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. 2016 Oct 27;50(1):e12310. doi: 10.1111/cpr.12310

Role of alpha‐ and beta‐adrenergic receptors in cardiomyocyte differentiation from murine‐induced pluripotent stem cells

Xiao‐Li Li 1,†, Di Zeng 1,†, Yan Chen 1,2, Lu Ding 1, Wen‐Ju Li 1, Ting Wei 1, Dong‐Bo Ou 1, Song Yan 1, Bin Wang 1, Qiang‐Sun Zheng 1,✉
PMCID: PMC6529101  PMID: 27790820

Abstract

Objectives

Induced pluripotent stem cell (iPSC)‐derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized.

Materials and methods

Murine‐induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated.

Results

Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac‐specific markers, GATA4, Nkx2.5 and Tnnt2. Although β‐AR signalling is the foremost signalling pathway in cardiomyocytes, EPI‐enhanced cardiac differentiation depended more on α‐AR signalling than β‐AR signalling. In addition, selective activation of α1‐AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α2‐ or β‐AR signalling induced no or less differentiation, respectively. EPI‐ and α1‐AR‐dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK‐ERK1/2 pathway and regulation of miPS cell‐cycle progression.

Conclusions

These results demonstrate that activation of ARs, particularly of α1‐ARs, promoted miPSC differentiation into cardiac lineages via MEK‐ERK1/2 signalling.

Abbreviations

ARs

adrenergic receptors

ClO

clonidine

CPC

cardiac progenitor cell

EBs

embryoid bodies

EPI

epinephrine

ERK1/2

extracellular signal‐regulated kinases 1 and 2

ESCs

embryonic stem cells

iPS‐CMs

iPSC‐derived cardiomyocytes

ISO

isoproterenol

MEK

mitogen‐activated protein extracellular kinase

miPSC

mouse‐induced pluripotent stem cell

PE

phenylephrine

1. Introduction

Heart failure, a complex clinical syndrome, which generally results from myocardial injury leading to the impairment of contractile functions, is a major global health issue.1 As the end stage for various heart diseases and due to the limited capability of injured myocardium to undergo extensive regeneration and repair, heart failure results in high mortality.2, 3, 4 Therefore, novel, innovative treatments for regenerating myocardium are urgently required. Stem cell–based regeneration provides a promising approach to regenerate myocardium and treat end‐stage heart failure. Stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are characterized by limitless proliferation capacity and multipotency and can be differentiated into functional cardiomyocytes in vitro.5, 6, 7, 8 However, the widespread application of ESC‐based therapies is limited by ethical concerns and technical limitations, including the potential for graft rejection.9 The ability to derive patient‐specific‐induced pluripotent stem cells (iPSCs) from somatic cells and differentiate them in vitro circumvents these issues.10, 11, 12, 13 Transplantation of cardiomyocytes derived from iPSCs (iPS‐CMs) in animal models of myocardial infarction improves cardiac function.14, 15 However, cardiomyocyte differentiation from iPSCs is inefficient, limiting their application in regenerative heart disease therapies. Moreover, though various signalling molecules and transcriptional factors are known to be essential for cardiomyocyte differentiation from iPSCs, such as BMP4,16, 17 WNT,18, 19, 20 FAK21 and collagen/β1 integrin,22, 23 the mechanisms of cardiomyocyte differentiation from iPSCs have not been fully elucidated.

Adrenergic receptors (ARs) belong to the family of G‐protein‐coupled receptors and are composed of seven transmembrane domains, an extracellular N‐terminal region and an intracellular C‐terminal region. ARs bind to endogenous catecholamine hormones, including epinephrine (EPI) and norepinephrine (NE), and regulate various metabolic changes.24, 25 ARs are classified into two main groups: α‐ARs and β‐ARs. β‐ARs comprise three subclasses, β1, β2 and β3, and are the canonical ARs in cardiomyocytes. β‐AR signalling regulates G‐proteins, which in turn can activate adenylyl cyclase and stimulate cAMP formation.26 Cyclic AMP can modulate various immunological functions, including the proliferation of lymphocytes, secretion of antibodies and production of pro‐inflammatory mediators. TSENG et al. discovered that the β‐ARs are involved in the regulation of neonatal cardiomyocyte proliferation and that this mitogenic control may be mediated via the p70 S6K pathway.27 In addition, β‐ARs regulate mouse mesenchymal stem cell osteogenesis28 and ESC‐cardiac differentiation.29 However, it is unclear whether these knowledge and methods learned from ESC differentiation can be fully applied to iPSCs. Kattman et al. found distinct responses of iPSCs to cytokine stimulation compared with ESCs.17 Thus, systematic and detailed analyses of the responses of iPSCs to β‐ARs are required.

In contrast, the physiological roles of α‐ARs in cardiac differentiation have been less well characterized. The α‐ARs comprise two subclasses, α1 and α2, with α1‐ARs being the predominant α‐AR expressed. A variety of secondary messenger molecules are regulated by α1‐AR signalling, which includes the mobilization of intracellular calcium through the activation of phospholipases C and A2, and stimulation of calcium influx via voltage‐dependent and voltage‐independent calcium channels.30 The α1‐ARs protect the heart by stimulating adaptive cardiomyocyte hypertrophy, preventing cardiomyocyte death and pathological remodelling after heart failure and enhancing contractile functions.25 Results from clinical trials have indicated that the absence of α1‐ARs exacerbates heart failure.31 However, the exact roles of α‐ARs in the cardiac differentiation of iPSCs and the underlying mechanisms have not been assessed.

