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Modulation of human embryonic stem cell-derived cardiomyocyte growth: a testbed for studying human cardiac hypertrophy?
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PMID:  21047517     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Human embryonic stem cell-derived cardiomyocytes (hESC-CM) are being developed for tissue repair and as a model system for cardiac physiology and pathophysiology. However, the signaling requirements of their growth have not yet been fully characterized. We showed that hESC-CM retain their capacity for increase in size in long-term culture. Exposing hESC-CM to hypertrophic stimuli such as equiaxial cyclic stretch, angiotensin II, and phenylephrine (PE) increased cell size and volume, percentage of hESC-CM with organized sarcomeres, levels of ANF, and cytoskeletal assembly. PE effects on cell size were separable from those on cell cycle. Changes in cell size by PE were completely inhibited by p38-MAPK, calcineurin/FKBP, and mTOR blockers. p38-MAPK and calcineurin were also implicated in basal cell growth. Inhibitors of ERK, JNK, and CaMK II partially reduced PE effects; PKG or GSK3β inhibitors had no effect. The role of p38-MAPK was confirmed by an additional pharmacological inhibitor and adenoviral infection of hESC-CM with a dominant-inhibitory form of p38-MAPK. Infection of hESC-CM with constitutively active upstream MAP2K3b resulted in an increased cell size, sarcomere and cytoskeletal assembly, elongation of the cells, and induction of ANF mRNA levels. siRNA knockdown of p38-MAPK inhibited PE-induced effects on cell size. These results reveal an important role for active protein kinase signaling in hESC-CM growth and hypertrophy, with potential implications for hESC-CM as a novel in vitro test system. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".
Authors:
Gábor Földes; Maxime Mioulane; Jamie S Wright; Alexander Q Liu; Pavel Novak; Béla Merkely; Julia Gorelik; Michael D Schneider; Nadire N Ali; Sian E Harding
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-11-01
Journal Detail:
Title:  Journal of molecular and cellular cardiology     Volume:  50     ISSN:  1095-8584     ISO Abbreviation:  J. Mol. Cell. Cardiol.     Publication Date:  2011 Feb 
Date Detail:
Created Date:  2011-01-31     Completed Date:  2011-04-29     Revised Date:  2014-05-13    
Medline Journal Info:
Nlm Unique ID:  0262322     Medline TA:  J Mol Cell Cardiol     Country:  England    
Other Details:
Languages:  eng     Pagination:  367-76     Citation Subset:  IM    
Copyright Information:
Copyright © 2010 Elsevier Ltd. All rights reserved.
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MeSH Terms
Descriptor/Qualifier:
Angiotensin II / pharmacology
Cardiomegaly / chemically induced,  metabolism*,  pathology*
Cell Cycle / drug effects
Cell Differentiation / drug effects
Cell Line
Cell Proliferation / drug effects
Cell Size / drug effects
Cells, Cultured
Embryonic Stem Cells / cytology*
Gene Expression Regulation / drug effects
Humans
MAP Kinase Kinase 3 / metabolism
Myocytes, Cardiac / cytology*,  drug effects,  metabolism*
Phenylephrine / pharmacology
Protein Kinase Inhibitors / pharmacology
Signal Transduction / drug effects
Vasoconstrictor Agents / pharmacology
p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
Grant Support
ID/Acronym/Agency:
G0600373/1//National Centre for the Replacement, Refinement and Reduction of Animals in Research; G0901467//Medical Research Council; RG/08/007/25296//British Heart Foundation; //Wellcome Trust
Chemical
Reg. No./Substance:
0/Protein Kinase Inhibitors; 0/Vasoconstrictor Agents; 11128-99-7/Angiotensin II; 1WS297W6MV/Phenylephrine; EC 2.7.1.-/MAP2K3 protein, human; EC 2.7.11.24/p38 Mitogen-Activated Protein Kinases; EC 2.7.12.2/MAP Kinase Kinase 3
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Journal Information
Journal ID (nlm-ta): J Mol Cell Cardiol
ISSN: 0022-2828
ISSN: 1095-8584
Publisher: Academic Press
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© 2011 Elsevier Ltd.
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Received Day: 29 Month: 6 Year: 2010
Revision Received Day: 12 Month: 10 Year: 2010
Accepted Day: 26 Month: 10 Year: 2010
pmc-release publication date: Month: 2 Year: 2011
Print publication date: Month: 2 Year: 2011
Volume: 50 Issue: 2-4
First Page: 367 Last Page: 376
ID: 3034871
PubMed Id: 21047517
Publisher Id: YJMCC6950
DOI: 10.1016/j.yjmcc.2010.10.029

Modulation of human embryonic stem cell-derived cardiomyocyte growth: A testbed for studying human cardiac hypertrophy?
Gábor Földesab Email: g.foldes@imperial.ac.uk
Maxime Mioulanea
Jamie S. Wrighta
Alexander Q. Liua
Pavel Novaka
Béla Merkelyb
Julia Gorelika
Michael D. Schneidera
Nadire N. Alia1
Sian E. Hardinga1
aNational Heart and Lung Institute, Imperial College London, UK
bHeart Center, Semmelweis University, Budapest, Hungary
Corresponding author. National Heart and Lung Institute, Imperial College London, Flowers Building, Armstrong Road, London, UK, SW7 2AZ. Tel.: +44 207 594 3009; fax: +44 20 7823 3392. g.foldes@imperial.ac.uk
1These authors contributed equally.

