Abstract
Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca2+ channels of different cell types. Moreover, influx of Ca2+ through L-type Ca2+ channels (LTCCs) activates Ca2+ 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 Ca2+ 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 Ca2+ channel α1C (CaV1.2) 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 Ca2+ concentration and pathways are essentially required for cardiac gene expression, differentiation and function.
Highlights
Embryonic stem (ES) and induced pluripotent stem cells are cells able to self-renew and give rise to cells found in all three embryonic germ layers [1,2,3]
Cardiac Differentiation of ES and induced pluripotent stem (iPS) Cells ES and iPS cells were differentiated to CMs in the presence of DMSO, nifedipine, verapamil (LTCCs blockers) or BayK8644 (opener of all L-type channels (CaV1.2 family))
Most of the embryoid body (EB) cultured under control conditions expressed larger and brighter enhanced green fluorescence protein (EGFP)-positive areas as compared with those obtained under nifedipine treatment
Summary
Embryonic stem (ES) and induced pluripotent stem (iPS) cells are cells able to self-renew and give rise to cells found in all three embryonic germ layers (ectoderm, mesoderm and endoderm) [1,2,3]. The induction of pluripotent stem cell differentiation towards cardiac fate is governed by multiple developmental steps such as mesoderm induction and maturation of early cardiomyocytes [7]. Cardiac development is a dynamic process that is tightly orchestrated by the sequential expression of multiple signal transduction proteins and transcription factors working in a combinatory manner [8], and even subtle perturbation of this events can have catastrophic consequences in the form of congenital disease such as heart failure. Entry of cells into the cardiac lineage is dependent upon appropriate external signals coupled to the expression of a set of transcription factors that initiates and activates the network for cardiac gene expression, cardiomyocytes differentiation and maturation [4], [5], [9]. Previous studies mainly focused on the analysis of chemical biology, in which small molecules are identified and used to regulate cell fate or modulate cell reprogramming [10], [11]
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