ARs are expressed in mouse embryonic stem cells, but the relationship between adrenergic receptor signalling and stem cell‐derived cardiomyocyte differentiation remains unclear. Here, we investigated the potential role and mechanisms of adrenergic receptor signalling in murine iPSC‐derived cardiomyocyte differentiation using the AR agonist, EPI and other specific AR agonists and antagonists.

2. Materials and methods

2.1. Culture of murine iPSCs and differentiation into cardiomyocytes

Undifferentiated Oct4‐GFP+ murine iPSCs (kindly provided by Duanqing Pei, Chinese Academy of Sciences) were cultured on MEF feeders in undifferentiated iPSC culture medium consisting of 85% knockout high‐glucose glutamine‐free Dulbecco's modified Eagle's minimal essential medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 15% (vol/vol) knockout serum replacement (Invitrogen), 0.1 mmol/L non‐essential amino acids (Invitrogen), 2 mmol/L GlutaMAX (Invitrogen), 0.1 mmol/L β‐mercaptoethanol (Sigma‐Aldrich, St. Louis, MO, USA) and 1000 U/mL murine leukaemia inhibitory factor (Chemicon, Temecula, CA, USA), as described previously.32 Culture medium was exchanged daily, and cells were passaged every 2–3 days to maintain their undifferentiated state. Before the initiation of differentiation, miPSC colonies were passaged up to three times on gelatin‐coated dishes without feeder cells to eliminate contaminating MEFs.

Induced PSCs were differentiated using the classical hanging‐drop method. Briefly, embryoid bodies were generated in hanging drops of 20 μL of differentiation medium, consisting of 80% DMEM (Invitrogen), 20% foetal bovine serum (Invitrogen), 2 mmol/L GlutaMAX (Invitrogen) and 0.1 mmol/L non‐essential amino acid stock (Invitrogen), which was seeded with ~800–1000 cells. After 3 days, EBs were transferred to gelatin‐coated culture plates and cultured in differentiation medium for 15 days. Differentiation medium was exchanged every 2 days.

2.2. Treatment with AR agonists and antagonists

To characterize the effect of EPI on cardiomyocyte differentiation from murine iPSCs, different concentrations of EPI (0.001, 0.005, 0.01, 0.05, 0.1 and 0.2 μmol/L) were added at different time points post‐differentiation. The different schedules of EPI treatments are shown in Fig. 1C. As a negative control, the solvent alone, which was used to dissolve EPI, was added. To further elucidate the signalling pathways underlying EPI‐dependent cardiac differentiation, the α‐AR antagonist, phentolamine (10 μmol/L; Sigma‐Aldrich), the β‐AR antagonist, propranolol (5 μmol/L; Sigma‐Aldrich),33 the selective α1‐AR agonist, phenylephrine (PE) (10 μmol/L; Sigma‐Aldrich), the selective α2‐AR agonist, clonidine (ClO) (10 μmol/L; Sigma‐Aldrich),34 the β‐AR agonist, isoproterenol (ISO) (1 μmol/L; Sigma‐Aldrich)29 and the MEK1/2 inhibitor, PD98059 (10 μmol/L; Selleckchem, Houston, TX, USA),35 were also used.

Figure 1.

Figure 1

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Expression profiles of adrenergic receptors during cardiomyocyte differentiation from murine‐induced pluripotent stem cells (miPSCs). (A) Morphology of undifferentiated miPSC colonies expressing Oct4‐GFP+ (green) cultured on gelatin‐coated plates. Scale bars=100 μm. (B) Positive staining of undifferentiated miPSC colonies for the pluripotency markers, Oct4 and Nanog. Merged phase‐contrast and fluorescent micrographs are shown (e‐h). Scale bars=100 μm. (C) Schematic of cardiomyocyte differentiation protocol from miPSCs. Different doses of epinephrine (EPI) were added at different time points post‐differentiation. (D) Vehicle control‐ or EPI‐treated EBs at 3, 5, or 9 days post‐differentiation. Insets: fluorescent micrographs of miPSC‐derived EBs expressing Oct4‐eGFP. Scale bar=200 μm. (E) Gene expression profiles of ARs from semi‐quantitative RT‐PCR analysis starting from day 3 to day 15 post‐differentiation are shown for vehicle control‐ and EPI‐treated EBs

