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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

The presence of nifedipine in differentiation medium altered the cardiac ICaL density of ES cell-derived CMs.(A–C), Representative L-type Ca2+ current traces recorded as indicated in protocol (A) from CM cultured in control (B) and nifedipine-treated (C) conditions. (D) Current densities recorded at the maximal peak of the current (10 mV) of cells generated in control and under nifedipine-treatment at days 8 and 12 of differentiation. (E–F) Current-voltage relationships (E) and activation curves (F) of ICaL recorded in control (open symbols) and nifedipine-treated (filled symbols) CMs. (G), Steady-state inactivation curves of control (open symbols) and nifedipine-treated (filled symbols) CMs. (H–I) shows the time constants of fast τf (H) and slow τs (I) phases of ICaL inactivation recorded in ES cell-derived CMs from control and nifedipine-treated cultures. (J) Immunolocalization of L-type calcium CaV1.2 subunit (α1C) in undifferentiated ES cells suggests enrichment in the cell periphery and cellular membrane. (K–L) Immunocytochemistry of L-type calcium CaV1.2 subunit (α1C) in CMs generated under both control (K) and nifedipine-treated (L) conditions. Insert are positive control experiments from the same bath of cells performed with mouse anti-α actinin. Hoechst 33342 was used to stain nuclei (blue). (Scale bars: 20 µm). * denote significant differences to control and † significant differences to day 12 of differentiation.
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pone-0053407-g006: The presence of nifedipine in differentiation medium altered the cardiac ICaL density of ES cell-derived CMs.(A–C), Representative L-type Ca2+ current traces recorded as indicated in protocol (A) from CM cultured in control (B) and nifedipine-treated (C) conditions. (D) Current densities recorded at the maximal peak of the current (10 mV) of cells generated in control and under nifedipine-treatment at days 8 and 12 of differentiation. (E–F) Current-voltage relationships (E) and activation curves (F) of ICaL recorded in control (open symbols) and nifedipine-treated (filled symbols) CMs. (G), Steady-state inactivation curves of control (open symbols) and nifedipine-treated (filled symbols) CMs. (H–I) shows the time constants of fast τf (H) and slow τs (I) phases of ICaL inactivation recorded in ES cell-derived CMs from control and nifedipine-treated cultures. (J) Immunolocalization of L-type calcium CaV1.2 subunit (α1C) in undifferentiated ES cells suggests enrichment in the cell periphery and cellular membrane. (K–L) Immunocytochemistry of L-type calcium CaV1.2 subunit (α1C) in CMs generated under both control (K) and nifedipine-treated (L) conditions. Insert are positive control experiments from the same bath of cells performed with mouse anti-α actinin. Hoechst 33342 was used to stain nuclei (blue). (Scale bars: 20 µm). * denote significant differences to control and † significant differences to day 12 of differentiation.

Mentions: As, the expression level of voltage-gated Ca2+ channels at the plasma membrane is a key regulator of Ca2+ homeostasis in excitable cells, and of downstream effects such as calcium-dependent transcription, we next studied the biophysical characteristics of ICaL. Whole cell ICaL of CMs was assessed using protocol illustrate in Figure 6A. As shown by representative recording traces (Figure 6B and 6C) and confirmed by the statistic analysis (Figure 6D), ICaL was found to be markedly reduced from 11.6±2.8 pA/pF (n = 9) in control CMs to 5.2±0.69 pA/pF (n = 8, P<0.01) in CMs generated under nifedipine treatment at day 12 of differentiation. As also shown in Figure 6D, we found an increase of ICaL density during differentiation with significance difference between untreated and nifedipine-treated CMs at day 8 (7.2±1.4 vs 3.4±1.1 pA/pF, P<0.01) and 12 (11.6±2.8 vs 5.2±0.6 pA/pF, P<0.01) of differentiation, suggesting the maturation of ICaL density during differentiation. Additionally, the mean I/V relationship at the maximum of ICaL was decreased by approximately 75% and shifted the maximum peak of I/V curve from 0 mV to +10 mV (Figure 6E) of CMs at day 12 of differentiation, suggesting at least in part a phosphorylation of the α1C subunit of the LTCC in CMs generated under nifedipine. To evaluate any alteration of the activation/inactivation process of ICaL, steady-state activation and inactivation were recorded and calculated as described in Supplemental material online. The half-activation voltage (V½) was more positive in CMs from nifedipine group (−6.9±0.6 mV, n = 5 versus –11.5±0.5 mV, n = 6; P<0.01, Figure 6F). The slope factor (k) (5.4±0.4 mV, n = 5 versus 6.4±0.5 mV, n = 6; P>0.05) as well as the channel availability (steady-state inactivation curve, Figure 6G) were not significantly different. The analysis of the time constant of the fast (τf) and slow (τs) phase of ICaL inactivation revealed that τf (Figure 6H) was clearly faster in control (5.9±1.5 ms at 0 mV, n = 8) than in nifedipine-treated CMs (11.08±2.1 ms at 10 mV, n = 6) (P<0.05). However, nifedipine treatment did not significantly affect τs (Figure 6I). Immunolocalization of LTCCs α1C subunit revealed the expression of this protein already in undifferentiated ES cells (day 0) (Figure 6J). In addition immunostaining of the LTCCs α1C subunit (CaV1.2) in CMs derived from untreated (Figure 6K) and nifedipine-treated (Figure 6L) groups also showed an expression of CaV1.2. However, untreated CMs revealed more homogeneous distribution pattern and presumably higher expression of CaV1.2 in comparison to CMs derived from nifedipine-treated cultures, suggesting that, nifedipine-treatment may result in mislocalization of Cav1.2, causing inefficient excitation-contraction of CMs.


