Medical & Biological Engineering & Computing

, 47:41

Neuregulin-1 enhances differentiation of cardiomyocytes from embryonic stem cells

Authors

  • Zhi Wang
    • Department of Cardiology, The First People’s HospitalShanghai Jiaotong University School of Medicine
  • Guotong Xu
    • Key Laboratory of Stem Cell Biology, Institute of Health Sciences and Institute of Biochemistry and Cell BiologyShanghai Institute for Biological Science, Chinese Academy of Science
  • Yalan Wu
    • Key Laboratory of Stem Cell Biology, Institute of Health Sciences and Institute of Biochemistry and Cell BiologyShanghai Institute for Biological Science, Chinese Academy of Science
  • Yuan Guan
    • Key Laboratory of Stem Cell Biology, Institute of Health Sciences and Institute of Biochemistry and Cell BiologyShanghai Institute for Biological Science, Chinese Academy of Science
  • Lu Cui
    • Key Laboratory of Stem Cell Biology, Institute of Health Sciences and Institute of Biochemistry and Cell BiologyShanghai Institute for Biological Science, Chinese Academy of Science
  • Xia Lei
    • Key Laboratory of Stem Cell Biology, Institute of Health Sciences and Institute of Biochemistry and Cell BiologyShanghai Institute for Biological Science, Chinese Academy of Science
  • Jingfa Zhang
    • Key Laboratory of Stem Cell Biology, Institute of Health Sciences and Institute of Biochemistry and Cell BiologyShanghai Institute for Biological Science, Chinese Academy of Science
  • Lisha Mou
    • Key Laboratory of Stem Cell Biology, Institute of Health Sciences and Institute of Biochemistry and Cell BiologyShanghai Institute for Biological Science, Chinese Academy of Science
  • Baogui Sun
    • Department of Cardiology, The First People’s HospitalShanghai Jiaotong University School of Medicine
    • Department of Cardiology, The First People’s HospitalShanghai Jiaotong University School of Medicine
Original Article

DOI: 10.1007/s11517-008-0383-2

Cite this article as:
Wang, Z., Xu, G., Wu, Y. et al. Med Biol Eng Comput (2009) 47: 41. doi:10.1007/s11517-008-0383-2

Abstract

Neuregulin-1 (NRG-1) is a multifunctional regulator that acts through receptor tyrosine kinases of the epidermal growth factor (EGF/ErbB) receptor family in diverse tissue. ErbB receptors are expressed in developing embryoid bodies (EBs), and the importance of the NRG-1/ErbB signaling axis in heart development has been investigated, but the underlying molecular mechanism is poorly studied. NRG-1 treatment at 100 ng/ml significantly increased the number of beating EBs of differentiated murine embryonic stem cells (ESCs). Furthermore, NRG-1 up-regulated the expression of the cardiac-restricted transcription factors Nkx2.5 and GATA-4 and factors involved in differentiated cardiac cells (α-MHC, β-MHC and α-actinin); NRG-1-induced increase of Nkx2.5 transcription was inhibited by treatment with the PI3 K inhibitor or ErbB receptor inhibitor. Western blot analysis confirmed that the expression of phospho-Akt in the beating foci was increased in the presence of NRG-1. Our results suggest that NRG-1 promotes cardiomyocyte differentiation of ESCs and the ErbB/PI3 K/Akt signaling pathway is one of the underlying molecular mechanisms.

Keywords

Neuregulin-1Embryonic stem cellsCardiomyocytesPhosphatidylinositol 3-kinase

1 Introduction

Because the regenerative capacity of adult cardiac tissue is limited, any substantial cell loss or dysfunction, such as that occurring during myocardial infarction, is largely irreversible and may lead to progressive heart failure, a leading cause of mortality around the world. Cell therapy for the replacement of lost or damaged tissue in the dysfunctional zone is a promising therapeutic approach to restoring cardiac function. Animal studies have used various types of cells for transplantation, including fetal and neonatal cardiomyocytes (CMs), adult stem cells, such as fibroblasts and bone marrow-derived cells [16, 29, 30, 34]. The assumed capacity of trans-differentiation of adult stem cells into other lineages in vivo might simply represent a fusing with existing cell types rather than direct conversion. Fetal and neonatal CMs appear to be promising candidates for transplantation because of their ability to integrate into the host tissue and improve heart function. However, the clinical application of such cell transplantation therapy is hampered by the lack of an available source of donor cells.