2.3. Immunofluorescence analysis

Cells were enzymatically dissociated using 0.05% trypsin‐EDTA and then plated onto coverslips for 48 h to gain the cell climbing slices. Cells were fixed with 4% (vol/vol) paraformaldehyde for 15 min at 37°C, permeabilized with 0.1% (vol/vol) Triton X‐100 for 10 min, and then blocked with 5% (wt/vol) BSA for 30 min. Samples were incubated with the following primary antibodies at 37°C for 120 min at the indicated dilutions: mouse monoclonal anti‐Oct4 (Oct4; 1:150), rabbit polyclonal anti‐Nanog (Nanog; 1:200), mouse monoclonal anti‐cardiac troponin‐T (cTnT; 1:200), rabbit polyclonal anti‐α‐actinin (α‐actinin; 1:200), rabbit polyclonal anti‐connexin 43 (Cx43; 1:200) and rabbit polyclonal anti‐myosin light chain 2 (MLC2v; 1:140). After washing with PBS, the bound antibodies were visualized by incubating samples with the appropriate secondary antibody at the indicated dilutions: Alexa 488 (1:200) and Cy3 (1:200). Secondary antibodies were incubated at 37°C for 40 min. Primary and secondary antibodies were purchased from Abcam (Cambridge, MA, USA). Samples were mounted and nuclei were counterstained with Prolong Gold Antifade Reagent with DAPI, which was used at the indicated dilution (1:1000; Sigma, St. Louis, MO, USA). Samples were imaged using an Olympus IX71 fluorescence microscope (Tokyo, Japan).

2.4. Semi‐quantitative RT‐PCR and quantitative real‐time PCR

Total RNA was isolated with the RNeasy mini kit (QIAGEN, Duessedorf, Germany) and treated with DNase (QIAGEN) to remove genomic DNA contamination. RNA (500 ng/μL) was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The resulting cDNA was amplified by polymerase chain reaction (PCR) using Go tag Master Mix (QIAGEN), and PCR products were separated and analysed on a 2% (wt/vol) agarose gel by electrophoresis. SYBR Green real‐time PCR studies were performed using Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Transcript levels of GAPDH were used for within‐experiment signal normalization. Quantification of relative transcript levels was performed using the 2−△△CT method. All experiments were conducted in triplicate. Primer sequences and PCR conditions are detailed in Table S1.

2.5. Western blot analysis

Differentiated cells were homogenized in RIPA lysis buffer (Biomiga) for Western blotting analysis. Homogenized samples were centrifuged at 3000 × g for 10 min, and the clarified supernatant was used for subsequent analysis. The total protein concentration of the clarified supernatant sample was determined using a BCA protein assay kit (Nanjing KeyGEN Biotech. Co. Ltd, Nanjing, China) following the manufacturer's instructions. Protein samples (100 μg/sample) were electrophoresed on SDS‐polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked at room temperature for 3 h with 5% non‐fat milk in 1× Tris buffered saline and 0.1% Tween‐20. Membranes were then incubated overnight with primary antibodies against cTnT, α‐actinin, Nkx2.5, GATA4, phosphorylated ERK1/2 (p‐ERK1/2), total ERK1/2, phosphorylated MEK (p‐MEK), total MEK and GAPDH at 4°C. Anti‐cTnT, anti‐α‐actinin, anti‐Nkx2.5 and anti‐GATA4 primary antibodies were purchased from Abcam. Anti‐p‐ERK1/2 and anti‐ERK1/2 antibodies were purchased from Sigma‐Aldrich. Anti‐p‐MEK, anti‐MEK and anti‐GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). After three washes, blots were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies at RT for 2 h and washed again. Chemiluminescence signals were detected using the Molecular Imager ChemiDoc™ XRS+ system (Bio‐Rad, Hercules, CA, USA). Band intensities were quantified using Image‐Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA).

2.6. Flow Cytometry

Fifteen days post‐differentiation, cells were dissociated into single cells, fixed with 1% (vol/vol) paraformaldehyde for 20 min at RT, and permeabilized with ice‐cold 90% methanol for 30 min on ice. Cells were washed twice with FACS buffer (PBS with 0.1% BSA and 0.1% Triton X‐100) and then incubated overnight with the primary antibody (mouse monoclonal anti‐cTnT, 1:200; Abcam) in FACS buffer at 4°C. After washing cells twice with 1 mL of FACS buffer, cells were incubated with Alexa 488‐conjugated goat anti‐mouse antibody (1:500; Abcam) for 30 min in the dark at RT. Data were collected on a FASCaliber flow cytometer (Beckton Dickinson, Franklin Lakes, NJ, USA) and analysed using FACS DiVa.

2.7. Proliferation and apoptosis assays

Proliferation status of the cells was determined by measuring the incorporation of BrdU. Cells were incubated with 10 μmol/L BrdU, and BrdU labelling was detected by fluorescence microscope or flow cytometry using an APC‐conjugated anti‐BrdU antibody (BD Pharmingen™; BrdU Flow Kits San Diego, CA, USA), following the instruction manual. Staining of samples without BrdU addition was used as negative control. Double staining of BrdU with cTnT and Nkx2‐5 was performed by Nkx2‐5 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and cTnT (mouse monoclonal anti‐cTnT, 1:200; Abcam) antibody. The apoptosis status of the cells was determined by TUNEL staining with the in situ Cell Death Detection kit (Roche, Mannheim, Germany) according to the manufacturer's instruction and analysed and quantified by flow cytometry. Cell‐cycling properties of the cells was determined by propidium iodide (PI) staining staining with the BD Cycletest™ Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instruction and analysed by flow cytometry.

2.8. Statistical analysis

Results are expressed as mean±standard error from at least three independent experiments. Statistical analyses were performed using SPSS 18.0 software (SPSS Inc.). The statistical significance of differences among groups was analysed using ANOVA followed by a LSD‐t post hoc test. P values<0.05 were considered statistically significant.