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 differentiation medium altered the cardiac ICaL density of ES cell-derived CMs.(A–C), Representative L-type Ca2+ current traces recorded as indicated in protocol (A) from CM cultured in control (B) and nifedipine-treated (C) conditions. (D) Current densities recorded at the maximal peak of the current (10 mV) of cells generated in control and under nifedipine-treatment at days 8 and 12 of differentiation. (E–F) Current-voltage relationships (E) and activation curves (F) of ICaL recorded in control (open symbols) and nifedipine-treated (filled symbols) CMs. (G), Steady-state inactivation curves of control (open symbols) and nifedipine-treated (filled symbols) CMs. (H–I) shows the time constants of fast τf (H) and slow τs (I) phases of ICaL inactivation recorded in ES cell-derived CMs from control and nifedipine-treated cultures. (J) Immunolocalization of L-type calcium CaV1.2 subunit (α1C) in undifferentiated ES cells suggests enrichment in the cell periphery and cellular membrane. (K–L) Immunocytochemistry of L-type calcium CaV1.2 subunit (α1C) in CMs generated under both control (K) and nifedipine-treated (L) conditions. Insert are positive control experiments from the same bath of cells performed with mouse anti-α actinin. Hoechst 33342 was used to stain nuclei (blue). (Scale bars: 20 µm). * denote significant differences to control and † significant differences to day 12 of differentiation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0053407-g006: The presence of nifedipine in differentiation medium altered the cardiac ICaL density of ES cell-derived CMs.(A–C), Representative L-type Ca2+ current traces recorded as indicated in protocol (A) from CM cultured in control (B) and nifedipine-treated (C) conditions. (D) Current densities recorded at the maximal peak of the current (10 mV) of cells generated in control and under nifedipine-treatment at days 8 and 12 of differentiation. (E–F) Current-voltage relationships (E) and activation curves (F) of ICaL recorded in control (open symbols) and nifedipine-treated (filled symbols) CMs. (G), Steady-state inactivation curves of control (open symbols) and nifedipine-treated (filled symbols) CMs. (H–I) shows the time constants of fast τf (H) and slow τs (I) phases of ICaL inactivation recorded in ES cell-derived CMs from control and nifedipine-treated cultures. (J) Immunolocalization of L-type calcium CaV1.2 subunit (α1C) in undifferentiated ES cells suggests enrichment in the cell periphery and cellular membrane. (K–L) Immunocytochemistry of L-type calcium CaV1.2 subunit (α1C) in CMs generated under both control (K) and nifedipine-treated (L) conditions. Insert are positive control experiments from the same bath of cells performed with mouse anti-α actinin. Hoechst 33342 was used to stain nuclei (blue). (Scale bars: 20 µm). * denote significant differences to control and † significant differences to day 12 of differentiation.
Mentions: As, the expression level of voltage-gated Ca2+ channels at the plasma membrane is a key regulator of Ca2+ homeostasis in excitable cells, and of downstream effects such as calcium-dependent transcription, we next studied the biophysical characteristics of ICaL. Whole cell ICaL of CMs was assessed using protocol illustrate in Figure 6A. As shown by representative recording traces (Figure 6B and 6C) and confirmed by the statistic analysis (Figure 6D), ICaL was found to be markedly reduced from 11.6±2.8 pA/pF (n = 9) in control CMs to 5.2±0.69 pA/pF (n = 8, P<0.01) in CMs generated under nifedipine treatment at day 12 of differentiation. As also shown in Figure 6D, we found an increase of ICaL density during differentiation with significance difference between untreated and nifedipine-treated CMs at day 8 (7.2±1.4 vs 3.4±1.1 pA/pF, P<0.01) and 12 (11.6±2.8 vs 5.2±0.6 pA/pF, P<0.01) of differentiation, suggesting the maturation of ICaL density during differentiation. Additionally, the mean I/V relationship at the maximum of ICaL was decreased by approximately 75% and shifted the maximum peak of I/V curve from 0 mV to +10 mV (Figure 6E) of CMs at day 12 of differentiation, suggesting at least in part a phosphorylation of the α1C subunit of the LTCC in CMs generated under nifedipine. To evaluate any alteration of the activation/inactivation process of ICaL, steady-state activation and inactivation were recorded and calculated as described in Supplemental material online. The half-activation voltage (V½) was more positive in CMs from nifedipine group (−6.9±0.6 mV, n = 5 versus –11.5±0.5 mV, n = 6; P<0.01, Figure 6F). The slope factor (k) (5.4±0.4 mV, n = 5 versus 6.4±0.5 mV, n = 6; P>0.05) as well as the channel availability (steady-state inactivation curve, Figure 6G) were not significantly different. The analysis of the time constant of the fast (τf) and slow (τs) phase of ICaL inactivation revealed that τf (Figure 6H) was clearly faster in control (5.9±1.5 ms at 0 mV, n = 8) than in nifedipine-treated CMs (11.08±2.1 ms at 10 mV, n = 6) (P<0.05). However, nifedipine treatment did not significantly affect τs (Figure 6I). Immunolocalization of LTCCs α1C subunit revealed the expression of this protein already in undifferentiated ES cells (day 0) (Figure 6J). In addition immunostaining of the LTCCs α1C subunit (CaV1.2) in CMs derived from untreated (Figure 6K) and nifedipine-treated (Figure 6L) groups also showed an expression of CaV1.2. However, untreated CMs revealed more homogeneous distribution pattern and presumably higher expression of CaV1.2 in comparison to CMs derived from nifedipine-treated cultures, suggesting that, nifedipine-treatment may result in mislocalization of Cav1.2, causing inefficient excitation-contraction of CMs.

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