FRS2α-mediated FGF signals suppress premature differentiation of cardiac stem cells through regulating autophagy activity

Abstract

Rationale: Although the fibroblast growth factor (FGF) signaling axis plays important roles in heart development, the molecular mechanism by which the FGF regulates cardiogenesis is not fully understood.

Objective: To investigate the mechanism by which FGF signaling regulates cardiac progenitor cell differentiation.

Methods and results: Using mice with tissue-specific ablation of FGF receptors and FGF receptor substrate 2α (Frs2α) in heart progenitor cells, we demonstrate that disruption of FGF signaling leads to premature differentiation of cardiac progenitor cells in mice. Using embryoid body cultures of mouse embryonic stem cells, we reveal that FGF signaling promotes mesoderm differentiation in embryonic stem cells but inhibits cardiomyocyte differentiation of the mesoderm cells at later stages. Furthermore, we also report that inhibiting FRS2α-mediated signals increases autophagy and that activating autophagy promotes myocardial differentiation and vice versa.

Conclusions: The results indicate that the FGF/FRS2α-mediated signals prevent premature differentiation of heart progenitor cells through suppressing autophagy. The findings provide the first evidence that autophagy plays a role in heart progenitor differentiation.

Fig. 1. Double ablation of Fgfr1/r2 causes premature differentiation of SHF progenitor cells

A&B, Sagittal sections of E8.5 embryos were immunostained with anti-smooth muscle α-actin (SMA) and sarcomeric α-actin (Sar) antibodies. Nuclei were counter-stained with To-Pro3 iodine (TP3). Inserts in (B) were high magnification views of the boxed areas. SM, splanchnic mesoderm; OFT, outflow tract; f, floxed alleles; cn, conditional null alleles; Nkx, Nkx2.5^Cre.

Fig. 2. Ablation of Frs2α leads to premature differentiation of SHF progenitor cells

A&D. Sagittal sections of E8.5 embryos were immunostained with the antibody against SMA, Sar, and serum response factor (SRF). Numbers in panels c’ and f’ are fluorescent intensities quantitated by the Image J software, which represent SRF expression levels in the SM. B. Whole-mount immunostaining of E9.0 embryos with the MF20 antibody. C. Real-time RT-PCR analyses of cells isolated from E9.5 pharyngeal arches. Data are normalized to GAPDH and expressed as folds of changes over wildtype samples. D. Sagittal sections of E8.5 embryos were immunostained with anti-SMA or Sar antibodies as indicated. E. The migration activity of cells isolated from E8.75 pharyngeal arches were assessed with the Transwell assays. SHF cells were identified with X-Gal staining. The ratios of migrated SHF cells over total SHF cells were calculated from 3 individual samples and expressed as mean±sd. Mef, Mef2C^Cre; SKA, skeletal α-actin; CAA, cardiac α-actin; w, wildtype Frs2α allele; *, p≤0.05.

Fig. 3. SHF differentiation is independent of its proliferation activity and is negatively regulated by both MAP kinase and PI3K/AKT pathways

A&B. The embryo cultures were treated with 2 mM excessive thymidine. Sagittal sections were immunostained with anti-cyclin D2 (A) or MF20 antibodies (B) to demonstrate that cell cycle arrest did not affect SHF differentiation. C. The embryos were cultured in the presence or absence of 5 μM MEK or 3 μM PI3K inhibitors. Sagittal sections were immunostained with the indicated antibodies demonstrating that inhibition of MEK or PI3K induced SHF differentiation. -, DMSO as a solvent control; Mi, MEK inhibitor; Ai, PI3K/AKT inhibitor.

Fig. 4. The FGF suppresses cultured SHF progenitor cells differentiation

Cells isolated from E9.5 pharyngeal arches were cultured in the presence or absence of FGF2 (25 ng/ml), MEK inhibitor (5 μM), PI3K inhibitor (3 μM), or FGFR inhibitors (250 nM) as indicated, and then were immunostained with anti-Isl1 (A) or MF20 (B) antibodies as indicated. The positively stained cells are indicated with arrows. Statistical analyses of MF20 positive cells in three independent experiments were presented in (C) as mean±sd. Ri, FGFR inhibitor; *, P≤0.05

Fig. 5. The FGF signaling axis regulates cardiogenesis in a developmental stage-specific manner