3. Results

3.1. Adrenergic receptors (ARs) are expressed throughout the process of miPSC differentiation into cardiomyocytes

To eliminate contaminating MEFs, miPSCs were passaged up to three times on gelatin‐coated dishes without feeder cells. As shown in Fig. 1A and B, miPSC colonies cultured on gelatin‐coated dishes remained undifferentiated, as indicated by the high expression of Oct4‐GFP+ (Fig. 1A) and positive staining for the pluripotency markers, Oct4 and Nanog (Fig. 1B). Cardiomyocytes were differentiated from miPSCs using the traditional hanging‐drop method, in which embryoid bodies are first formed before inducing their differentiation. After inducing differentiation, Oct4‐GFP+ expression decreased over time, indicating the loss of pluripotency (Fig. 1D).

The potential regulation of cardiac differentiation by epinephrine (EPI) necessitates the expression of ARs on undifferentiated miPSCs; therefore, we first characterized the expression profiles of α‐ and β‐ARs during the process of cardiomyocyte differentiation. Semi‐quantitative RT‐PCR analysis of α‐ and β‐AR subtypes showed that both are expressed in undifferentiated miPSCs and that the expression of both increased with time after the induction of differentiation (Fig. 1E). The expression of both AR subtypes suggests that ARs may regulate the differentiation of cardiomyocytes from miPSCs.

3.2. Treatment with the AR agonist, epinephrine (EPI), enhances cardiomyocyte differentiation from miPSCs

To determine whether AR activation affects cardiomyocyte differentiation from miPSCs, cells were treated with EPI at different concentrations, ranging from 0.001 to 0.2 μmol/L, for 7 days starting on the day that differentiation was induced. The relative expression level of the cardiac‐specific gene, cardiac myofilament protein T (Tnnt2), increased with EPI concentration and reached a peak at 0.05 μmol/L (Fig. 2A). This effect was further validated by the increased protein expression of troponin‐T (cTnT) at day 15 post‐differentiation (Fig. 2B). Thus, subsequent studies were performed with 0.05 μmol/L EPI.

Figure 2.

Figure 2

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Epinephrine enhances miPSC differentiation into cardiomyocytes. (A–B) Results of dose–response experiments to determine the optimal concentration of EPI that promotes cardiac differentiation from miPSCs. Cardiac differentiation was monitored by gene (A) and protein (B) expression levels of cardiac troponin‐T (cTnT). (C) Gene expression profiles of pluripotency markers (Oct3/4, Nanog) and cardiac‐specific markers (Tnnt2, Nkx2.5, GATA4, MLC2a and MLC2v) analysed by semi‐quantitative RT‐PCR (at day 0–15 post‐differentiation). (D) Quantitative RT‐PCR analysis of cardiac‐specific markers (Tnnt2, Nkx2.5, GATA4, GATA6, MLC2a and MLC2v) at day 15 post‐differentiation indicated that EPI promoted cardiomyocyte differentiation from miPSCs. (E, F) Quantitative RT‐PCR analysis of the cardiac mesoderm marker, Brachyury (T) (E), and the late phase cardiomyocyte marker, Tnnt2 (F), in vehicle control‐ or EPI‐treated EBs. (G–J) Protein expression levels of cardiac troponin‐T (cTnT), α‐actinin and Nkx2.5, as measured by western blot analysis, in vehicle control‐ or EPI‐treated EBs at day 15 post‐differentiation. Results are expressed as mean±SEM, n=3. **P<0.01 relative to control

To further characterize the time course of cardiomyocyte differentiation after EPI treatment, we examined the gene expression profiles of other cardiac‐specific genes, including cardiac myofilament protein T (Tnnt2), NK2 transcription factor‐related locus 5 (Nkx2.5), GATA‐binding protein 4 (GATA4), and atrial and ventricular transcripts of myosin light chain 2 (MLC2a, and MLC2v), with semi‐quantitative RT‐PCR and real‐time PCR analysis. EPI treatment significantly increased the expression of all of the above cardiac‐specific markers (Fig. 2C,D). Because the cardiac mesoderm is important for cardiomyocyte differentiation, we also characterized the expression profile of the cardiac mesoderm marker, Brachyury (T), during the process of cardiac differentiation. EPI not only increased the expression of the late phase cardiomyocyte‐specific marker, Tnnt2, but also that of the mid‐phase cardiomyocyte‐specific marker, Brachyury (T) (Fig. 2E,F). Western blot analysis of the expression of cardiac troponin‐T (cTnT), the cardiac‐specific cytoskeletal protein, α‐actinin and Nkx2.5 revealed that these proteins were expressed 2‐ to 4‐fold higher in EPI‐treated compared with vehicle control‐treated embryoid bodies at day 15 post‐differentiation (Fig. 2G–J).

Next, we examined the sarcomeric organization of iPSC‐CMs in response to EPI treatment by double‐immunostaining cells with antibodies against cardiac‐specific proteins, cTnT, MLC2v and α‐actinin, and the gap junction protein, Cx43, at day 15 post‐differentiation. Cross‐striated myofilaments were better organized in miPSC‐derived cardiomyocytes treated with EPI than in control cardiomyocytes that were not treated with EPI, further validating that EPI treatment augments not only the cardiomyocyte population derived from iPSC enhanced but also sarcomeric organization and structural maturation of miPS‐CMs. (Fig. 3).