Embryonic stem cells (ESCs) are uniquely endowed with the capacity of self-renewal and the potential to give rise to all possible cells of all three germ lineages, including beating CMs [35]. Several reports demonstrated that only ~5% of the cells within embryoid bodies (EBs) spontaneously differentiate into ESC-derived CMs (ESCMs) [19, 25, 27]. Therefore, much effort has been invested in developing strategies for differentiation of early CMs from ESCs, and further development of these cells for clinical application in heart diseases is warranted [5, 20].

Increasing evidence has shown that neuregulin-1/heregulin-1 (NRG-1/HRG-1), a member of the epidermal growth factor (EGF) family, is a vital factor involved in heart development. Mice carrying homozygous mutations in NRG-1 died at mid-gestation due to embryonic cardiac failure and lacked trabeculae in the ventricular chambers. This finding indicated that NRG-1 might promote maturation of the embryonic heart tissue [10, 21]. All NRG isoforms that contain the EGF-like domain are capable of binding to and activating one set of the EGF family receptor (ErbB) dimers, which could trigger the tyrosine phosphorylation of the ErbB receptor [21, 38]. ErbB receptor expression in developing EBs has been investigated [36]. Therefore, we aimed to observe the role of exogenous NRG-1 during ESC differentiation and the underlying molecular mechanism.

2 Methods

2.1 Cell culture and differentiation

Murine undifferentiated stem cells of the ES-D3 line (ATCC, CRL 1934) were grown on mitotically inactivated mouse embryonic fibroblast (MEF) feeder layers in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/BRL) supplemented with 15% fetal bovine serum (FBS; HyClone), 50 U/ml penicillin, 50 μg/ml streptomycin (Gibco/BRL), 2 mM l-glutamine (Gibco/BRL), 10 μM β-mercaptoethanol (β-ME, Gibco/BRL), 0.1 mM non-essential amino acids (NEAAs, Gibco/BRL), and 1,000 U/ml leukemia inhibitory factor (LIF, Chemicon, USA). MEF layers were obtained from embryos of 13.5-day pregnant ICR mice (Slaccas, Shanghai Institute for Biological Sciences, China). The study was approved by the Ethics Committee of Shanghai Jiaotong University School of Medicine. Animal handling followed the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals.

An ESC suspension of 2 × 104 cells per ml was prepared in DMEM supplemented with 20% FBS, 50 U/ml penicillin, 50 μg/ml streptomysin, 2 mM l-glutamine, 10 μM β-ME, and 0.1 mM NEAAs. Cell drops (400 cells/drop) were placed on the lid of bacterial Petri dishes for 2 days to form EBs. EBs were transferred to suspension in new bacterial Petri dishes containing fresh differentiation medium. On day 7, the individual EBs were transferred to gelatin-coated 24-well tissue culture plates for terminal differentiation. To examine the effect of NRG-1 on cardiac differentiation, the recombinant peptide containing the β variant of the EGF-like domain of NRG-1 (Peprotech) was added to the differentiation medium from day 3 to 7 at the concentrations indicated. The ErbB receptor inhibitor AG1478 and PI3 K inhibitor Wortmannin (both Sigma, St. Louis) were used at a final concentration of 1 μM each.