Introduction

Human embryonic stem cells (hESC) are presently the stem cell type with the greatest proven capacity for producing phenotypically authentic cardiomyocytes (hESC-CM). While their use for cardiac repair faces a number of logistical problems, they are widely held to have great promise as a potential human-based in vitro cardiomyocyte model system for the cardiac researcher and the pharmaceutical industry. This potential has been enhanced by the realization that both hESC and their close cousins, the induced pluripotent stem cells (iPSC), can be obtained with disease-specific genotypes [1]. hESC-CM are stable in long-term culture and show relative ease of genetic manipulation compared to adult primary cardiomyocytes. Based on their gene expression patterns and electrophysiological, morphological, and contractile properties, the majority of hESC-CM initially resemble human immature cardiomyocytes but have the capacity to develop in a number of respects [2–5]. Acute contractile and electrophysiological characteristics of hESC-CM show promise in terms of reflecting the adult human phenotype [4,6,7], and models of arrhythmia generation have already been described [8,9]. However, it is less obvious whether longer term responses of hypertrophy, proliferation, and apoptosis, important for both cardiac pathology studies and toxicology, would have similar fidelity.

In this study, we have focused on hypertrophic responses in hESC-CM. We have used canonical inducers of both pathological and physiological hypertrophy (phenylephrine, angiotensin II, and stretch) and quantitated the output in terms of a wide range of hypertrophic markers. Importantly, we have used high-content automated microscopy to gather a number of these measurements, pointing the way towards high-throughput assays. We have interrogated the mechanism underlying the hypertrophic changes, initially using a broad screen of small molecule inhibitors for some of the most widely known hypertrophic pathways. Selecting the most active stimulus/inhibitor combination, we have verified the result using overexpression of upstream activators or dominant-negative constructs and downregulation using siRNA. Our results form a basis for the use of hESC-CM as a hypertrophic model system for cardiac research and drug discovery/toxicology.


Materials and methods
2.1  Differentiation and isolation of human embryonic stem cell-derived cardiomyocytes

Cardiomyocytes were derived from human ESC line H7, which was grown on Matrigel (BD Sciences)-coated plates with daily changes of mouse embryonic fibroblast (MEF)-conditioned medium, supplemented with 8 ng/ml recombinant basic human fibroblast growth factor (bFGF, Invitrogen) and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin). MEFs were isolated from 13 dpc MF-1 strain mouse embryos and treated with mitomycin C (0.01 mg/ml, Sigma) at passage 4. MEF-CM was prepared from mitotically inactive MEFs by daily feeding/collecting hESC medium containing 80% KnockOut DMEM (KO-DMEM), 20% KOSR, 1 mM L-glutamine, 10 mM non-essential amino acids, antibiotics, 0.1 mM β-mercaptoethanol, and 4 ng/ml bFGF (all from Invitrogen) for up to a week (150 ml/18.8 × 106 cells/T225 flask). Human ESC were differentiated via embryoid bodies (EBs) by mechanically breaking up the colonies after 3–10 min of collagenase IV (Invitrogen) treatment to remove spontaneously differentiated cells, followed by culturing in suspension culture in low adherence plates for 4 days in differentiation medium (hESC medium in which 20% KOSR was replaced by non-heat-inactivated foetal calf serum) [6,10]. The EBs were plated out onto gelatine (0.5%)-coated plastic dishes, and spontaneously beating areas, which appeared from day 9 after EB formation, were microdissected from EB outgrowths at around day 30 (range 25–40 days). In some experiments, cells were isolated from beating clusters at other time points after differentiation. Differentiated hESC in T175 flasks or 10-cm culture dishes were removed from the surface by treatment with trypsin-EDTA (Sigma-Aldrich) for 5 min and collagenase IV for 10 min, counted and plated onto 96-well plates coated with 0.5% gelatin. These were grouped either as 15 to 40 days (early), 41 to 60 days (intermediate) and 61–180 days (late) after differentiation. For high-content measurements, cells were generated from dense hESC monolayers, which were treated with human recombinant Activin A (100 ng/ml, R&D Systems) (day 0–1), and bone morphogenetic protein 4 (BMP4, 10 ng/ml, R&D Systems) (days 1–5) in RMPI-B27 medium (Sigma) [11]; spontaneously beating areas appeared within 1–2 weeks after BMP4 withdrawal. Following dissociation of clusters or monolayers into single cells, cells were seeded onto gelatinized dishes and subjected to treatments after overnight attachment in differentiation medium.

