Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue-specific expression of the green fluorescent protein

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the in itiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the co rresponding stage are very small, and cardiac precursor cells cannot be ide ntified because of the lack of cross striation and spontaneous contractions . In the present study, we have used the murine embryonic stem (ES, D3 cell l ine) cell system for the in vitro differentiation of cardiomyocytes. To ide ntify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (G FP) under control of the cardiac alpha-actin promoter. First, fluorescent a reas in ES cell-derived cell aggregates (embryoid bodies [EBs]) were detect ed 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2release sites were built up in these GFP-labeled cardiac precursor cells an d early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments prov ed the functional expression of the L-type Ca2+ current (I-Ca) starting fro m day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, I- Ca was expressed at very low densities 4 pA/pF. The biophysical and pharmac ological properties of I-Ca proved similar to terminally differentiated car diomyocytes. In cardiac precursor cells, I-Ca 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 stimulatio n. The adenylyl cyclase activator forskolin was without effect. IP3-sensiti ve 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 I-Ca. Coexpression of bot h I-Ca and ryanodine-sensitive Ca2+ stores at day 10 of development coincid ed with the beginning of spontaneous contractions in most EBs. Thus, the fu nctional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a ha llmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca 2+-ATPases are expressed before the initiation of cardiomyogenesis. Interes tingly, the functional expression of ryanodine receptors/sensitive stores i s delayed as compared with VDCC.