Ghrelin promotes cardiomyocyte differentiation of adipose tissue‑derived mesenchymal stem cells by DDX17‑mediated regulation of the SFRP4/Wnt/β‑catenin axis

Abstract

Adipose tissue‑derived mesenchymal stem cells (ADMSCs) differentiate into cardiomyocytes and may be an ideal cell source for myocardial regenerative medicine. Ghrelin is a gastric‑secreted peptide hormone involved in the multilineage differentiation of MSCs. To the best of our knowledge, however, the role and potential downstream regulatory mechanism of ghrelin in cardiomyocyte differentiation of ADMSCs is still unknown. The mRNA and protein levels were measured by reverse transcription‑quantitative PCR and western blotting. Immunofluorescence staining was used to show the expression and cellular localization of cardiomyocyte markers and β‑catenin. RNA sequencing was used to explore the differentially expressed genes (DEGs) that regulated by ghrelin. The present study found that ghrelin promoted cardiomyocyte differentiation of ADMSCs in a concentration‑dependent manner, as shown by increased levels of cardiomyocyte markers GATA binding protein 4, α‑myosin heavy chain (α‑MHC), ISL LIM homeobox 1, NK2 homeobox 5 and troponin T2, cardiac type. Ghrelin increased β‑catenin accumulation in nucleus and decreased the protein expression of secreted frizzled‑related protein 4 (SFRP4), an inhibitor of Wnt signaling. RNA sequencing was used to determine the DEGs regulated by ghrelin. Functional enrichment showed that DEGs were more enriched in cardiomyocyte differentiation‑associated terms and Wnt pathways. Dead‑box helicase 17 (DDX17), an upregulated DEG, showed enhanced mRNA and protein expression levels following ghrelin addition. Overexpression of DDX17 promoted protein expression of cardiac‑specific markers and β‑catenin and enhanced the fluorescence intensity of α‑MHC and β‑catenin. DDX17 upregulation inhibited protein expression of SFRP4. Rescue assay confirmed that the addition of SFRP4 partially reversed ghrelin‑enhanced protein levels of cardiac‑specific markers and the fluorescence intensity of α‑MHC. In conclusion, ghrelin promoted cardiomyocyte differentiation of ADMSCs by DDX17‑mediated regulation of the SFRP4/Wnt/β‑catenin axis.

Keywords: Wnt/β‑catenin signaling pathway; adipose tissue‑derived mesenchymal stem cell; cardiomyocyte differentiation; dead‑box helicase 17; ghrelin; secreted frizzled‑related protein 4.

Figure 1.

Ghrelin promotes cardiomyocyte differentiation of ADMSCs. (A) Protein expression and (B) quantification of cardiomyocyte markers in ghrelin (1, 5, 10, 50 and 100 nM)-treated ADMSCs. (C) Immunofluorescence staining of GATA4 (green) and α-MHC (red) in ADMSCs. Scale bar, 50 µm. Data are expressed as mean ± SD (n=3). *P<0.05, **P<0.01 vs. control; ^##P<0.01 vs. induction. ADMSC, adipose tissue-derived mesenchymal stem cell; GATA4, GATA binding protein 4; MHC, myosin heavy chain; ISL1, ISL LIM homeobox 1; Nkx2.5, NK2 homeobox 5; TNNT2, Troponin T2, cardiac type.

Figure 2.

Ghrelin promotes cardiomyocyte differentiation of ADMSCs via regulation of SFRP4/Wnt/β-catenin axis. (A) Protein expression and (B) immunofluorescence of β-catenin in ADMSCs. (C) Protein expression of SFRP4 in treated ADMSCs. Scale bar: 50 µm. Data are expressed as mean ± SD (n=3). **P<0.01 vs. control; ^##P<0.01 vs. induction. ADMSC, adipose tissue-derived mesenchymal stem cell; SFRP4, secreted frizzled-related protein 4.

Figure 3.

RNA-seq analysis and DEGs. (A) PCA of ADMSC RNA-seq data. (B) Hierarchical clustering analysis was performed for all DEGs. Induction and induction + ghrelin groups exhibited clustering. (C) Volcano map of DEGs. seq, sequencing; DEG, differentially expressed gene; PCA, principal component analysis; ADMSC, adipose tissue-derived mesenchymal stem cell; FC, fold-change.