2.2 Semi-quantitative RT-PCR

Total RNA from EBs on day 7 of suspension culture (n = 8 each) and beating EB outgrowths from day 8 to 16 of expansion (n = 8 each) was extracted by use of TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. An amount of 2 μg total RNA was reverse transcribed by use of M-MLV reverse transcriptase (Invitrogen) with random primers according to the manufacturer’s recommended protocol. The reverse-transcriptase product (1 μl) was then amplified by PCR. The specific primers were designed by use of Primer Premier 5.0 (Shanghai DNA Biotechnology Corp, China). PCR amplification conditions were an initial denaturation at 95°C for 5 min, followed by 36 cycles of denaturation at 94°C for 30 s, annealing at 56–67.5°C for 40 s, a 30-s extension at 72°C, and a final extension at 72°C for 10 min. The corresponding primer set sequences with annealing temperature and expected product length are in Table 1. PCR products were separated electrophorectically on 2% agarose gels with use of 0.001% ethidium bromide. The fluorescent densities of the resulting bands were determined by use of quantity one software (Bio-Rad, USA) and normalized to that of β-actin.
Table 1

Primers used for RT-PCR: sequence, annealing point, and expected length of the PCR product

Gene

Primer

Product size (bp)

Annealing point (°C)

Nkx2.5

For 5′-CGACGGAAGCCACGCGTGCT-3′

181

60

Rev 5′-CCGCTGTCGCTTGCACTT-3′

GATA-4

For 5′-CTC GATATGTTTGATGACTTCT-3′

345

56

Rev 5′-CGTTTTCTGGTTTGAATCCC-3′

α-MHC

For 5′-GGAAGAGTGAGCGGCGCATCAAGG-3′

301

67.5

Rev 5′-CTGCTGGAGAGGTTATTCCTCG-3′

β-MHC

For 5′-GCCAACACCAACCTGTCCAAGTTC-3′

205

67.5

Rev 5′-TGCAAAGGCTCCAGGTCTGAGGGC-3′

β-actin

For 5′-GAAATCGTGCGTGACATCAAAG-3′

216

60

Rev 5′-TGTAGTTTCATGGATGCCACAG-3′

α-MHCα-myosin heavy chain, β-MHCβ-myosin heavy chain, bp base pair, For forward, Rev reverse

2.3 Immunohistochemistry

Whole EBs were plated on 1% gelatin-coated coverslips. Fixation involved 4% paraformaldehyde for 15 min at room temperature. Samples were permeabilized with 0.25% Triton-X100 (Sigma, St. Louis) for 10 min. Blocking was performed in 1% bovine serum albumin (BSA, Sigma, St. Louis). An antibody against sarcomeric α-actinin (diluted 1:100, Santz Cruz Biotechnology) was incubated overnight at 4°C. After an extensive washing, EBs were exposed to the Cy3-conjugated secondary antibody (diluted 1:100, Solomon Biotechnology Corp, China) at room temperature for 60 min. DAPI was used to stain nuclei. Samples were mounted with aqueous mounting medium (DAKO, USA). Fluorescence imaging involved use of a Nikon imaging system (Yokohama, Japan). Quantification of the immunostained areas involved Image-Pro Plus 5.0 software, Media Cybernetics, USA, with normalization to the total surface of each EB.

2.4 Western blot analysis

Spontaneously contracting foci within EBs were dissected and collected in modified RIPA buffer [50 mM Tris-HCl, pH 7.4, 1% NP40, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 1 mM PMSF], and allowed to lyse for 30 min on ice. The cell extracts were centrifuged at 12,000g for 15 min at 4°C. Protein concentration was measured in supernatant by use of a Bio-Rad protein assay kit. Samples containing equal amounts of proteins underwent 10% SDS-PAGE and were transferred to nitrocellulose membranes (Bio-Rad, USA). After being blocked, the membranes were incubated overnight with anti-p-Akt (diluted 1:500) or anti-β-actin (diluted 1:1,000, both Signalway Antibody). After a washing with TBST, the membranes were incubated for 1 h with secondary antibody horseradish peroxidase-conjugated anti-rabbit IgG, then underwent detection by the chemiluminescence system. Protein expression was quantified by use of quantity one software (Bio-Rad, USA).

2.5 Statistical analysis

Data are expressed as mean ± SEM of at least three independent experiments. Statistical analyses involved use of one-way ANOVA or Student’s t test. P < 0.05 was considered statistically significant.

