differentiation (2 days after plating), less than 2% of the plated EBs show spontaneously CAs in control cultures. The percentage of EBs contained such CAs continued to rise until 64% at day 11 of differentiation, whereas very few EBs (5, 7%) with spontaneously CAs were observe on day 10 of differentiation in nifedipine-treated cultures. The percentage of EBs with CAs increases slightly and remains low until day 12 of differentiation. After day 12, the percentage continued to increase until 41, 7% of the EBs show CAs on day 14 of differentiation. However, the maximum percentage of EBs with CAs were seen on day 10 (one day before as compared to control cultures) in BayK8644-treated cultures and were remain unchanged and continued to beat vigorously until day 20 (the longest period observed) of differentiation (Figure 2A). EGFP-positive cells were seen to have more synchronized contracting in BayK8644 and control treatment group through real-time imaging of the beating Ebs. This opposite and positive effect of BayK8644 suggests that it may increases Ca2+ current in a way that should allow extra Ca2+ influx to be used efficiently during cardiomyogenesis.

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