Figure 4.

Functional enrichment analysis of DEGs. (A) GO and KEGG enrichment analysis of all DEGs. (B) DEGs in the MSCs differentiation and Wnt signaling pathway related GO terms. (C) DEGs in some classical signaling pathways of KEGG analysis. DEG, differentially expressed gene; MSCs, mesenchymal stem cells; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, biological process; CC, cellular component; MF, molecular function.

Figure 5.

Potential genes that regulated by ghrelin in the process of the cardiomyocyte differentiation of ADMSCs. (A) The mRNA expression of TRIP6, SOX30, TRIM36, DDX17, WNK1, TCF3 and CCN1 in the induction medium-cultured ADMSCs in the presence or absence of ghrelin. (B) Protein expression of DDX17 in ADMSCs. Data are expressed as mean ± SD (n=3). *P<0.05, **P<0.01 vs. induction. ADMSCs, adipose tissue-derived mesenchymal stem cells; TRIP6, thyroid hormone receptor interactor 6; SOX30, SRY-box transcription factor 30; TRIM36, tripartite motif containing 36; DDX17, Dead-box helicase 17; WNK1, WNK lysine deficient protein kinase 1; TCF3, transcription factor 3; CCN1, cellular communication network factor 1; ADMSC, adipose tissue-derived mesenchymal stem cell.

Figure 6.

DDX17 promotes cardiomyocyte differentiation of ADMSCs and β-catenin nuclear accumulation. (A) mRNA and (B) protein expression levels of DDX17 in DDX17 knockdown ADMSCs. DDX17 was knocked down in the induction medium-cultured ADMSCs. (C) Protein expression of GATA4 and α-MHC. (D) IF staining of α-MHC in treated ADMSCs. (E) Protein expression and (F) IF staining of β-catenin in ADMSCs. Scale bar, 50 µm. Data are expressed as mean ± SD (n=3). **P<0.01 vs. LV-Empty. DDX17, Dead-box helicase 17; ADMSC, adipose tissue-derived mesenchymal stem cell; GATA4, GATA binding protein 4; MHC, myosin heavy chain; IF, immunofluorescence; LV, lentivirus.

Figure 7.

DDX17, upregulated by ghrelin, promotes ADMSC cardiomyocyte differentiation by downregulating SFRP4. (A) Protein expression of SFRP4 in DDX17-overexpressing ADMSCs. **P<0.01 vs. LV-Empty. (B) Protein expression and (C) quantification of GATA4, α-MHC, ISL1, Nkx2.5 and TNNT2. (D) Immunofluorescence staining of α-MHC in ADMSCs. Scale bar, 50 µm. Data are expressed as mean ± SD (n=3). *P<0.05, **P<0.01 vs. control; ^##P<0.01 vs. induction; ^$$P<0.01 vs. induction + ghrelin. DDX17, Dead-box helicase 17; ADMSC, Adipose tissue-derived mesenchymal stem cell; SFRP4, secreted frizzled-related protein 4; LV, lentivirus; GATA4, GATA binding protein 4; MHC, myosin heavy chain; ISL1, ISL LIM homeobox 1; Nkx2.5, NK2 homeobox 5; TNNT2, Troponin T2, cardiac type.

Figure 8.

Role of ghrelin in promotion of ADMSCs cardiomyocyte differentiation. Ghrelin promotes differentiation of ADMSCs into cardiomyocyte via activation of the Wnt/β-catenin pathway. DDX17, an upregulated differentially expressed gene after ghrelin treatment, promotes nuclear translocation of β-catenin and inhibits protein expression of SFRP4. DDX17 is involved in ghrelin-induced cardiomyocyte differentiation of ADMSCs, which is associated with regulation of the SFRP4/Wnt/β-catenin axis. ADMSC, adipose tissue-derived mesenchymal stem cell; DDX17, Dead-box helicase 17; SFRP4, Secreted frizzled-related protein 4; GATA4, GATA binding protein 4; MHC, myosin heavy chain; Nkx2.5, NK2 homeobox 5; ISL1, ISL LIM homeobox 1; TNNT2, Troponin T2, cardiac type.