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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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Nifedipine reduced spontaneous Ca2+ transient activity in ES cell-derived CMs.(A) Representative tracings of spontaneous Ca2+ transients recorded in spontaneously beating CMs from control and nifedipine-treated cultures. Fluorescence images (labeled with Fura 2 AM) of the recorded cells are shown on the left panel. (B) Left, spontaneous Ca2+ transients recorded from CMs derived under untreated (top) and nifedipine-treated (bottom) condition before and after caffeine application. Right, caffeine-induced peak amplitude (top, right) of the Ca2+ signals and Fractional release (bottom, right) calculated as the ratio of peak Ca2+ concentration under control condition to peak Ca2+ concentration induced by caffeine. (C–F) Comparison of the frequency (C), amplitude (D), maximum upstroke (E) and decay (F) velocity of spontaneous Ca2+ transients on day 12 in controls (n = 10) and nifedipine-treated (n = 8) cultures.
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pone-0053407-g007: Nifedipine reduced spontaneous Ca2+ transient activity in ES cell-derived CMs.(A) Representative tracings of spontaneous Ca2+ transients recorded in spontaneously beating CMs from control and nifedipine-treated cultures. Fluorescence images (labeled with Fura 2 AM) of the recorded cells are shown on the left panel. (B) Left, spontaneous Ca2+ transients recorded from CMs derived under untreated (top) and nifedipine-treated (bottom) condition before and after caffeine application. Right, caffeine-induced peak amplitude (top, right) of the Ca2+ signals and Fractional release (bottom, right) calculated as the ratio of peak Ca2+ concentration under control condition to peak Ca2+ concentration induced by caffeine. (C–F) Comparison of the frequency (C), amplitude (D), maximum upstroke (E) and decay (F) velocity of spontaneous Ca2+ transients on day 12 in controls (n = 10) and nifedipine-treated (n = 8) cultures.

Mentions: Since Ca2+ transients are important events in the regulation of contraction and relaxation of CMs, we, therefore, determine the consequences on overall Ca2+ homeostasis by investigating the Ca2+ transients of single CMs at different time points of differentiation. Spontaneously evoked Ca2+ transients of shorter amplitude were detected in CMs generated under nifedipine-treatment at day 12 of differentiation (Figure 7A). To determine whether the reduction of Ca2+ transients in CMs generated under nifedipine is due to reduced SR Ca2+ stores load that release Ca2+ via functional RyRs or due to reduced LTTC currents, we measured caffeine mobilization of store Ca2+ and its effect on [Ca2+]i transients by applying caffeine (10 mM,) on to flura-2 AM loaded CMs. As can be observed in Figure 7B, caffeine application elicited an instantaneous, rapid, and large release of Ca2+ from the intracellular stores, resulting in a high amplitude caffeine-induced Ca2+ transient in control (n = 11) as well as in nifedipine-treated (n = 7) CMs (P<0.05). The maximum amplitude of caffeine-induced Ca2+ transient (indicating the SR Ca2+ content was significantly reduce in CMs generated under nifedipine-treated compared to untreated (Figure 7B, right top), which could fully explain the decrease of ICaL density in nifedipine-treated CMs. Interestingly, a significantly lower fractional release of Ca2+ was observed in nifedipine-treated CMs compared to untreated CMs, suggesting the presence of functional and less SR Ca2+ stores load in nifedipine-treated CMs and supporting a role for nifedipine-sensitive receptors in LTCCs-mediated Ca2+ influx. As also revealed by the spontaneous AP measurements, the frequency of Ca2+ transients was significantly decreased (0.95±0.3 Hz, n = 9 vs. 2.6±0.5 Hz, n = 12, P<0.01, Figure 7C) as well as the relative amplitude (0.34±0.09, n = 9 vs. 0.2±0.04, n = 12, P<0.01, Figure 7D) in the nifedipine group. Moreover the maximum upstroke velocity (Vmax, Figure 7E) of the Ca2+ transients was decreased by approximately 70% (Figure 7F). The Ca2+ transients changed time-dependingly during differentiation and displayed large differences in amplitude and frequency in both untreated and nifedipine-treated cells (Figure S3A).