2.2  Use of phenylephrine, angiotensin II and cyclic mechanical stretch

To determine the effect of hypertrophic G-protein-coupled receptor agonists, hESC-CM were incubated in differentiation medium containing 10 μM α-adrenergic phenylephrine or 1 μM angiotensin II (both Sigma) for 48 h. In separate sets of experiment, cultures of isolated hESC-CM were exposed to cyclic equiaxial mechanical stretch in the presence of normal medium. Frequency of cyclic stretch was 0.5 Hz with pulsation of 10–25% elongation of cells for 24 h. Cells were stretched by applying a cyclic vacuum suction under Bioflex plates with computer-controlled equipment (FX-2000; Flexcell International). Control cultures remained on the plate without stretch.

2.3  Small molecule inhibitors of hypertrophy

To determine the effect of protein kinase inhibition on growth in cell size and proliferation, selective small molecule p38 inhibitor SB202190 (1 μM, Sigma), PKG inhibitor KT5823 (1 μM), HDAC II inhibitor trichostatin A (0.25 μM), ERK inhibitor PD98059 (10 μM), JNK inhibitor SP600125 (1 μM), GSK3β inhibitor 1-azakenpaullone (10 μM), CaMK II inhibitor KN93 (10 μM), calcineurin inhibitor cyclosporine A (0.2 μM), mTOR inhibitor rapamycin (10 ng/ml), and calcineurin/FKBP inhibitor FK506 (0.1 μM) were administered to hESC-CM in the presence of absence of phenylephrine for 48 h. The effect of phenylephrine was also tested in the presence of cell cycle inhibitors: myosin II inhibitor blebbistatin (10 μM, for 48 h) and synthetic anti-tubulin agent nocodazole (50 ng/ml, for 6 h). DMSO was used as control and did not affect cell size.

2.4  Targeting of p38–MAPK by dominant negative p38–MAPK and constitutively active MAP2K3b adenoviruses and p38 siRNA knockdown

For further characterization of p38–MAPK effects, we overexpressed a dominant-negative form of p38α (p38αDN, a gift from Dr. Yibin Wang) or constitutively active MAP2K3b (a gift from Dr. Michael Marber) in hESC-CM. p38α DN was mutated in its dual phosphorylation site (from T-G-Y to A-G-F), causing lack of kinase activity. Cells were infected on day 1 in culture by adding titered adenovirus to the culture medium at a multiplicity of infection (MOI) of 4 or greater. The gene transfer efficiency of cultures was determined through parallel infections with GFP adenovirus (Ad-CMV-GFP). For siRNA knockdown, p38 siRNA (Ambion Silencer pre-designed MAPK14, s3586, Applied Biosystems) transfection was performed using Oligofectamine reagent (Invitrogen, 1 μl/well, final incubation volume 50 μl) per manufacturer's instructions. Scrambled siRNA and mock transfection were used as negative controls.

2.5  Immunocytochemistry

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and labeled with anti-cardiac specific troponin I (cTnI, Santa Cruz, 1:200 dilution), anti-Ki67 (proliferation marker, Abcam, 1:100), anti-p38–MAPK (A-12, Santa Cruz, 1:100), anti-atrial natriuretic factor (ANF, a marker of hypertrophy, Santa Cruz, 1:300), Rhodamine–phalloidin (Invitrogen, 1:500), mTOR (Abcam, 1:100), anti-sarcomeric myosin heavy chain (MF20, Hybridoma Bank, 1:200), and anti-myosin heavy chain α/β (MHC α/β, clone 3-48, Abcam, 1:200) primary antibodies. Primary antibodies were detected with FITC- (Abcam), Alexa 488- (Invitrogen), Alexa 546- (Invitrogen), and Cy5- (Abcam) conjugated secondary antibodies (all 1:400). DNA was visualized with DAPI (0.5 μg/ml; Sigma). Images were acquired on Zeiss Axio Observer Z1 fluorescence microscopy.

2.6  Plate imaging

Combinations of immunocytochemistry markers were used to further characterize detailed phenotypic properties of hESC-CM culture. The hESC-CM cultures were dissociated into individual cells before treatment and plated at low density (up to 5000 cells per well of a 96-well plate). Plates were scanned on ArrayScan™ VTi automated microscopy and image analysis system (Cellomics Inc., Pittsburgh, PA, USA) using modified Target Activation, Cell Cycle, Morphology Explorer and Compartmental Analysis BioApplication protocols. Using the system of automated highly sensitive fluorescence imaging microscope with 10× objective and suitable filter sets, the stained cells were identified with DAPI in fluorescence channel 1, cTnI- or MHC α/β-Alexa 488 in channel 2 and ANF-, and Ki67-Alexa546 in channel 3, respectively. The arbitrary value calculated from the standard deviation of the intensity of the pixels under the channel measuring DAPI reflected the content of the intact and fragmented DNA. The maximal ratio of the MHC-positive cells versus the whole differentiated hESC population was 45.4 ± 3.5% (from n = 15 experiments, the average ratio was 20.4 ± 3.3%, e.g., Fig. 1A). An approximate estimate showed that ~ 4 × 105 initial undifferentiated ESC (after expansion and differentation) produced ~ 6 × 104 hESC-CM. From each well, 1000–1500 total cells were analyzed, giving a minimum of 100 cardiomyocytes (and on average, 2–300). Each treatment was tested in triplicate wells, and the experiments were repeated 3 times, except where indicated. Mean average intensities and percentage of responders (those objects deemed 2 standard deviations brighter than the average of the control cells) were recorded.