3 Results

3.1 Cardiomyocyte differentiation of ESCs in vitro

MEFs at passages 3–5 were used as feeder cells (Fig. 1a). Two to three days after seeding, undifferentiated ESC colonies were increased in size and showed well-defined edges (Fig. 1b). EBs were formed during suspension (Fig. 1c). Most EBs attached to culture plates within 24 h, became flattened and formed thin multilayered structures (Fig. 1d). Beginning at day 8 of differentiation, clusters of spontaneously beating ESCMs appeared in the outgrowths of several EBs. Compared to the number of spontaneously differentiating EBs, the number showing one or more beating foci at day 9 was significantly increased with NRG-1 treatment and further increased until day 12 (Fig. 2a). The beating frequency of EBs presenting at least one beating cluster was measured at day 10 of differentiation. Stimulation with the adrenoreceptor agonist isoprenaline (1 μM) induced a positive chronotropic response, whereas the muscarinic cholinoceptor agonist carbachol (1 μM) induced a negative chronotropic response in the spontaneous beating ESCMs, regardless of NRG-1 treatment (Fig. 2b).
https://static-content.springer.com/image/art%3A10.1007%2Fs11517-008-0383-2/MediaObjects/11517_2008_383_Fig1_HTML.jpg
Fig. 1

Cardiomyocyte differentiation of ESCs in vitro. Mouse embryonic fibroblasts at passage 3 (a). Undifferentiated ESC colonies with well-defined edges (b). Embryoid body formed during suspension (c). Attached EBs became flattened and formed thin multilayered structures; spontaneous beating ESCMs in the area of the EB (box) (d)

https://static-content.springer.com/image/art%3A10.1007%2Fs11517-008-0383-2/MediaObjects/11517_2008_383_Fig2_HTML.gif
Fig. 2

NRG-1 increased the number of beating EBs. EBs were treated with NRG-1 (100 ng/ml) for the times indicated (a). More than 48 EBs were counted per condition per time point. *< 0.05 versus control. NRG-1-treated and untreated groups showed no difference in change in frequency of beats after treatment with 1 μM isoprenaline (Iso) or 1 μM carbachol (Cch) (b)

3.2 Effect of NRG-1, ErbB receptor inhibitor, and PI3 K inhibitor on expression of early cardiac-restricted transcription factors

NRG-1 significantly up-regulated the mRNA levels of the early cardiac-restricted transcription factors Nkx2.5 and GATA-4 at day 7 in differentiating ESCs in a dose-dependent manner, with maximal activity at 100 ng/ml NRG-1 (Fig. 3). The PI3 K inhibitor Wormannin at 1 μM completely blocked the NRG-1-induced increase in Nkx2.5 transcription (Fig. 4). Furthermore, the ErbB receptor inhibitor AG1478 inhibited the NRG-1-induced increase in Nkx2.5 expression in differentiating ESCs. As well, Nkx2.5 expression was impaired in the presence of Wortmannin or AG1478 alone without NRG-1 treatment.
https://static-content.springer.com/image/art%3A10.1007%2Fs11517-008-0383-2/MediaObjects/11517_2008_383_Fig3_HTML.gif
Fig. 3

NRG-1 increased the mRNA expression of cardiac-restricted transcription factors Nkx2.5 and GATA-4 in a dose-dependent manner (a). Results expressed as integrated fluorescence density (IFD) after normalization to β-actin (b). *P < 0.05, **P < 0.01 versus control

https://static-content.springer.com/image/art%3A10.1007%2Fs11517-008-0383-2/MediaObjects/11517_2008_383_Fig4_HTML.gif
Fig. 4

NRG-1-induced increase in Nkx2.5 expression was impaired by co-treatment with inhibitors of ErbB (AG1478) or PI3 K (Wortmannin). RT-PCR analysis of EBs from day 7 in the presence of AG1478/Wortmannin with or without NRG-1 (a). Results expressed as IFD after normalization to β-actin (b). *P < 0.05 versus control; **P < 0.05 versus NRG-1