Figure 3.

Figure 3

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Epinephrine affects the sarcomeric organization of miPSC‐CMs. Vehicle control‐ or EPI‐treated cells were double immunostained for MLC2v (A), α‐actinin (B) or Cx43 (C) (green) and cTnT (red) to visualize the sarcomeric organization of cells at day 15 post‐differentiation. Nuclei in the same field were counterstained with DAPI (blue). Scale bar=20 μm

3.3. EPI enhances cardiomyocyte differentiation of miPSCs primarily through α‐AR signalling

Because both α‐ and β‐ARs are expressed on miPSCs, we investigated whether either α‐ARs, β‐ARs or both were required for EPI‐enhanced cardiomyocyte differentiation from miPSCs. Signalling through α‐ and β‐ARs was selectively inhibited by treating cells with phentolamine (α‐ARs antagonist) or propranolol (β‐ARs antagonist) on days 0–7 post‐differentiation. Both α‐ and β‐AR signalling were also blocked by adding both phentolamine and propranolol together. Treatment with the selective α‐AR antagonist, phentolamine, abolished the increase in cTnT protein expression normally induced by EPI, whereas treatment with the β‐AR antagonist, propranolol, only diminished the increase in cTnT protein expression induced by EPI (Fig. 4A). The importance of α‐ and β‐AR signalling to EPI‐dependent cardiomyocyte differentiation was further validated by the reduced expression of cardiac‐specific genes, Tnnt2, Nkx2.5, MLC2a and MLC2v, following treatment with phentolamine and propranolol, respectively (Fig. 4B–F). Interestingly, blocking α‐ARs resulted in greater inhibition of EPI‐enhanced cardiomyocyte differentiation than did blocking β‐ARs. Blocking both α‐ and β‐ARs resulted in greater inhibition of EPI‐enhanced cardiac‐specific gene and cTnT protein expression than did blocking either α‐ and β‐AR alone.

Figure 4.

Figure 4

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Adrenergic receptors are required for epinephrine‐enhanced cardiomyocyte differentiation from miPSCs. (A) Protein expression levels of cTnT in EB cardiomyocytes at day 15 post‐differentiation treated with or without EPI in combination with or without phentolamine and propranolol, as assessed by Western blotting analysis. (B) Gene expression levels of Tnnt2, Nkx2.5, GATA4, MLC2a and MLC2v, as determined by semi‐quantitative RT‐PCR analysis. (C–F) Quantitative RT‐PCR analysis of Tnnt2, Nkx2.5, MLC2a and MLC2v gene expression in miPSC‐CMs at day 15 post‐differentiation. (G, H) Percentage of miPSC‐derived cTnT+ cardiomyocytes at day 15 post‐differentiation after treatment with EPI and specific AR subtype inhibitors, as determined by flow cytometry. α‐AR blocker group: treated with phentolamine; β‐AR blocker group: treated with propranolol; α+β‐AR blocker group: treated with phentolamine and propranolol. Results are expressed as the mean±SEM, n=3, *P<0.05, **P<0.01 vs. corresponding values

These results were further confirmed with FACS analysis of cTnT+ cells at day 15 post‐differentiation. Quantitative FACS analysis revealed that EPI treatment increased the percentage of cTnT+ cardiomyocytes from 6.6% in the control group to 31.7% in the EPI‐treated group, representing a 4.8‐fold increase in the percentage of cTnT+ cells induced. The increase in the percentage of cTnT+ cardiomyocytes induced by EPI was reduced after blocking either α‐ or β‐ARs (Fig. 4G and 4H, respectively), but to a greater extent after blocking α‐ARs (7.4% vs. 25.6%, respectively, Fig. 4G). Blocking both α‐ and β‐ARs further reduced the percentage of cTnT+ cardiomyocytes to 5.8% (P<0.01 vs. control group). These results indicate that although both α‐ and β‐ARs are involved in the differentiation of miPSCs into cardiomyocytes, α‐AR signalling is the primary signalling pathway promoting cardiac differentiation.

3.4. α1‐ARs are the primary AR subtype involved in EPI‐induced cardiomyocyte differentiation from miPSCs

To determine which, if any, α‐AR subtype was involved in the enhancement of cardiomyocyte differentiation induced by EPI, cells were treated with selective AR subtype agonists, including the α1‐AR agonist, phenylephrine (PE), the α2‐AR agonist, clonidine (CIO), and the β‐AR agonist, isoproterenol (ISO), on days 0–7 post‐differentiation. As shown in Fig. 5A–E, treatment with the α1‐ARs agonist, phenylephrine, induced the expression of cardiac‐specific proteins, including cTnT, α‐actinin, Nkx2.5 and GATA4, whereas treatment with the selective α2‐AR agonist, clonidine, did not. Although treatment with the selective β‐AR agonist, isoproterenol, resulted in increased cTnT and GATA4 protein levels compared with those with vehicle control treatment, they were significantly lower than those observed with PE treatment. In addition, quantitative real‐time PCR analysis was performed to quantify differences in the gene expression of the early cardiac‐specific marker, NKX2.5, and late phase cardiomyocyte markers, Tnnt2, MLC2v and MLC2a, among all groups. PE‐treated embryoid bodies expressed NKX2.5, Tnnt2, MLC2v and MLC2a at levels that were 2‐ to 4‐fold higher than those of the vehicle‐control group and significantly higher than those of the CIO and ISO groups (Fig. 5G,J). These results were further validated with flow cytometry. The percentage of cTnT+ cells was significantly increased by PE treatment compared with vehicle‐control, CIO or ISO treatment (27.7% vs. 6.8% vs. 6.7% vs. 11.8%, respectively, Fig. 5K,L). However, the enhancement of cardiomyocyte differentiation by AR subtype‐specific agonists was significantly less than by EPI. These results indicate that α1‐ARs are the primary AR subtype involved in EPI‐enhanced cardiomyocyte differentiation, although β‐ARs play a minor but significant role.

