Wnt/β-catenin pathway induces cardiac dysfunction via AKAP6-mediated RyR2 phosphorylation and sarcoplasmic reticulum calcium leakage

Figures

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  Figure 1. Activation of the Wnt/β-catenin pathway disturbs calcium homeostasis in HL-1 cells. (A) Confocal images of HL-1 cells treated with or without Wnt3a for 6 h. Fluo4-AM dye and R-CEPIA1er are used to indicate cytosol and SR calcium signals, respectively. Fluorescence intensity (F.I.) was calculated to represent the calcium level. (B–E) Cytosol and SR calcium levels in HL-1 cells treated with the indicated compounds for 6 h. Wnt3a: 500 ng/ml; LiCl: 20 mM; CHIR: 5 μM; SFRP2: 10 μg/ml; XAV939: 1 μM; LF3: 1 μM; Wnt5a: 500 ng/ml; Box5: 10 μM; KN62: 1 μM. Tg (100 nM) was used as a positive control. (F and G) HL-1 cells preincubated with SFRP2 (10 μg/ml) for 15 min could eliminate the effects of canonical Wnt activators on cytosol and SR calcium levels. Error bars indicate SD (n = 6).

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  Figure 2. Downstream gene expression is essential for the canonical Wnt pathway affecting calcium homeostasis in HL-1 cells. (A and B) Cytosol and SR calcium levels in HL-1 cells treated with 5 μM CHIR for 5 min and 6 h. (C and D) Cytosol and SR calcium levels in HL-1 cells preincubated with H2O or cycloheximide (CHX, 1 μg/ml) for 15 min before 5 μM CHIR treatment for 6 h. Error bars indicate SD (n = 6).

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  Figure 3. AKAP6 is a target gene of the Wnt/β-catenin pathway. (A) Top 20 enriched pathways of KEGG pathway analysis for HL-1 cells treated with CHIR vs. DMSO for 6 h. The Wnt signaling pathway was highly activated (red frame). (B) Top 20 differentially expressed genes for HL-1 cells treated with CHIR vs. DMSO for 6 h. AKAP6 (red frame) was increased by 2.40-fold with a P-value < 0.0001. (C) Schematic illustration of potential TBEs in mouse AKAP6 promotor. TBE sequence: SCTTTGWWS. (D) Luciferase reporter assay for full-length AKAP6 promotor (AP) and truncated AKAP6 promotors (deletion of TBE1 in AP: TBEd1; deletion of TBE2 in AP: TBEd2; deletion of both TBE1 and TBE2 in AP: TBEd1&2). Error bars indicate SD (n = 6).

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  Figure 4. AKAP6 is important for the canonical Wnt pathway affecting cardiac calcium homeostasis via RyR2. (A–C) HL-1 cells were transfected with shAKAP6 (shAKAP6-1 and shAKAP6-2) or empty vector constructs and selected under 1000 μg/ml of G418. (A and B) Cytosol and SR calcium of the cells treated with 5 μM CHIR for 6 h. (C) Relative mRNA level of AKAP6 in the indicated cells. Error bars indicate SD (n = 6). (D–F) Western blot analysis of RyR2 and RyR2 Ser2808 phosphorylation levels in HL-1 cells transfected with mouse AKAP6 or treated with 5 μM CHIR for 6 h. Error bars indicate SD (n = 3). (G and H) Cytosol and SR calcium levels in HL-1 cells preincubated with tetracaine (1 μM) for 15 min before CHIR (5 μM) treatment for 6 h. Error bars indicate SD (n = 6).

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  Figure 5. Inhibition of canonical Wnt signaling or knockdown of AKAP6 could rescue ISO-induced cardiac cell dysfunction in HL-1 cells. (A–D) HL-1 cells were incubated with ISO (20 μM) for 72 h. (A) Relative mRNA levels of cardiac fibrosis marker genes ANP, BNP, and collagen 1. (B and C) Western blot analysis of GSK3β and phosphorylated GSK3β levels. (D) Relative mRNA levels of c-Myc, Axin2, and AKAP6. Error bars indicate SD (n = 3). (E and F) Cytosol and SR calcium levels in HL-1 cells or AKAP6 knockdown cells treated with or without ISO (20 μM) for 72 h, followed by SFRP2 (10 μg/ml) or CHIR (5 μM) treatment for 6 h. (G) Relative mRNA levels of ANP, BNP, and collagen 1 in ISO-induced HL-1 cells transfected with shAKAP6 or treated with SFRP2 for 6 h. Error bars indicate SD (n = 6).

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  Figure 6. SFRP2 knockout causes SR calcium leakage and cardiac fibrosis through AKAP6. (A) Relative mRNA (n = 6) and protein (n = 3) levels of SFRP2. (B) Relative mRNA levels of CCND1 and c-Myc (n = 6). (C and D) Western blot analysis of GSK3β and phosphorylated GSK3β levels (n = 3). (E) Relative mRNA levels of ANP, BNP, and collagen 1 (n = 6). (F) HE stain and Masson stain of mouse left ventricular tissues. Scale bar, 100 μm. (G) Relative mRNA levels of AKAP6 (n = 6). (H and I) Cytosol and SR calcium levels in primary cardiomyocytes of control or SFRP2−/− mice (n = 6). Error bars indicate SD.

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  Figure 7. Inhibition of canonical Wnt signaling could block TAC-induced heart fibrosis and dysfunction in mice. (A) HE stain. Scale bar, 100 μm. (B and C) Masson stain and quantitation for healthy fraction (ratio of red area/blue area). Scale bar, 100 μm. Error bars indicate SD (n = 3). (D–G) IHC of MMP9 and collagen 1 (Col1) and the relevant H-scores (higher H-score indicates higher fibrosis level). Scale bar, 100 μm. Error bars indicate SD (n = 5). (H) Echocardiography for EF, FS, LVID.d, and LVID.s. (I) Relative mRNA levels of c-Myc, CCND1, AKAP6, RyR2, and SERCA2a. Error bars indicate SD (n ≥ 3).