3.3 NRG-1 increased the level of α-MHC and β-MHC mRNA with further development of ESCs

The mRNA levels of cardiac-specific α-MHC and β-MHC in ESCs treated with or without NRG-1 increased from day 8 to 16 of differentiation. As compared with no NRG-1, NRG-1 at 100 ng/ml significantly increased the mRNA level of α-MHC in ESCs during an early to middle developmental stage, from day 8 to 13 of differentiation, and significantly increased the level of β-MHC from day 13 to 16 of differentiation (Fig. 5).
https://static-content.springer.com/image/art%3A10.1007%2Fs11517-008-0383-2/MediaObjects/11517_2008_383_Fig5_HTML.gif
Fig. 5

Treatment with NRG-1 (100 ng/ml) increased the mRNA expression of cardiac-specific α-MHC and β-MHC between day 8 and 16 of development (a). Results expressed as IFD after normalization to β-actin (b). *P < 0.05 versus control at the same day of differentiation

3.4 NRG-1 enhanced the expression of α-actinin and p-Akt within the EB beating outgrowths

Synchronously beating EBs in both groups with or without NRG-1 treatment exhibited characteristics of CMs as demonstrated by immunostaining with antibody against cardiac α-actinin. The area positive for α-actinin with NRG-1 treatment was greater than with control treatment (Fig. 6a). Western blot analysis of NRG-1-treated EB beating outgrowths showed increased expression of p-Akt (Fig. 6b).
https://static-content.springer.com/image/art%3A10.1007%2Fs11517-008-0383-2/MediaObjects/11517_2008_383_Fig6_HTML.gif
Fig. 6

Treatment with NRG-1 (100 ng/ml) increased the proportion of cardiac sarcomeric α-actinin in differentiated EBs at day 13 (a). NRG-1-treated EBs stained with actinin (red fluorescence, Ia); spontaneously differentiating EBs stained with actinin (IIa); Ib and IIb are double images of actinin and DAPI (blue fluorescence). Quantification of the area positive for actinin staining of the corresponding EB (a, right panel). Western blot analysis of NRG-1-treated EB beating outgrowth shows increased expression of p-Akt (b, left panel). Quantitative analysis of p-Akt (b, right panel). Densitometry for the control sample was set to 100%. *P < 0.05 versus control

4 Discussion

The importance of the NRG-1/ErbB signaling axis in heart development has been investigated [9, 10], but the underlying molecular mechanism is poorly understood. ESCs can differentiate into multiple lineages in vitro, including cardiac cells. So they are an ideal model to unravel the underlying molecular events during embryonic heart development. Spontaneous differentiation of ESCs into CMs in vitro normally requires an initial aggregation step to form EBs in hanging drops for 2 days then suspension for 5 days before they are plated onto gelatin-coated tissue culture dishes [2, 40]. Within 1–4 days after plating, some EBs present one or more beating foci, termed ESCMs, or CM syncytium. During embryogenesis, the expression pattern of early myocardiogenic progenitor cells is characterized by a high transcription level of the homeoprotein Nkx2.5, which is related to the co-expression of the zinc-finger transcription factor GATA-4 [3, 22, 23]. We found the mRNA levels of Nkx2.5 and GATA-4 dose-dependently up-regulated in the presence of NRG-1 (Fig. 3). Moreover, NRG-1 at an optimal concentration, 100 ng/ml, significantly increased the proportion of beating EBs (Fig. 2a) and increased the cardiac-specific transcription of α-MHC/β-MHC and the sarcomeric protein α-actinin (Figs. 5, 6a). These results suggest that exogenous NRG-1 could promote the commitment of ESCs toward a cardiac lineage. Although Suk et al. [36] previously reported that NRG-1 promoted CM differentiation of ESCs, the function of these ESCMs, such as the chronotropic feature, remained unknown [17]. In the present study, we further observed that spontaneous beating frequency in EBs with or without NRG-1 treatment was actively responsive to the β-adrenergic and muscarinic agonists (Fig. 2b), so regardless of NRG-1 treatment, these differentiated ESCMs were functional CMs.

