Neuregulin-1 enhances differentiation of cardiomyocytes from embryonic stem cells
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- Wang, Z., Xu, G., Wu, Y. et al. Med Biol Eng Comput (2009) 47: 41. doi:10.1007/s11517-008-0383-2
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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.
KeywordsNeuregulin-1Embryonic stem cellsCardiomyocytesPhosphatidylinositol 3-kinase
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 . 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 . Therefore, we aimed to observe the role of exogenous NRG-1 during ESC differentiation and the underlying molecular mechanism.
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
Primers used for RT-PCR: sequence, annealing point, and expected length of the PCR product
Product size (bp)
Annealing point (°C)
For 5′-CTC GATATGTTTGATGACTTCT-3′
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.
3.1 Cardiomyocyte differentiation of ESCs in vitro
3.2 Effect of NRG-1, ErbB receptor inhibitor, and PI3 K inhibitor on expression of early cardiac-restricted transcription factors
3.3 NRG-1 increased the level of α-MHC and β-MHC mRNA with further development of ESCs
3.4 NRG-1 enhanced the expression of α-actinin and p-Akt within the EB beating outgrowths
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.  previously reported that NRG-1 promoted CM differentiation of ESCs, the function of these ESCMs, such as the chronotropic feature, remained unknown . 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 . 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 . 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.  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 .
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.
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).