Evidence for a Critical Role of Catecholamines for Cardiomyocyte Lineage Commitment in Murine Embryonic Stem Cells

<div><p>Catecholamine release is known to modulate cardiac output by increasing heart rate. Although much is known about catecholamine function and regulation in adults, little is known about the presence and role of catecholamines during heart development. The present study aimed therefore to evaluate the effects of different catecholamines on early heart development in an <i>in vitro</i> setting using embryonic stem (ES) cell-derived cardiomyocytes. Effects of catecholamine depletion induced by reserpine were examined in murine ES cells (line D3, αPIG44) during differentiation. Cardiac differentiation was assessed by immunocytochemistry, qRT-PCR, quantification of beating clusters, flow cytometry and pharmacological approaches. Proliferation was analyzed by EB cross-section measurements, while functionality of cardiomyocytes was studied by extracellular field potential (FP) measurements using microelectrode arrays (MEAs). To further differentiate between substance-specific effects of reserpine and catecholamine action via α- and β-receptors we proved the involvement of adrenergic receptors by application of unspecific α- and β-receptor antagonists. Reserpine treatment led to remarkable down-regulation of cardiac-specific genes, proteins and mesodermal marker genes. In more detail, the average ratio of ∼40% spontaneously beating control clusters was significantly reduced by 100%, 91.1% and 20.0% on days 10, 12, and 14, respectively. Flow cytometry revealed a significant reduction (by 71.6%, n = 11) of eGFP positive CMs after reserpine treatment. By contrast, reserpine did not reduce EB growth while number of neuronal cells in reserpine-treated EBs was significantly increased. MEA measurements of reserpine-treated EBs showed lower FP frequencies and weak responsiveness to adrenergic and muscarinic stimulation. Interestingly we found that developmental inhibition after α- and β-adrenergic blocker application mimicked developmental changes with reserpine. Using several methodological approaches our data suggest that reserpine inhibits cardiac differentiation. Thus catecholamines play a critical role during development.</p></div

Primers for qRT-PCR and sqRT-PCR (Fig. 3A–F).

<p>Primers for qRT-PCR and sqRT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070913#pone-0070913-g003" target="_blank">Fig. 3A–F</a>).</p

Effect of reserpine on ectodermal and mesodermal marker expression during ES cell differentiation toward CMs.

<p>(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.</p

Blocking of α- and β-ARs mimics the reserpine-induced effect on cardiomyogenesis.

<p>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).</p

Effect of reserpine on proliferation and cardiomyocyte differentiation.

<p>(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.</p

Effect of catecholamines on spontaneously beating clusters of EBs after acute application of reserpine.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070913#pone-0070913-t002" target="_blank">Table 2</a>.</p

Time course of cell culture and drug application.

<p>(A) Schematic overview of catecholamine synthesis and corresponding enzymes (adopted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070913#pone.0070913-Catecholamine1" target="_blank">[3]</a>). (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).</p

Comparison of β-adrenergic and muscarinic modulation of beating rate in control and reserpine-treated ES cell-derived cardiomyocytes.

<p>(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)).</p

Effect of nifedipin and lidocain on spontaneous action potential generation.

<p>Application of the calcium channel blocker nifedipin completely stopped action potential generation in day 60 hiPSC-CMs (A) and day 60 hESC-CMs (B) at a concentration of 10 µM. Application of 1 µM nifedipin resulted in a strong shortening of the action potential. Application of lidocain could completely prevent action potentials in hiPSC-CMs at a concentration of 50 µM (C), in hESC-CMs 1000 µM abrogated action potential generation.</p

Recording of sodium and calcium currents.

<p>Sodium- (A) and L-type calcium- (B) currents were recorded in whole cell mode. Representative traces are shown. Cardiomyocytes from hiPSCs displayed significantly increased sodium currents on day 60 of differentiation as compared to hESC-CMs.</p