The neuregulins are part of a larger family of proteins whose members are all structurally related to EGF. The bioactivity and importance of only the EGF-like domain of the NRG has been fully appreciated. All NRG isoforms that contain the domain are capable of binding to and activating one set of ErbB receptor dimers, which could trigger the tyrosine phosphorylation of ErbB receptor. NRG was identified as a multifunctional regulator involved in cell fate choice [8, 24, 33]. The importance of NRG-1/ErbB signaling was shown during embryonic heart development and in the adult heart [9, 11]. PI3 K lies downstream of many receptor tyrosine kinases and G protein-coupled receptors [14]. It has been suggested to play a role in differentiation of several cell lineages, including skeletal myocytes, adipocytes, and erythroleukemia cells, which, like CMs, are derived from mesodermal cells [1, 37, 41]. Previously, signal transduction initiated by menadione and hepatocyte growth factor activated PI3 K in CM differentiation of ESCs was reported respectively [31, 32]. In our study, NRG-1-induced increase of Nkx2.5 transcription was inhibited in the presence of AG1478 or Wortmannin, an irreversible inhibitor of PI3 K (Fig. 4). Furthermore, western blot analysis of NRG-1-treated EBs showed increased expression of p-Akt (Fig. 6b), the downstream target of PI3 K. These results suggest that the PI3 K/Akt cascade may be required for transmission of NRG-1 signals during CM differentiation of murine ESCs. Moreover, Nkx2.5 expression was impaired in the presence of Wortmannin or AG1478 alone without NRG-1 treatment. Inhibition of PI3 K leading to severe impairment of cardiac differentiation was also reported previously [18, 28], which indicated the crucial involvement of PI3 K in heart development.

Akt is responsible for the activation of p300, a transcriptional co-activator, which can acetylate several DNA-binding transcription factors and enhance their DNA binding activities [4, 12]. Previous studies have shown GATA-4 as a target of p300, and subsequent p300-mediated acetylation of GATA-4 is known to participate in the differentiation of ESCs into CMs [6, 7, 13, 15, 42, 43]. Moreover, Akt positively regulates the transcriptional activity of GATA-4 in CMs by phosphorylation and subsequent inactivation of glycogen synthase kinase 3β [26]. Therefore, NRG-1 could regulate GATA-4 expression probably directly through Akt or through Akt-increased activation of p300; the transcriptional up-regulation of GATA-4 might be responsible for the significant increase in Nkx2.5 expression because Nkx2.5 expression is directly regulated by GATA-4 during heart development [3, 22, 23]. Identifying the complexity of NRG’s regulatory function in the heart is meaningful for new therapeutic opportunities targeting ErbB receptor-mediated signaling.

Recently, Kim et al. [17] reported on the MAPK/ERK signaling pathway mediated by HRG-1 in the development of CMs from ESCs, whereas our results suggest PI3 K/Akt as the key downstream factor in NRG-1 promoting cardiogenesis in ESCs. The differences in results between the studies may reflect differences in culture conditions of the differentiating ESCs, methods used to generate EBs, length of EB suspension, concentration of serum used for differentiation, and/or manner of administration of reagents to the EBs. We used the EB suspension system to observe the effect of NRG-1 on cardiogenesis of ESCs, but Kim et al. used an EB adhesion system. In our experiment, EBs were cultured with NRG-1 or kinase inhibitors in differentiation medium from day 3 to 7, whereas Kim et al. cultured 4-day-old EB adhesion cells with HRG-1 or kinase inhibitors for 4 to 5 days in serum-free medium. The process of cardiogenesis in a developing EB possibly requires many signals, and cross-talk between these signals should be explored in further research work. Moreover, we presumed that the microenvironment within the EB culture would influence the CM differentiation via intrinsic signal transduction pathways [39].

In conclusion, we demonstrate that NRG-1 promotes CM differentiation of mouse ESCs at an early stage of development and the ErbB/PI3 K/Akt signaling pathway is one of the underlying molecular mechanisms. Thus, ESCs treated with NRG-1 may be a useful source to produce a sufficient number of cardiac myocytes for cellular transplantation strategies and many other applications.

Acknowledgments

We thank Chunchun Zhuge, Jianfeng Shen, Hua Jiang, Chunliang Li, Yijun Shi and Lingjie Li for their valuable help in experiments. This work was supported by the Shanghai Science and Technology Committee Grant (No. 030121).

Copyright information

© International Federation for Medical and Biological Engineering 2008