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Caffeine-induced Ca2+ signaling as an index of cardiac progenitor cells differentiation

Basic Research in Cardiology volume 105, pages 737–749 (2010)Cite this article

Abstract

Cardiac progenitor cells (CPCs), migrating from heart tissue, in culture aggregate to form cardiospheres (CSs) in which replication and cardiogenic differentiation occur. However, the frequency of functional differentiation in CSs and the role of cell clustering in supporting it remain to be established. The aim of our study is to quantify differentiation of a muscle-type Ca2+ release mechanism in CS-derived cells, correlate it with cardiac differentiation markers and test its dependency on CS formation. CPCs migrating from murine cardiac explants were studied prior and after CSs formation (Pre-CS and Post-CS). Inducibility of RyR- and IP3-R-mediated Ca2+ transients in individual cells was tested by exposure to caffeine and ATP, respectively; expression of cardiac and non-cardiac lineage markers was assessed. Caffeine responsiveness was negligible in Pre-CS cells and increased by 7.5 fold in Post-CS cells (3.6 vs. 26.9%; p < 0.05), and was closely correlated with activation of the cardiac TnI gene promoter. ATP-induced responses, frequent in Pre-CS (86%), were slightly increased in Post-CS cells (94%; p < 0.05). Expression of cardiac-specific Ca2+-handling proteins (Cav1.2, NCX1, RyR2, SERCA2a) was either limited to the Post-CS stage, or markedly enhanced. CS beating was infrequent, but its pharmacology was compatible with cardiac excitation–contraction coupling. Expression of non-cardiac lineage was low in general, and similar between Pre- and Post-CS cells. Culture conditions inhibiting CSs formation prevented the increase in caffeine responders. In conclusion, clustering in CSs leads to the induction of a muscle-specific functional response in about 30% of CPCs; this is accompanied by development of a cardiac-specific expression pattern.

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Acknowledgments

We are particularly grateful to Prof. Gianluigi Condorelli and Dr. Pasquale Gallo for providing the TNNI3-LVV virus, Stemgen (Prof. A. Vescovi and Dr. G. Lamorte) for the technical supports microscopy imaging acquisition, Drs. Roberto Gaetani and Isotta Chimenti for their help in the cell culture. We thank Drs Schiaffino S. and Ausoni S., Università di Padova for kindly providing transgenic mice. This work was supported by grants from foundation Provincia Italiana. Congregazione Figli dell’Immacolata Concezione; grant CE n LSHB-CT2004- 502988 SC&CR; Cariplo Foundation grant 2007-5639 (A.Z.). Cenci-Bolognetti Foundation, Pasteur Institute, University of Rome”La Sapienza” (A.G.).

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Authors and Affiliations

  1. Department of Biotechnologies and Biosciences, University of Milano-Bicocca, p.za della Scienza 2, 20126, Milan, Italy

    C. Altomare, L. Barile, S. Marangoni, M. Rocchetti, M. Alemanni, G. Mostacciuolo & Antonio Zaza

  2. Department of Experimental Medicine, Pasteur Institute, Cenci-Bolognetti Foundation, University of Rome “La Sapienza”, viale Regina Elena 324, 00161, Rome, Italy

    L. Barile, A. Giacomello & E. Messina

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  1. C. Altomare
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  2. L. Barile
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  9. Antonio Zaza
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Correspondence to Antonio Zaza.

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C. Altomare and L. Barile contributed equally to this work.

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Supplementary material 1 (PDF 1317 kb)

Movie 1. spontaneous beating CS 5 days after plating on fibronectin (MPEG 1850 kb)

Movie 3. recording of twitch experiment on limited region of beating CS; superfused test drugs are listed as subtitles (MPEG 2604 kb)

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Altomare, C., Barile, L., Marangoni, S. et al. Caffeine-induced Ca2+ signaling as an index of cardiac progenitor cells differentiation. Basic Res Cardiol 105, 737–749 (2010). https://doi.org/10.1007/s00395-010-0111-6

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  • DOI: https://doi.org/10.1007/s00395-010-0111-6

Keywords

  • Ca2+ transients
  • Cardiac differentiation
  • Progenitor cells
  • Functional markers