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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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Functional characteristics of Na+ channel and depolarization-activated outward K+ currents.(A–B) Representative traces (A) and currents densities (B) of Na+ current recorded from a control and nifedipine-treated cells at days 4 (non-beating cell) and 12 (beating CM) of differentiation. (C–D) Representative traces and current-voltage relationships (I/V) of the peak depolarization-activated outward K+ current (Ipeak). (E–F) Effect of acute application of Na+ channel (E) blocker TTX (1 µM) and non-specific K+ channel (F) blocker TEA (10 mM). The dotted lines indicate the zero current level.
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pone-0053407-g005: Functional characteristics of Na+ channel and depolarization-activated outward K+ currents.(A–B) Representative traces (A) and currents densities (B) of Na+ current recorded from a control and nifedipine-treated cells at days 4 (non-beating cell) and 12 (beating CM) of differentiation. (C–D) Representative traces and current-voltage relationships (I/V) of the peak depolarization-activated outward K+ current (Ipeak). (E–F) Effect of acute application of Na+ channel (E) blocker TTX (1 µM) and non-specific K+ channel (F) blocker TEA (10 mM). The dotted lines indicate the zero current level.

Mentions: INa was not different between both untreated and nifedipine-treated groups (original traces, Figure 5A). Also INa density (Figure 5B) was similar (P>0.05) between both groups at day 4 (18.1±1.6 pA/pF, n = 12 versus 17.5±2.2 pA/pF, n = 18) and 12 (30.7±2.0 pA/pF, n = 9 versus 32.4±3.7 pA/pF, n = 7) of differentiation.


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)

Functional characteristics of Na+ channel and depolarization-activated outward K+ currents.(A–B) Representative traces (A) and currents densities (B) of Na+ current recorded from a control and nifedipine-treated cells at days 4 (non-beating cell) and 12 (beating CM) of differentiation. (C–D) Representative traces and current-voltage relationships (I/V) of the peak depolarization-activated outward K+ current (Ipeak). (E–F) Effect of acute application of Na+ channel (E) blocker TTX (1 µM) and non-specific K+ channel (F) blocker TEA (10 mM). The dotted lines indicate the zero current level.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0053407-g005: Functional characteristics of Na+ channel and depolarization-activated outward K+ currents.(A–B) Representative traces (A) and currents densities (B) of Na+ current recorded from a control and nifedipine-treated cells at days 4 (non-beating cell) and 12 (beating CM) of differentiation. (C–D) Representative traces and current-voltage relationships (I/V) of the peak depolarization-activated outward K+ current (Ipeak). (E–F) Effect of acute application of Na+ channel (E) blocker TTX (1 µM) and non-specific K+ channel (F) blocker TEA (10 mM). The dotted lines indicate the zero current level.
Mentions: INa was not different between both untreated and nifedipine-treated groups (original traces, Figure 5A). Also INa density (Figure 5B) was similar (P>0.05) between both groups at day 4 (18.1±1.6 pA/pF, n = 12 versus 17.5±2.2 pA/pF, n = 18) and 12 (30.7±2.0 pA/pF, n = 9 versus 32.4±3.7 pA/pF, n = 7) of differentiation.

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