2.7  Cellular protein content

Cells were fixed with 10% trichloroacetic acid (30 min), stained with 0.1 % (w/v) Naphthol Yellow S (Sigma, 30 min), and washed with 1% acetic acid (30 min) [12]. DNA was visualized with DAPI. Cellular total protein to DNA content ratio was analyzed on Cellomics platform by Target Activation Bioapplication. Protein to DNA ratio was further verified by measuring the ratio of absorptions at 260 vs. 280 nm wavelengths with spectrophotometer (NanoDrop 8000, Thermo Fisher).

2.8  Isolation of RNA

Undifferentiated and differentiated hESC cultures were lysed in RLT buffer for total RNA extraction as per manufacturer's protocol (Qiagen, CA). Total RNA was obtained from human left ventricular tissue explanted during transplant surgery. The RNA was purified using RNeasy columns (Qiagen, CA), quantified, and checked for quality on a denaturing 1% agarose gel. To generate double-stranded cDNA, 1 μg of total RNA was used for RT2 First Strand Kit (SABiosciences, MD) or High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA), according to published protocols.

2.9  Quantitative RT-PCR and PCR array

For quantifying mRNA levels of ANF, p38–MAPK, αMHC, βMHC, SERCA2a, and the ryanodine receptor2 in differentiated hESC cultures, real-time PCR analyses were performed with TaqMan Gene Expression Assays (Hs00383230_g1, Hs01047706_m1, Hs00411887_m1, Hs01110632_m1, Hs01566028_g1, and Hs00892842_m1, respectively, Applied Biosystems, CA). GAPDH Endogenous Control (FAM/MGB probe) was used as a housekeeping control. For PCR array, the cDNA was hybridized in a 96-well format against the Gene Array PAHS-018 with RT2 qPCR Master Mix, which contained SYBR green dye (RT2 Profiler™ PCR Array System, SABiosciences) as per the manufacturer's instructions. The array contains primers for ELK1, FOS (c-Fos), JUN, and NFκB. The PCR was performed with ABI 5700 (Applied Biosystems) and Rotor-Gene 3000 (Corbett Research) real-time PCR instruments, and the relative expression was determined by ΔΔCt method in which fold increase = 2−ΔΔCt.

2.10  Volume measurements of hESC-CM

Hopping mode scanning ion conductance microscopy was used to estimate the volume of live and fixed isolated hESC-CM [13]. A glass micropipette probe filled with electrolyte is connected to a high-impedance, head-stage current amplifier and mounted on a computer-controlled three-axis translation stage. Control electronics drive the translation stage to scan the cells under the micropipette probe. The position of probe tip, in relation to the sample surface, influences the ion current through the pipette. The ion current provides a signal for the feedback loop, which controls the vertical axis of the positioning system. Whole-cell volume was estimated as described earlier [14].

2.11  Statistics

Results are expressed as mean SEM. The data were analyzed by unpaired Student's t test or one-way analysis of variance and the Fisher's protected least significant difference test for multiple comparisons. Differences at the level of P < 0.05 were considered statistically significant.


Results
3.1  hESC-derived cardiomyocytes increase in size during prolonged culture

Embryoid bodies derived from H7 line showed spontaneous contractile activity from 9 to 15 days after induction of differentiation. hESC-CM isolated from EBs stained positive for atrial natriuretic factor (ANF), and sarcomeric proteins typical of myocytes, such as sarcomeric myosin heavy chains (clone 3–48 and MF20) as well as cardiac troponin I (Fig. 1A). In long-term cultures (for a period up to 6 months), hESC-CM increased in cell size and beating rate and showed a modest elongation after a steady-state period of 40 days (all P < 0.01) (Fig. 1BCD). The percentage of hESC-CM with organized sarcomere structures was similar in early (15 to 40 days after differentiation), intermediate (41 to 60 days), and late cultures (> 60 days) (29 ± 3%, 27 ± 1% and 28 ± 2%, each n = 6 cultures, respectively, P > 0.05, ANOVA) (Fig. 1E).

3.2  Expressions of hypertrophy-related genes are upregulated in differentiated hESC cultures

To investigate the temporal expression of hypertrophy-related structure proteins, transcription factors, calcium modulators, and atrial natriuretic factor (ANF), total RNA was collected from undifferentiated hESC and early, intermediate, and late differentiated cultures. As assessed by quantitative RT-PCR, mRNA levels of growth- and hypertrophy-related transcription factor genes (c-fos: 74-fold, P < 0.001, elk-1: 9.5-fold, P < 0.0001, and NFκB: 2.3- fold, P < 0.001, at 1 month after differentiation) were significantly upregulated in differentiated hESC as compared with undifferentiated hESC. Expression levels of α and βMHC showed a marked increase after 68 days bringing αMHC expression to the range of the adult failing sample used, although βMHC was still somewhat lower. The α/β ratio was higher in hESC-CM than in the adult failing sample, or than has been reported for normal human ventricle where αMHC comprises only 30% of total [15] (Supplementary Fig. 1ABC). The mRNA levels of calcium handling cardiac ryanodine receptor (RyR2) and sarcoplasmic reticulum Ca ATPase (SERCA2a) were upregulated in later stage cultures compared to early hESC-CM (Supplementary Fig. 1DE). The mRNA levels of ANF were robustly induced in the early, intermediate, and late stage beating clusters as compared with undifferentiated hESC (Supplementary Fig. 1F).