Figure 5.

Figure 5

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α1‐ARs are the primary AR subtype responsible for epinephrine‐enhanced cardiomyocyte differentiation. (A–E) Representative immunoblots show the expression of cardiac‐specific proteins, including cTnT, α‐actinin, Nkx2.5 and GATA4. (F–I) Quantitative RT‐PCR analysis of cardiac markers after treatment with vehicle control, PE, ClO or ISO. Expression of cardiac markers is increased after PE treatment compared with after vehicle control, CIO or ISO treatment. (J, K) Percentage of cTnT + cardiomyocytes in miPSC‐derived EBs at day 15 post‐differentiation after treatment with specific AR subtype agonists. PE, α1‐AR agonist, phenylephrine; ClO, α2‐AR agonist, clonidine; ISO, β‐AR agonist, isoproterenol. Results are expressed as the mean±SEM, n=3, *P<0.05, **P<0.01 vs. corresponding values

3.5. Enhancement of cardiomyocyte differentiation by AR activation requires MEK‐ERK1/2 signalling

Previous studies have indicated that both α1‐ and β‐ARs can activate ERK signalling upon stimulation with catecholamines.34, 36 To determine whether the effects of EPI‐ and α1‐AR on cardiomyocyte differentiation require MEK‐ERK1/2 signalling, the phosphorylation status of MEK and ERK1/2 were examined by Western blotting analysis after stimulation with the AR agonists, EPI, PE, CIO or ISO, at day 5 post‐differentiation. Stimulation with EPI and PE induced significant ERK phosphorylation/activation, whereas stimulation with the β‐AR agonist, isoproterenol (ISO), induced weaker MEK‐ERK1/2 phosphorylation/activation. Stimulation with the α2‐AR agonist, clonidine, did not activate MEK‐ERK1/2 (Fig. 6A,B). These results demonstrate that MEK‐ERK1/2 activation by EPI results primarily from α1‐AR signalling.

Figure 6.

Figure 6

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Epinephrine‐enhanced cardiomyocyte differentiation requires MEK‐ERK1/2 signalling. (A, B) Relative levels of phosphorylated MEK (A) and ERK1/2 (B) in EBs at day 5 post‐differentiation treated with or without EPI, the α1‐AR agonist, phenylephrine (PE), the α2‐AR agonist, clonidine (ClO) and the β‐AR agonist, isoproterenol (ISO), respectively, as determined by Western blot analysis. (C, D) MEK and ERK1/2 phosphorylation levels in cells treated with PD98059 (PD) were measured by Western blot analysis. (E, F) Expression levels of cTnT (E) and GATA4 (F) in miPSC‐derived cardiomyocytes at day 15 post‐differentiation after treatment with the small‐molecule MEK inhibitor, PD98059. Results are expressed as mean±SEM, n=3, *P<0.05, **P<0.01 vs. corresponding values

The critical role of MEK‐ERK1/2 signalling to cardiomyocyte differentiation was further validated by inhibiting MEK‐ERK1/2 signalling with a small‐molecule inhibitor of MEK, PD98059. As shown in Fig. 6, MEK‐ERK1/2 signalling was activated by PE treatment, and this effect was suppressed by pretreatment with PD98059. Importantly, inhibition of MEK‐ERK1/2 signalling prevented the increased expression of cardiac‐specific proteins, such as cTnT and α‐actinin (Fig. 6E,F), by PE, confirming that MEK‐ERK1/2 signalling involves in EPI‐ and α1‐AR‐dependent augment of cardiomyocyte population from miPSCs.

3.6. α1‐AR‐dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK‐ERK1/2 pathway

