Figure 1.
Time course of cell culture and drug application.
(A) Schematic overview of catecholamine synthesis and corresponding enzymes (adopted from [3]). (B) Scheme of culture protocols: Yellow arrows indicate the days of medium changes (20% IMDM) and addition of DMSO (1∶1000) or reserpine (10 µM in 1∶1000 DMSO) as used for the ‘d4’ Protocol. (1) Colony of undifferentiated pluripotent D3 αPIG44 murine ESCs on MEFs preceding the start of differentiation (scale bar: 50 µm). (2–4) Control (untreated), solvent control (DMSO; 1∶1000) and reserpine-treated (10 µM in 1∶1000 DMSO) day 11 EBs plated on 0.1% gelatine-coated culture dishes (scale bars 200 µm).
Table 1.
Primers for qRT-PCR and sqRT-PCR (Fig. 3A–F).
Figure 2.
Effect of reserpine on proliferation and cardiomyocyte differentiation.
(A) Microscopy images of EBs at day 10 and 14 of differentiation (day 14 EBs were purified with puromycine). eGFP positive CMs containing clusters in control, DMSO (solvent control) and reserpine-treated EBs are shown (green arrow in reserpine-treated). Scale bars: 200 µm. (B) Time course of appearing spontaneously beating clusters within plated EBs at indicated time points. (C) EB proliferation was analyzed based on cross sectional areas (mm2) from phase contrast pictures captured at days 2, 4 and 10 after differentiation start (n = 3). (D) Percentage of eGFP positive CMs from control, DMSO and reserpine treated EBs at day 10 (n = 11; *** p<0.01) and day 14 (n = 7) of differentiation derived from flow cytometry. (E) Immunocytochemical staining of EBs at day 11 of differentiation for cardiac marker proteins: eGFP positive cardiomyocytes (green), Hoechst 33342 stained nuclei (blue) and cardiac αActinin (red; top panel) or cardiac TroponinC (red; bottom panel). Top row represents overlay pictures. Scale bars: 50 µm.
Figure 3.
Quantitative real-time PCR analysis during ES cell differentiation.
Time course of relative expression of (A) pluripotency genes (Zfp42, Oct4, Nanog) and differentiation marker (Fgf5), (B) cardiac specific genes (Myh6, Mlc2a) and mesodermal markers (Nkx2-5, Gata4 and T-bra), (C) catecholamine synthesis genes (TH, Ddc, Dbh, Pnmt), (D), endodermal marker genes (Sox17, Fox2a, Hnf4a, Alb, Afp), (E) ectodermal and neuronal markers (Map2, Tubb3, Nes). 14+P: d14 puromycin purified clusters; MEF: mouse embryonic fibroblasts. Values represent relative quantities (RQ) plotted on a log2 scale. Primers are listed in Table 1. (F) Semiquantitative reverse-transcription PCRs from ES cells (ES), control and reserpine-treated EBs during differentiation from day 2 to day 10 representative of the gene expression of α- and β-AR subtypes (α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, β3; Gapdh was used as house-keeping control). L: DNA Ladder. Amplified fragment sizes are stated on the right of each pane.
Figure 4.
Effect of reserpine on ectodermal and mesodermal marker expression during ES cell differentiation toward CMs.
(A) Representative phase contrast image of reserpine-treated EB showing cells with typical neuronal morphology. Scale bar: 50 µm. (B) Immunolocalization of (left) neuronal marker β-III-Tubulin (red) and (right) catecholamine synthesis enzyme dopamine-β-hydroxylase (DBH; violet) in day 11 EBs: eGFP positive cardiomyocytes (green), Hoechst 33342 stained nuclei (blue). Top row represents overlay pictures.
Figure 5.
Comparison of β-adrenergic and muscarinic modulation of beating rate in control and reserpine-treated ES cell-derived cardiomyocytes.
(A) Spontaneously contracting EB plated on MEA. (B) Representative beating frequencies demonstrating effects of ß-adrenergic agonist isoprenaline (ISO, 1 µM) and muscarinic agonist carbachol (CCH, 1 µM) on cardiac clusters generated under control (top panel) and reserpine-treated (bottom panel) conditions. Original FP traces (10 sec) of each indicated condition are showcased (on top of each plot). (C) Statistical analysis of FP frequencies in MEA measurements (n = 4). (xp<0.05: significant difference between baseline and 1 µM ISO in ctrl EBs; *p<0.05: significant difference between ctrl and reserpine-treated EB under ISO (1 µM)).
Figure 6.
Effect of catecholamines on spontaneously beating clusters of EBs after acute application of reserpine.
Representative FP frequencies of day 11 to 12 beating cardiac clusters under acute presence of reserpine (10 µM) and after recovery with adrenergic receptor agonists (A) ISO (1 µM), (B) epinephrine (EPI, 100 nM) and (C) norepinephrine (NE, 100 nM). EBs were incubated with reserpine until maximum negative chronotropic effects were observed (between 8–30 min) and then co-applied with the adrenergic agonist as stated. Depicted are frequency-time plots of representative 60 sec intervals during the experiment. 10 sec FP traces are showcased on top of each. Note: After rescue with epinephrine no washout could be recorded. Full summary of all EBs measured is shown in Table 2.
Table 2.
Summary of all acute reserpine application experiments and adrenergic receptor (AR) agonist stimulated recovery (from Fig. 6A–C).
Figure 7.
Blocking of α- and β-ARs mimics the reserpine-induced effect on cardiomyogenesis.
Fluorescence pictures of EBs treated with the unspecific α- and β-blockers phentolamine (10 µM) and propranolol (5 µM) and the combination of both showing the respective expression of GFP positive CMs on days 8 to 14 of differentiation. Left and the second to left column representing control and reserpine-treated EBs differentiated from the same passage, respectively. (Scale bars: 200 µm).