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© The Rockefeller University Press, 0021-9525/1998//2045 $5.00
The Journal of Cell Biology, Volume 143, Number 7, , 1998 2045-2056

Regular Articles

Functional Characteristics of ES Cell–derived Cardiac Precursor Cells Identified by Tissue-specific Expression of the Green Fluorescent Protein



E. Kolossov*, B.K. Fleischmann*, Q. Liu*, W. Bloch{ddagger}, S. Viatchenko-Karpinski*, O. Manzke§, G.J. Ji*, H. Bohlen§, K. Addicks{ddagger}, and J. Hescheler*

* Institute of Neurophysiology, {ddagger} Institute of Anatomy I, and § Department of Internal Medicine I, University of Cologne, D-50931 Cologne, Germany

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions.

In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac {alpha}-actin promoter. First, fluorescent areas in ES cell–derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1–2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs.

Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.

Key Words: GFP • cardiac {alpha}-actin promoter • in vitro cardiomyogenesis • ion channels • Ca2+

Abbreviations used in this paper: ANP, atrial natriuretic peptide; CICR, calcium-induced calcium release; DAB, 3,3-diaminobenzidine-tetrahydrochloride; EB, embryoid body; ES, embryonic stem; GFP, green fluorescent protein; PKA, cAMP-dependent protein kinase A; SR, sarcoplasmic reticulum; Tg, thapsigargin; VDCC, voltage-dependent L-type Ca2+ channels.

E. Kolossov and B.K. Fleischmann contributed equally to the manuscript.


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