Next, we attempted to elucidate further why activation of MEK‐ERK1/2 by α1‐AR promotes cardiac differentiation of miPSCs. It has already been shown that α1‐AR participates in heart development and function and affect cell proliferation by ERK signalling.25 However, whether MEK‐ERK1/2 activation mediated by α1‐AR affects proliferation of iPSC‐CMs is unclear. We evaluated the proliferation of day‐15 miPS‐CMs with double immunostaining of cTnT/BrdU. As shown in Fig. 7A, the percentages of cells double‐positive for cTnT/BrdU were not significantly different across all groups. This result suggests that activation of α1‐AR did not affect the proliferation of cardiomyocytes. As the effect of PE is exerted over days 0–7, a crucial time for CPC (cardiac progenitor cell) specification, we then investigated the proliferation of CPCs by Nkx2.5/BrdU double staining of cells at day 7. As shown in Fig. 7C–E, PE‐treated cells showed a markedly increased proportion of both Nkx2‐5+ and proliferating BrdU+/Nkx2‐5+ cells, suggesting that PE induced the proliferation of CPCs. The PE‐promoting effects were fully abrogated by PD98059, suggesting that the MEK‐ERK1/2 pathway is involved in the α1‐AR‐dependent proliferation of CPCs. We also examined apoptosis via TUNEL staining of cells using FACS analysis and found that the apoptotic index of the PE group was not affected compared with the vehicle‐treated group, whereas stimulation with PD98059 increased apoptosis relative to the vehicle‐treated control group. Taken together, these data indicate that α1‐AR‐dependent proliferation is restricted in CPCs via the MEK‐ERK1/2 pathway.

Figure 7.

Figure 7

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α1‐AR causes the proliferation of CPCs and cell‐cycle alterations in a MEK‐ERK1/2 pathway. (A) Immunostaining of cTnT and BrdU in day‐15 iPS‐CMs. Data were quantified from three random fields. Scale bars=50 μm. Nuclei were counterstained with DAPI. (B) TUNEL staining analysis showed apoptotic indexes of miPSC at differentiation day 15. (C‐E) Double staining of Nkx2‐5 and BrdU at day 7. (F, G) TUNEL staining analysis showed apoptotic indexes of miPSC at differentiation day 7. (H‐J) Cell‐cycle assessment by flow cytometry at differentiation for 24 h. Results are expressed as mean±SEM, n=3, *P<0.05, **P<0.01 vs. corresponding values

3.7. α1‐AR regulates miPS cell‐cycle progression

It is long known that stem cell differentiation correlates with a lengthening of the cell cycle, in particular G1.37 Whether α1‐AR can influence the cell cycle and promote differentiation of miPS cells is unclear. To address this question, we analysed the cell‐cycle profile in miPSCs pretreated with PE and PD98059 for 24 h using propidium iodide (PI) staining. It is notable that the fraction of G1 cells increased after stimulation with PE, and the S fraction also decreased. In contrast, when the cells were treated with PD98059, cell cycling was is remodelled, as shown by the increased proportion of cells in the S stage and decreased proportion of cells in the G1 stage (Fig. 7H–J). Taken together, these results suggest that α1‐AR potentially permits the initiation of myocardial differentiation by modulating miPS cell cycle in a MEK‐ERK1/2‐dependent mechanism

4. Discussion

In this study, we investigated the role of α‐ and β‐adrenergic receptors in the differentiation of cardiomyocytes from miPSCs in vitro. Our findings indicate that: (i) treatment with the AR agonist, EPI, robustly enhances cardiomyocyte differentiation from miPSCs; (ii) EPI regulates cardiomyocyte differentiation primarily through α1‐AR signalling; (iii) EPI‐ and α1‐AR‐dependent cardiomyocyte differentiation from miPSCs by specifically promoting the proliferation of CPCs via the MEK‐ERK1/2 pathway and regulating miPS cell‐cycle progression. Our study provides new insight into the various mechanisms that promote the differentiation of iPSC‐derived cardiac lineages and implicates α‐AR subtype in cardiac differentiation.

Cardiovascular diseases are the most frequent cause of death in adults and main non‐infectious cause of death in children around the world. Due to the limited capacity of adult cardiomyocytes (CMs) to proliferate, cell losses of failing heart are irreversible and strategies for replacement of damaged cells by various types of exogenous cells are being developed.38 Patient‐specific origin of pluripotent iPSCs represents an attractive source of clinically useful CMs because it is easily accessible and expandable in culture, and has broad developmental potential and high capacity to reproducibly differentiate into spontaneously beating cardiac cells in vitro.39 The major effort of many investigators in recent years has been to develop methods for enrichment of iPS cell‐derived CMs in vitro in order to improve their yield, purity and safety.21, 40 However, more data are required to establish safety, efficacy and long‐term behaviour of iPSC‐derived grafts.

ARs are indispensable for the regulation of adult heart function and several other physiological processes, including inotropy, chronotropy, bronchodilation, vasoconstriction, sedation and analgesia in combination with other neuronal and hormonal systems. In particular, the β‐ARs, the predominant adrenergic receptors subtype (about 90% of total adrenergic receptors in the heart), are primarily responsible for the sympathetic regulation of both cardiac chronotropy and inotropy.41, 42 In addition, α‐ARs play a role in the regulation of physiological hypertrophy, survival signalling, contractility and ischaemic preconditioning. Among the α‐AR subtypes, G‐protein‐coupled α1‐ARs are the predominant subtype expressed in the myocardium of most species. In the cardiovascular system, α1‐AR signalling exerts important protective effects through the activation of cellular growth pathways, which result in hypertrophy and proliferation.25 Treatment with the canonical α1‐AR agonist, PE, has robust and specific effects on a wide range of hypertrophy markers, such as cell size and volume, ANF expression, sarcomere alignment and protein/DNA ratio.43 α2‐AR subtypes lower sympathetic outflow and blood pressure and are essential for autocrine feedback inhibition of catecholamine secretion from the adrenal gland.44

