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Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP

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Abstract

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists β,γ-methylenadenosine 5′-triphosphate (β,γ-MetATP) and 8-bromoadenosine 5′-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca2+]i) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca2+]i transients induced by ADP/ATP were abolished by the phospholipase C-β (PLC-β) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca2+]i transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca2+]i response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca2+]i transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

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Acknowledgments

We would like to thank Dr. Yahya Asadi Habibabadi, Ms. Desiré Möhner, Mrs. Martina Voigt, and Ms. Constance Danzer for their generous supports to accomplish this research project. This work was supported by the von Behring-Röntgen Foundation, Marburg and the Excellence Cluster Cardiopulmonary System (ECCPS) of the German Research Foundation.

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Authors and Affiliations

  1. University Heart Center, Clinic of Internal Medicine I, Friedrich Schiller University Jena, Jena, Germany

    Safoura Mazrouei, Mohamed M. Bekhite, Hans-Reiner Figulla & Maria Wartenberg

  2. Department of Physiology, Faculty of Medicine, Justus Liebig University, Giessen, Germany

    Fatemeh Sharifpanah & Heinrich Sauer

  3. Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt

    Mohamed M. Bekhite

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  1. Safoura Mazrouei
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  2. Fatemeh Sharifpanah
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  3. Mohamed M. Bekhite
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  4. Hans-Reiner Figulla
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  5. Heinrich Sauer
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  6. Maria Wartenberg
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Correspondence to Maria Wartenberg.

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Supplemental Fig. 1

Flow cytometry analysis of the number of α-actinin-positive cells presented as dot plots. Dots inside high-lighted (blue) frames indicate α-actinin-positive cardiac cells in the untreated control and the ATP or ADP-treated samples. (PPTX 389 kb)

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Mazrouei, S., Sharifpanah, F., Bekhite, M.M. et al. Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP. Purinergic Signalling 11, 491–506 (2015). https://doi.org/10.1007/s11302-015-9468-1

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