3.3  Phenylephrine, cyclic stretch, and angiotensin II induce cellular hypertrophy of hESC-CM

Next we investigated the effects of putative hypertrophic stimuli on hESC-CM. Administration of an α-adrenoceptor/Gq agonist phenylephrine (PE, for 48 h) resulted in a significant increase in cell area (1.8-fold, P < 0.0001), number of hESC-CM with organized sarcomere structure (3.8-fold, P < 0.0001), and perinuclear immunoreactive ANF intensities (2.1-fold, P < 0.001) and ANF mRNA (Fig. 2C). Hopping mode ion conductance scanning microscopy showed that PE markedly increased average volume of hESC-CM (2-fold, P < 0.001, Fig. 2AC). Administration of PE resulted in a cytoskeletal rearrangement such as altered cellular distribution of F-actin, indicative of myofibril thin filaments (as well as stress fiber formation) (Fig. 2BC). Total cellular protein to DNA content was significantly higher in PE-treated cells (7-fold higher for the whole culture by Nanodrop, and 2.1-fold higher, P < 0.0001, n = 55, for MHC-positive cells, Fig. 2C). Similarly to PE, stretching hESC-CM for 24 h resulted in an increase in cell size (1.6-fold, P < 0.001, Fig. 3A). Cyclic stretch resulted in a significant increase in the percentage of hESC-CM with organized sarcomere structure after 24 h (P < 0.001, Fig. 3A). αMHC, βMHC, and ANF mRNA levels were increased in response to cyclic stretch (P < 0.05 and 0.001 vs. unstretched control, respectively, triplicate determinations). The incubation with angiotensin II (100 μM, 48 h) resulted in a more modest but significant increase in cell size (1.2-fold, P < 0.05) as well as a greater percentage of hESC-CM with organized sarcomeres and raised αMHC, βMHC, and ANF mRNA levels (Fig. 3B). The α/β-MHC ratio did not change significantly in response to stretch, phenylephrine, or angiotensin II after 48-h treatment.

3.4  Small molecule hypertrophy inhibitors decreases cell size in hESC-CM

To identify the canonical hypertrophy pathways mediating effects of PE, we treated hESC-CM with small molecule protein kinase inhibitors for 48 h. Comparison with PE alone showed significant differences in cell size after inhibition of p38–MAPK (SB202190), HDAC II (trichostatin A), ERK (PD98059), JNK (SP600125), CaMK II (KN93), mTOR (rapamycin), calcineurin (cyclosporine A), and calcineurin/FKBP (FK506) (Fig. 4A). A further analysis additionally comparing control hESC-CM size revealed that there remained significant differences after ERK, JNK, and CaMK II inhibition, indicating that PE-induced growth was not completely abolished. Inhibition of PKG (KT5823) and GSK3β (1-azakenpaullone) had no significant effect on cell size in PE groups (P = 0.06 and 0.7, respectively), and these remained significantly increased compared to DMSO control. Inhibition of p38–MAPK, calcineurin, and mTOR also reduced basal cell size in the absence of PE (P < 0.05). The majority of inhibitors reduced PE-induced increase in ANF mRNA levels, although levels were too variable to distinguish the strength of individual effects (Fig. 4B). In line with earlier studies [16,17], ANF expression remained stimulated despite rapamycin treatment.

3.5  Effect of p38–MAPK inhibition on hESC-CM growth

The p38 MAPK inhibitor SB202190 showed the strongest inhibition of spontaneous cell growth (which may be driven by serum or the continuous beating) and reduced the effects of PE. Similarly, the cyclic stretch-induced increase in cell size and sarcomere alignment were abolished by SB202190 (P < 0.001) (Supplementary Fig. 2AB). We therefore used more stringent methods to confirm the role of p38–MAPK in basal cell growth and PE-induced hypertrophy of hESC-CM. We found that the mRNA levels of p38–MAPK were strongly upregulated in differentiated cultures (up to 26,000-fold, P < 0.0001 vs hESC). In addition to earlier microarray surveys of differentiating hESC [3,18], our quantitative RT-PCR array showed that differentiation was associated with a transient upregulation of proximal regulatory kinases of p38–MAPK such as MAP2K3 (1.4-fold, P < 0.01). Silencing of p38–MAPK by siRNA abolished PE-induced increase in cell size (P < 0.001) (Fig. 5A). Inhibition of p38–MAPK had tonic effects to decrease hESC-CM size even in the absence of added hypertrophic stimuli (Supplementary Fig. 3A). Administration of either SB202190 or SB203580, another inhibitor with different specificity and off-target effects, caused a modest elongation of the cells (+ 7% and + 11% vs. control hESC-CM, respectively, P < 0.05) (Supplementary Fig. 3B). Beating rate of hESC-CM was similar in all groups (P = 0.85) (Supplementary Fig. 3C). Similarly, infection of hESC-CM with a dominant-negative form of p38α lacking kinase activity decreased cell size after 48 h (at MOI: 5, by 29 ± 5% vs. GFP adenovirus-infected cells, P < 0.001) (Supplementary Fig. 3A) although without changing shape (P = 0.80) (Supplementary Fig. 3B). The percentage of hESC-CM with organized sarcomeric structure in unstimulated cultures was unchanged by SB202190 or dominant-negative p38–MAPK (P = 0.96).

