Secreted frizzled related protein 1 protects H9C2 cells from hypoxia/re-oxygenation injury by blocking the Wnt signaling pathway

Abstract

Background: In animal models, secreted frizzled related protein 1 (Sfrp1) inhibition of the Wnt signaling pathway is beneficial because Sfrp1 reduces myocardial apoptosis and prevents heart failure. The mechanisms mediating the cellular survival effect of Sfrp1 has not been completely elucidated. The present study was designed to investigate the possible protective actions of Sfrp1 on cardiac muscle cells using an in vitro model of ischemia/reperfusion, and to evaluate the possible involvement of the Wnt signaling pathway.

Methods: We used a recombinant AAV9 vector to deliver the Sfrp1 gene into H9C2 rat cardiomyoblasts and adopted an in vitro model of ischemia/reperfusion. Cell vitality was measured by CKK-8 and the trypan blue exclusion assay. Western blot was used to evaluate the expression of Dvl-1, β-catenin, c-Myc, Bax, and Bcl-2. Flow cytometry analysis of cardiomyocyte apoptosis was performed.

Results: We confirmed that Sfrp1 significantly increased cell viability (assayed by trypan blue and CKK-8) and decreased apoptosis (assayed by flow cytometry analysis and the Bax/Bcl-2 ratio). These effects were partly attributable to the ability of Sfrp1 to down-regulate Wnt signaling pathway (assayed by Western blot to evaluate the expression of Dvl-1, β-catenin, and c-Myc). Indeed, reactivation of the Wnt signaling pathway activity with the specific activator, Licl, reduced Sfrp1-induced cardioprotection during hypoxia and reoxygenation.

Conclusions: The present study demonstrated that Sfrp1 directly protected H9C2 cells from hypoxia and reoxygenation-induced reperfusion injury and apoptosis through inhibition of the Wnt signaling pathway, and added new mechanistic insight regarding the cardioprotective role of Sfrp1 on ischemic damage.

Keywords: Apoptosis; H9C2; Hypoxia reoxygenation injury; Sfrp1; Wnt signaling pathway.

Fig. 1

RT-PCR showing Sfrp1 mRNA in H9C2 cells

Fig. 2

Evaluation of H9C2 cell viability by CKK-8 (a) and trypan blue exclusion (b) assay. Hypoxia and reoxygenation (H+R) cause a marked reduction in the number of viable cells. This effect was antagonized by Sfrp1-transfected cells before hypoxia and reoxygenation (Sfrp1+H+R). The cytoprotective effects of Sfrp1 were significantly reduced by the Wnt signaling pathway activator, Licl. Median values with interquartile range (box) and range (whiskers). Post-hoc analyses of group differences were assessed with Kruskal-Wallis pairwise comparison. Statistical significance was set at the standard level (p <0.05), and the Bonferroni correction was applied in post-hoc analyses (α = 0.05/6 = 0.0083). (Note:* vs. control P <0.001; # vs. H+R P <0.001; vs. Sfrp1+H+R P <0.001)

Fig. 3

a Western blot shows that the expression of the anti-apoptotic protein, Bcl2 was reduced and Bax was enhanced by hypoxia and reoxygenation (H+R). These changes were antagonized by Sfrp1-transfected cells before hypoxia and reoxygenation (Sfrp1+H+R). The Wnt signaling pathway activator, Licl, reduced the effects of Sfrp1. b Median values with interquartile range (box) and range (whiskers). Post-hoc analyses of group differences were assessed with Kruskal-Wallis pairwise comparison. Statistical significance was set at the standard level (p<0.05), and the Bonferroni correction was applied in post-hoc analyses (α=0.05/6=0.0083). (Note:* vs. control P<0.001; # vs. H+R P<0.001; vs. Sfrp1+H+R P<0.001)

Fig. 4

AnnexinV-FITC/PI double staining flow cytometry to detect the apoptosis rate. The apoptosis rates of H9C2 cells were increased by hypoxia and reoxygenation (H+R). These changes were antagonized by Sfrp1-transfected cells before hypoxia and reoxygenation (Sfrp1+H+R). The Wnt signaling pathway activator, Licl, reduced the effects of Sfrp1. a Control group. b H+R group. c Sfrp1+H+R group. d Sfrp1+H+R+Licl group. e Median values with interquartile range (box) and range (whiskers). Post-hoc analyses of group differences were assessed with Kruskal-Wallis pairwise comparison. Statistical significance was set at the standard level (p<0.05), and the Bonferroni correction was applied in post-hoc analyses (α=0.05/6=0.0083). (Note:* vs. control P<0.001; # vs. H+R P<0.001; vs. Sfrp1+H+R P<0.001) (Additional files 1, 2, 3 and 4)

Fig. 5

a Western blot detected the expression of Wnt signaling pathway key molecules. Hypoxia and reoxygenation (H+R) caused a marked increase in the protein levels of Dvl, β-catenin, and c-Myc. These changes were antagonized by Sfrp1-transfected cells before hypoxia and reoxygenation (Sfrp1+H+R); however, pre-treatment with the Wnt signaling pathway activator, Licl, increased the expression of Dvl, β-catenin, and c-Myc. b The median value of Dvl/GADPH with interquartile range (box) and range (whiskers). c The median value of β-catenin/GADPH with interquartile range (box) and range (whiskers). d The median value of c-Myc/GADPH with interquartile range (box) and range (whiskers). Post-hoc analyses of group differences were assessed with Kruskal-Wallis pairwise comparison. Statistical significance was set at the standard level (p<0.05), and the Bonferroni correction was applied in post-hoc analyses (α=0.05/6=0.0083). (Note:* vs. control P<0.001; # vs. H+R P<0.001; vs. Sfrp1+H+R P<0.001)