A&B. Embryo bodies (EBs) were treated with FGFR inhibitor (250 nM) from day 0 to day 4, and the percentage of beating embryoid bodies (EBs) was counted and presented as means±sd of three independent experiments (A); expression of mesoderm markers and early cardiac markers was analyzed with RT-PCR at the indicated time points (B). C-E. EB cultures were treated with 25 ng/ml FGF2, the MEK inhibitor (5 μM), PI3K inhibitor (3 μM), or FGFR inhibitors (250 nM) at day 6 to day 10. The percentage of beating EBs were scored and presented as mean±sd from three independent experiments (C); expression of cardiac markers in day 10 EBs was assessed by Western blot (D) or real-time RT-PCR analyses (E). β-actin was used as a loading control; *, p<0.05.

Fig. 6. The FGF signaling axis inhibits ESCs undergoing myocardiac differentiation via suppressing autophagy

A. EB cultures of mouse ESCs were treated with 250 nM FGFR inhibitor from day 6. The abundance of LC3, Beclin 1, and P27 was analyzed by Western blot at day 7. B, EB cultures were treated with 100 nM rapamycin, 5 mM NH₄Cl, 5 nM bafilomycin A1, 250 nM FGFR inhibitor, 25 ng/ml FGF2 as indicated, or the indicated combinations, and the beating foci were scored at day 8. Data were mean±sd of three independent experiments. C. EB cultures were treated with 5 μM MEK inhibitor or 3 μM PI3K inhibitor from day 6. The abundance of LC3, Beclin 1, and P27 was assessed with Western blot (panels a&b), and the beating foci were counted from three independent experiments and expressed as mean±sd (panel c). D. EB cultures were treated with siRNA at day 6 and the cells were lysed at day 8. Expression of the indicated molecules were assessed by real-time RT-PCR analyses. E. E8.5 embryos were cultured in the presence or absence of 100 nM rapamycin, and the expression of SMA and myosin was analyzed with immunostaining. β-actin was used as a loading control; Rap, rapamycin; NH₄, NH₄Cl; BFA, bafilomycin A; *, p≤0.05; BECN1, Beclin 1.

Fig. 7. Ablation of Frs2α promotes autophagy in the OFT myocardium

A. Sagital sections of E9.5 Frs2α^cn/Nkx or Frs2α^f/f embryos expressing the GFP-LC3 transgene were immunostained with anti-GFP antibody. Data are confocal images demonstrating increased LC3 positive punctate foci that represented autophagosomes (arrows) in the Frs2α^cn/Nkx OFT myocardium. Average numbers of punctate foci per cell were presented as mean±sd in panel c. B. Frs2α^cn/Nkx/ or Frs2α^f/f/GFP-LC3 embryos from chloroquine injected dam were analyzed as in A, further demonstrating increase autophagy influx in Frs2α^cn/Nkx OFT myocardium. C. Double immunostaining of E9.5 wildtype/GFP-LC3 embryos demonstrating that only a fraction of autophagosomes contained mitochondria (arrows).

Fig. 8. The FRS2α-mediated signals in the myocardium promote proliferation and suppress maturation

A. E12.5 embryos were subjected to immunostaining analyses with anti-phosphorylated histone H3 and MF20 antibodies. Statistical data from three independent experiments were presented in panel c. Nuclei were counterstained with To-Pro3. Inserts were high magnification views of the boxed areas. B. Heart sections of E14.5 embryonic were subjected to H&E staining. The average thickness of compact zones of the ventricle from 5 individual hearts was shown in panel c. C. Sections from E12.5 embryos were immunostained with anti-Sar and MF20 antibodies. Nuclei were counterstained with To-Pro3. D. Western blot analyses of E12.5 wildtype or Frs2α^cn/Nkx hearts showed increased expression of Sarcomeric actin and myosin in mutant hearts. β-actin was used as a loading control. E&F. E11.5 heart cultures from Frs2α floxed (E), or indicated genotypes embryos were treated with 100 nM rapamycin or 5 mM NH₄Cl as indicated, and total RNAs were extracted for real-time RT-PCR analyses. Data were normalized with GAPDH and expressed as folds of changes from control samples. G. Cardiac mesenchymal cells isolated from neonatal hearts were cultured in the presence or absence of FGF2. The cells were then immunostained with anti-SMA antibodies. Nuclei were counterstained with hematoxylin. Average ratios of SMA positive cells from three individual experiments were shown in panel c as means±sd. MHC, myosin heavy chain; *, p≤0.05.