3.6  Constitutively active MAP2K3 induces hypertrophic growth of hESC-CM

To investigate directly the effects of activating p38–MAPK, we infected hESC-CM with recombinant adenovirus expressing a constitutively active form of the upstream activator MAP2K3b (Fig. 5B). Infection resulted in an increased cell size, significantly higher percentage of hESC-CM with organized sarcomeres, cytoskeletal rearrangement (all P < 0.0001 vs. GFP adenovirus-infected cells, 48 h), elongation of the cells (+ 12% vs. GFP control, P = 0.02), and induced ANF mRNA levels (1.7-fold, P < 0.05). Furthermore, constitutive activation of MAP2K3b resulted in formation of binuclear hESC-CM (relative increase in binuclear hESC-CM percentage at 2 MOI: 4-fold, 5 MOI: 2.8-fold, and 10 MOI: 2.1-fold vs. GFP control, all P < 0.001). This together suggests that hESC-CM infected with constitutively active MAP2K3b are undergoing cellular hypertrophy. Activation of MAP2K3b did not change absolute cell numbers (P = 0.9) or the percentage of cells which were Ki67+/MHC+ (P = 0.3). Infection with GFP adenovirus did not change basal cell size, or the response to phenylephrine (data not shown).

3.7  Phenylephrine regulates cell size independently of cell cycle

To investigate whether the effect of hypertrophic agonist PE on cell size can be dissected from cell cycle checkpoints, hESC-CM were treated with cell cycle inhibitors. At 30 days after differentiation, the ratio of Ki67-positive hESC-CM was similar in PE-treated and control cells (P = 0.34), suggesting that PE did not modulate proliferative capacity. Administration of blebbistatin, a myosin class II inhibitor, blocked cytokinesis of proliferating hESC-CM, resulting in formation of binuclear cells (Fig. 6AC). Blebbistatin augmented the PE-induced increase in cell size (P < 0.01, Fig. 6B). The percentage of Ki67-positive hESC-CM was similar in the control and blebbistatin-treated cultures, whereas blebbistatin increased Ki67 labeling in PE-treated cells (Fig. 6D). As assessed by Arrayscan analysis, the distribution of cells in G2/M and G1/G0 cell cycle phases in the control and blebbistatin-treated cultures was comparable (Fig. 6E). Cells treated with another cell cycle inhibitor, nocodazole, arrested with a G2/M-phase DNA content (Fig. 6E). Nocodazole had no effect on PE-induced changes in cell size (data not shown), further suggesting that PE can increase cell size independently from cell cycle.

The number of hESC-CM and the distribution of cells in cell cycle phases were similar in control and SB202190-treated groups (P = 0.66) (Supplementary Fig. 3D). Similarly, dominant-negative p38–MAPK vs. GFP control groups had comparable Ki67 ratios (P = 0.84).


Discussion

We have demonstrated that hESC-CM undergo growth either spontaneously with prolonged time in culture or more markedly after the canonical physiological or pathological stimuli, phenylephrine (PE), angiotensin II, or stretch. Use of increase in cellular area as a marker was supported by similar increases in volume and protein/DNA ratio. Modest changes in length/width ratio were occasionally observed, but it is not surprising that the adult elongated cardiomyocyte morphology did not develop in the absence of an anisotropic stimulus. Relating this to current models of hypertrophy, further effects observed were an increase in the number of hESC-CM with organized sarcomeric structures and a rearrangement in cytoskeletal organization. Despite significant basal levels, hypertrophic stimuli produced a marked increase in ANF in early-stage hESC-CM. A variety of interventions demonstrated the independence of the effects on growth from those on cell cycle progression. The range of morphological and expression markers that are altered in response to the Gq agonists and stretch was well matched between hESC-CM and rat neonatal cardiomyocyte models [19].

