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The cardiac differentiation of ECC proceeds in hanging drops (Fig. 1) [11]. Cells were cultured in differentiation medium supplemented with 1% dimethyl sulfoxide (DMSO) (Sigma–Aldrich, Germany) as inducer of cardiac differentiation. Drops of differentiation medium (20 μl) with 400 PD 153035 hydrochloride each were placed onto the lids of Petri dishes filled with PBS and cultured for 2 d. The ECC aggregated and formed so-called embryoid bodies (EBs). During the 48 h of EB formation the cells were cultured with low glucose (5 mM), standard glucose (25 mM) and high glucose concentrations (40 mM or 100 mM). Subsequent culture from day 2 to day 5 + 10 was performed under standard conditions (25 mM glucose). The cell aggregates formed were transferred into non-adhesive bacteriological grade Petri dishes and maintained for 3 days in suspension in differentiation medium. The 5-d-old EBs were placed onto 0.1% gelatine-coated dishes and cultured for another 5–10 d (5 + 5 d, 5 + 10 d). The first spontaneously beating clusters appeared 1 d after plating.