In addition to ARs roles in adult cardiac function, accumulating evidence suggests that ARs are essential for cardiac development.26 The most studies about cardiac development and differentiation approaches to date are those focusing on the regulation of β‐ARs. It is also demonstrated that β‐ARs are vital for cardiac differentiation of mouse embryonic stem cells.45 Less is known about cardiac α‐ARs, but studies from the last 30 years indicate that long‐term activation of cardiac α1 ‐ARs activates beneficial trophic signalling in the developing heart and that these α1‐AR‐mediated trophic effects in the adult, in many ways, counteract the negative effects of overstimulation of β1‐ARs in heart failure. Here, we demonstrate, for the first time, the conclusive and universal role of ARs in cardiac differentiation of miPSCs and provide novel insight into the underlying mechanism. Our results indicate that EPI regulates cardiomyocyte differentiation primarily through α1‐AR signalling, although β‐AR signalling is also involved.

The MEK‐ERK1/2 plays an important role in cardiac lineage commitment.46, 47 Extracellular signal‐regulated kinases (ERKs) are now one of the most widely studied signalling pathways in cellular biology. At the cellular level, ERK1/2 regulates cell‐cycle progression, proliferation, cytokinesis, transcription, differentiation, senescence, cell death, migration, GAP junction formation, actin and microtubule networks, and cell adhesion.48 Previous studies have indicated that both α1‐ARs and β‐ARs activate ERK upon stimulation with catecholamines.49 Similarly, Glennon et al. demonstrated that ERK signalling is required for α1‐AR‐mediated survival signalling and hypertrophy.50 Furthermore, in neonatal rat ventricular myocytes, inhibition of ERK activation with the inhibitor, PD98059, prevented the effects of α1‐AR activation on myofibrillar organization and c‐fos gene expression.36, 51 Our study confirmed that both α1‐ARs and β‐ARs are implicated in promoting MEK‐ERK activation and discovered that the α1‐ARs are the primary ARs responsible for MEK‐ERK1/2 activation. PE elevated the expression of MEK‐ERK1/2 phosphorylation, while specific MEK inhibitor PD98059 could constrain such promotion of expression, suggesting that MEK‐ERK1/2 signalling is required for EPI‐ and α1‐AR‐dependent cardiomyocyte differentiation from miPSCs.

The most successful cardiac differentiation approaches to date are those focusing on the induction of CPCs. iPSC‐derived multipotent CPCs, which possess higher proliferation capacity and can differentiate into multiple cardiac lineages, might offer an advantage over cardiomyocytes, as they contribute to both muscularization and vascularization. However, one of the major limitations for their utilization is the difficulty of expansion. Here, we observed that α1‐AR not only increased the fraction of iPSC‐derived CPCs but also specifically promoted their proliferation via the MEK‐ERK1/2 pathway, further confirming the importance of manipulating CPCs to guide efficient cardiac differentiation.

The cell cycle has been found to influence cell fate decisions, and a correlation between differentiation and cell‐cycle lengthening has been reported in several cell lineages in vitro and in vivo.37 PSCs exhibit an unusual mode of cell‐cycle regulation with a truncated G1 and a large percentage of S phase cells.52 As PSCs differentiate, the cell cycle is remodelled, such that G1 is lengthened and the relative amount of time associated with S phase cells is reduced.53 Recent reports further suggested that the extending the S and G2 phases of the hES cell cycle may dominantly impede pluripotent state dissolution and maintain ESC identity, whereas the presence of G1 phase potentially permits the initiation of differentiation.54 Together, these studies point towards a direct relationship between the cell cycle and differentiation, consistent with earlier reports describing the ability of PSCs to initiate their differentiation programme from G1 phase.53 Mek/Erk signalling plays a pivotal role in pluripotency maintenance. Inhibition of Mek/Erk signalling constrains the differentiation of mouse ESCs.55 Mouse ESCs can be derived and maintained in medium supplemented with inhibitors of Mek and Gsk3 signalling.56 Similarly, the Mek inhibitor PD98059 is used in establishing and maintaining human ground‐state pluripotent stem cells.57, 58 In our study, PE was demonstrated to stimulate the differentiation of miPSCs by significant enrichment of cells in the G1 phase and also decrease the fraction of cells in the S phase, while specific MEK inhibitor PD98059 could reverse the effect of PE, suggesting that α1‐AR potentially permits the initiation of myocardial differentiation by extending the G1 phases of the miPS cell cycle in a MEK‐ERK1/2‐dependent mechanism.

In conclusion, we demonstrated that epinephrine promotes cardiac differentiation from miPSCs primarily through α1‐AR and MEK‐ERK1/2 signalling. The results of our study suggest that activation of α1‐ARs or MEK‐ERK1/2 signalling may represent complementary strategies to promote the differentiation of miPSCs into cardiac lineages, which will contribute to the establishment of a highly efficient and facile differentiation programme to obtain functional cardiomyocytes for iPSC‐based cardiovascular regenerative medicine.

Conflicts of interest

The authors declare no conflicts of interest.

Supporting information

  

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Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (no. 31271039 and 31400832). We thank Prof. Duanqing Pei from Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (GIBH), for providing murine iPSC line. We also thank members of the present stem cell research group for their camaraderie, collegiality and passion.

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