An initial broad sweep of the major pathways implicated in hypertrophy was made using small molecule inhibitors at optimal concentrations taken from literature on current rat and mouse models. This is an equivalent strategy to current medium- or high-throughput industry screens to generate initial targets. Complete inhibition of PE-induced cell size change was seen with inhibitors of p38–MAPK and calcineurin/FKBP and mTOR. p38–MAPK and calcineurin were also implicated in spontaneous development, since inhibitors decreased cell size in the absence of PE. Inhibitors of HDAC II, ERK, JNK, and CaMK II reduced PE effects but did not completely abolish them. Differences could be concentration-related: typically the more complex screens will use a range of inhibitor concentrations to determine IC50 values and so assess specificity and maximum response. PKG or GSK3β inhibitors had no effect on basal or PE-induced increases in hESC-CM size: it is interesting that neither demonstrated the potentiation of PE effects that might be expected given the role of these pathways in opposing hypertrophy [20,21].

We went on to verify the observations from the broad screen, which had identified the p38 MAPK inhibitor as most potent in reducing basal and PE-induced cell growth. The role of p38–MAPK in the spontaneous increase in hESC-CM size in culture was confirmed by an additional pharmacological inhibitor and adenoviral transfection of a dominant-inhibitory form of p38–MAPK. We further showed that infection with recombinant adenovirus containing constitutively active form of upstream MAP2K3b resulted in an increased cell size, sarcomere and cytoskeletal assembly, elongation of the cells, and induction of ANF mRNA levels. Of note, the ratio of binuclear hESC-CM was significantly higher in the constitutive active MAP2K3b group. siRNA knockdown of p38–MAPK inhibited PE-induced effects on cell size. These characteristics strongly suggest that active p38–MAPK signaling causes hypertrophic growth of hESC-CM in vitro. The p38–MAPK pathway fulfils a number of roles, which change with development in hESC, since it has been implicated in the direction of differentiation to favor cardiomyogenesis [22–24] and previous studies indicated the presence of p38–MAPK in hESC as well as in differentiating embryoid bodies [24,25]. However, the involvement of p38–MAPK in hypertrophic remodeling has been controversial. Reduction of hypertrophy by small molecule inhibitors of p38–MAPK has been seen in various rat, mouse, and hamster models [26–28], but transgenic manipulation has not generally supported such a role (though the embryonic lethality from p38α knockout has made these experiments technically difficult) [29].

When results differ in this way between animal models and hESC-CM/iPSC-CM, with their human genetic background, it will be interesting to see how the balance of evidence is weighed. One reason for differences may be the immature phenotype of the hESC-CM especially at early time points. In the present study, we showed that expression of myosin heavy chain isoforms, SERCA2, and ryanodine receptor 2 increased strongly 2 months after differentiation of hESCs. These changes would tend to be associated with maturation, and this is approximately the duration over which other indicators of development are seen, such as upregulation of repolarization-related K+ channels, resistance to arrhythmias, and development of intracellular Ca2+ stores [4]. However, the immaturity of hESC-CM must remain a significant caveat.

Obviously, we have not been exhaustive in exploring all the known hypertrophic pathways and have not even considered those newly identified from genomic, transcriptomic, or proteomic arrays. However, it is clear from both this study and the wealth of literature [30,31] that there are multiple interacting pathways controlling aspects of hypertrophy. Numerous reviews show the interconnections and redundancy of the control systems and the emergent properties that arise from these complex relations [32,33]. The challenge is now to apply network systems biology to identify key control points that coordinate multiple signal inputs and then produce graded outputs of cardiac growth [34]. The techniques and data described here show that one advantage of hESC-CM is their suitability for high-throughput methodologies, which will match functional cellular outputs to array-generated information. An exciting aspect of hESC-CM or iPSC-CM is the ability to compare cells directly with individual patient responses for particular mutations or haplotypes. For example, iPSC-CM have recently been generated from patients with LEOPARD syndrome, which include a hypertrophic phenotype [35]. In vitro these iPSC-derived cardiomyocytes had a greater cell size, more sarcomeric organization, and high nuclear NFATC4 than controls, although ANF, protein/DNA ratio and volume were not assessed. Efforts such as these, as well as the comparisons being undertaken by pharmaceutical industries for clinical predictivity of hESC-CM, relative to current in vivo and in vitro screens, will allow an understanding of the fidelity of response of hESC-CM to adult heart. Ultimately, only clinical trials can assess whether a given model has been of use in predicting compounds that will be effective against human disease.


Conclusions

HESC-CM have continued capacity to increase in size after differentiation, and growth is stimulated by classical physiological and pathological hypertrophic agents. Effects of small molecule inhibitors indicated the involvement a number of known hypertrophic pathways in this process, including p38–MAPK, calcineurin, FKBP, mTOR, HDAC II, ERK, JNK, and CaMK II. These results represent the basis for development of hESC-CM as a tool for the cardiac researcher or pharmaceutical industry, while the methodologies used may lead to high-throughput small molecule or RNAi screens to investigate hypertrophic and anti-hypertrophic responses. Establishment of the phenotypic fidelity of hESC-CM and their subsequent incorporation into humanized, high-throughput, genotype-specific models could produce a step change in productivity for the cardiac researcher.

Supplementary materials related to this article can be found online at doi:10.1016/j.yjmcc.2010.10.029.