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 reduced spontaneous Ca2+ transient activity in ES cell-derived CMs.(A) Representative tracings of spontaneous Ca2+ transients recorded in spontaneously beating CMs from control and nifedipine-treated cultures. Fluorescence images (labeled with Fura 2 AM) of the recorded cells are shown on the left panel. (B) Left, spontaneous Ca2+ transients recorded from CMs derived under untreated (top) and nifedipine-treated (bottom) condition before and after caffeine application. Right, caffeine-induced peak amplitude (top, right) of the Ca2+ signals and Fractional release (bottom, right) calculated as the ratio of peak Ca2+ concentration under control condition to peak Ca2+ concentration induced by caffeine. (C–F) Comparison of the frequency (C), amplitude (D), maximum upstroke (E) and decay (F) velocity of spontaneous Ca2+ transients on day 12 in controls (n = 10) and nifedipine-treated (n = 8) cultures.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539992&req=5

pone-0053407-g007: Nifedipine reduced spontaneous Ca2+ transient activity in ES cell-derived CMs.(A) Representative tracings of spontaneous Ca2+ transients recorded in spontaneously beating CMs from control and nifedipine-treated cultures. Fluorescence images (labeled with Fura 2 AM) of the recorded cells are shown on the left panel. (B) Left, spontaneous Ca2+ transients recorded from CMs derived under untreated (top) and nifedipine-treated (bottom) condition before and after caffeine application. Right, caffeine-induced peak amplitude (top, right) of the Ca2+ signals and Fractional release (bottom, right) calculated as the ratio of peak Ca2+ concentration under control condition to peak Ca2+ concentration induced by caffeine. (C–F) Comparison of the frequency (C), amplitude (D), maximum upstroke (E) and decay (F) velocity of spontaneous Ca2+ transients on day 12 in controls (n = 10) and nifedipine-treated (n = 8) cultures.
Mentions: Since Ca2+ transients are important events in the regulation of contraction and relaxation of CMs, we, therefore, determine the consequences on overall Ca2+ homeostasis by investigating the Ca2+ transients of single CMs at different time points of differentiation. Spontaneously evoked Ca2+ transients of shorter amplitude were detected in CMs generated under nifedipine-treatment at day 12 of differentiation (Figure 7A). To determine whether the reduction of Ca2+ transients in CMs generated under nifedipine is due to reduced SR Ca2+ stores load that release Ca2+ via functional RyRs or due to reduced LTTC currents, we measured caffeine mobilization of store Ca2+ and its effect on [Ca2+]i transients by applying caffeine (10 mM,) on to flura-2 AM loaded CMs. As can be observed in Figure 7B, caffeine application elicited an instantaneous, rapid, and large release of Ca2+ from the intracellular stores, resulting in a high amplitude caffeine-induced Ca2+ transient in control (n = 11) as well as in nifedipine-treated (n = 7) CMs (P<0.05). The maximum amplitude of caffeine-induced Ca2+ transient (indicating the SR Ca2+ content was significantly reduce in CMs generated under nifedipine-treated compared to untreated (Figure 7B, right top), which could fully explain the decrease of ICaL density in nifedipine-treated CMs. Interestingly, a significantly lower fractional release of Ca2+ was observed in nifedipine-treated CMs compared to untreated CMs, suggesting the presence of functional and less SR Ca2+ stores load in nifedipine-treated CMs and supporting a role for nifedipine-sensitive receptors in LTCCs-mediated Ca2+ influx. As also revealed by the spontaneous AP measurements, the frequency of Ca2+ transients was significantly decreased (0.95±0.3 Hz, n = 9 vs. 2.6±0.5 Hz, n = 12, P<0.01, Figure 7C) as well as the relative amplitude (0.34±0.09, n = 9 vs. 0.2±0.04, n = 12, P<0.01, Figure 7D) in the nifedipine group. Moreover the maximum upstroke velocity (Vmax, Figure 7E) of the Ca2+ transients was decreased by approximately 70% (Figure 7F). The Ca2+ transients changed time-dependingly during differentiation and displayed large differences in amplitude and frequency in both untreated and nifedipine-treated cells (Figure S3A).

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