The following are the supplementary materials related to this article. Supplementary Fig. 1

Quantitative RT-PCR data of mRNA levels of αMHC (A), βMHC (B), αMHC vs. βMHC ratio (C), SERCA2a (D), ryanodine receptor 2 (RyR2) (E), and ANF (F) in early (11 to 33 days after differentiation), intermediate (34 to 67 days), and late differentiated hESC cultures (> 68 days): fold difference compared to adult left ventricular tissue (A to E) or undifferentiated hESC (F) (n = 3). The mRNA levels in non-beating areas of differentiated hESC cultures were not detectable. Results are shown as mean + SEM on a log-scale (one-way ANOVA, *P < 0.05, and ***P < 0.001 vs. early differentiated hESC group (A–E) or hESC (F)).


Click here for additional data file (mmc1.ppt)

Supplementary Fig. 2

Cyclic stretch-induced increase in cell size and sarcomere alignment is mediated via p38–MAPK pathway. The hESC-CM (~ 30d) underwent cyclic stretch (0.5 Hz with pulsation of 10–25% elongation of cells, 24 h) in the presence of small molecule p38 inhibitor SB202190 (1 μM) or DMSO. (A) Bar graphs show cell size of hESC-CM relative to control. (B) For quantitation of sarcomere organization, hESC-CM were scored for the presence or absence of highly organized sarcomeres (n > 100 MHC-positive cells analyzed per well, mean ± SEM of triplicate wells, repeated in n = 3 preparations. *P < 0.05 vs. control group, ***P < 0.001 vs. control, #P < 0.001, and $P < 0.05 vs. respective vehicle-treated group).


Click here for additional data file (mmc1.ppt)

Supplementary Fig. 3

Effect of pharmacological and genetic inhibition of p38–MAPK on hESC-CM growth and proliferation. Bar graphs showing cell size (A), cell shape expressed as length to width ratio (B), beating rate (C), and percentage of Ki67-positive hESC-CM (D) treated with DMSO, SB202190 (1 μM) or SB203580 (1 μM) or infected with a dominant-negative form of p38α lacking kinase activity or GFP-control adenovirus (MOI: 5) at 30 days after differentiation. Results are shown as mean ± SEM. *P < 0.05, *P < 0.01, and ***P < 0.001 vs. control group. The Cellomics Cell Cycle BioApplication classified hESC-CM into their cell cycle phase based on the total nuclear intensity of DNA binding DAPI (E). Cell cycle distribution presented as histogram where the Y-axis represents the number of instances and the X-axis represents the total nuclear intensity. The positions of the 2 N and 4 N DNA contents as well G0/G1, G2/M, and S phases are indicated (n = 600 from 3 experiments).


Click here for additional data file (mmc1.ppt)


Disclosures

None declared.


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Glossary
Cell proliferation The term is used in the contexts of cell development and cell division. It refers to growth of cell populations, where one cell grows and divides to produce two.
Differentiation The process whereby an unspecialized embryonic stem cell acquires the features of a specialized cell, such as a heart, liver, or muscle cell.
Embryoid bodies (EBs) Clumps of cellular structures that arise when embryonic stem cells are cultured. Embryoid bodies contain tissue from all three germ layers: endoderm, mesoderm, and ectoderm. Embryoid bodies are not part of normal development and occur only in vitro.
Embryonic stem cells Primitive undifferentiated cells derived from the early embryo that have the potential to become a wide variety of specialized cell types.

Acknowledgments

G.F. was supported by BHF, Wellcome Trust Value in People Award, Hungarian Scientific Research Fund (OTKA F67919; MB08A 81237) and National Development Agency (TÁMOP 4.2.2-08/1/KMR-2008-0004). S.E.H. and N.N.A. were supported by the NC3Rs, BHF, and Rosetrees Trust. The H7 line used in these studies was donated by Geron Corporation (Menlo Park, CA, USA) under a collaborative agreement without further financial benefit to the authors. We thank Aphiwat Luangsomboon for his support in the angiotensin experiments.


Article Categories:
  • Original Article

Keywords: Abbreviations ANF, atrial natriuretic factor, bFGF, basic human fibroblast growth factor, CaMK II, Ca2+/calmodulin-dependent kinase II, EB, embryoid body, ERK, extracellular signal-regulated kinases, GSK3, glycogen synthase kinase 3, HDACII, histone deacetylase, FKBP, FK506 binding protein, hESC, human embryonic stem cells, hESC-CM, human embryonic stem cell-derived cardiomyocytes, JNK, c-Jun N-terminal kinases, MAP2K4 and MAP2K3, MAPK kinase 4 and 3, respectively, MEF, mouse embryonic fibroblast, MHC, myosin heavy chains, MOI, multiplicity of infection, mTOR, mammalian target of rapamycin, p38–MAPK, p38 mitogen-activated protein kinase, PKG, protein kinase G, Ryr2, cardiac ryanodine receptor 2, and SERCA2, sarco/endoplasmic reticulum Ca-ATPase..
Keywords: Keywords Embryonic stem cells, Cardiomyocytes, Human, Protein kinases, Hypertrophy.

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