Fig 1.
Glypican-6 expression is increased in the failing mouse heart.
Schematic of the aortic banding (AB) heart failure model with heart regions indicated (A). Chronic pressure overload was induced in adult mice by banding of the ascending aorta. RA: right atrium, RV: right ventricle, LA: left atrium, LV: left ventricle. Relative LV mRNA levels of GPC1 (B), GPC2 (C), GPC3 (D), GPC4 (E) and GPC6 (F) after 24h, 1, 3, 16 and 18 weeks of AB or sham-operation in male mice (n = 3–10). See Table 1 for animal characteristics. mRNA expression was normalized to ribosomal protein L32 (RPL32) expression. Representative immunoblots and quantitative data of full length GPC6 (GPC6FL; Mw ≈62kDa) in LV protein lysates from AB- and sham-operated control mice analyzed under reducing conditions (+ dithiothreitol). For immunoblotting of heparan sulfate (HS) proteoglycans in tissue, proteoglycans were methanol (MetOH) precipitated prior to digestion with heparan sulfate degrading enzymes [8, 36](G and H; n = 3 at all time-points). Recombinant human GPC6 produced in E.coli was used as positive control (Rec.hGPC6). Data are presented as mean ± S.E.M. Unpaired Student’s t-test was used for statistical testing vs. controls at respective time-points. *P<0.05; **P<0.01; ***P<0.001. Pearson correlations of LV GPC6/RPL32 mRNA vs. LV weight/tibia length (TL)(I), ACTA1/RPL32 mRNA (J), lung weight/TL (K) and NPPA/RPL32 mRNA (L) in AB- and sham-operated mice (24h-18 weeks).
Table 1.
Characterization of mice subjected to aortic banding.
Fig 2.
Glypican-6 expression is upregulated in the failing human heart.
Relative GPC6 mRNA levels in left ventricular biopsies from end-stage heart failure patients (n = 18) compared to controls (n = 7) (A). See Table 2 for patient characteristics. GPC6 mRNA levels were normalized to ribosomal protein L32 (RPL32) expression and data presented as individual data points. Unpaired Student’s t-test was used to test for statistical significance. Pearson correlation between left ventricular GPC6 mRNA level and ejection fraction (EF) in end-stage heart failure patients (n = 18) (B).
Table 2.
Characterization of patients with end-stage heart failure.
Fig 3.
Glypican-6 is mainly produced by cardiac fibroblasts in the heart.
Relative GPC6 mRNA in rat heart neonatal fibroblasts (NFB) and cardiomyocytes (NCM; A), n = 13–15 from three separate cell cultures. Immunoblot and quantification of the N-terminal GPC6 (GPC6N; Mw ≈35kDa) from NFB and NCM cell lysates analyzed under reducing conditions (+ dithiothreitol; B and C), n = 3. Recombinant human GPC6 produced in E.coli was used as positive control (Rec.hGPC6). Immunoblot of glycanated GPC6 (GPC6glyc; Mw>150kDa) in protein lysates from NFB and NCM transduced with an adenovirus encoding human GPC6 (AdhGPC6) run under non-reducing conditions (-dithiothreitol; D). Data are presented as mean ± S.E.M. Unpaired Student’s t-test (A and C) was used to test for statistical significance. **P<0.01; ***P<0.001; significantly different from other cell type.
Fig 4.
Glypican-6 expression is regulated by BMP4 in cardiac fibroblasts.
Relative GPC6 mRNA in neonatal rat cardiac fibroblasts (NFB), adult mouse cardiac fibroblasts (AFB) and NIH 3T3 fibroblasts treated with bone morphogenetic protein (BMP)4, compared to non-stimulated controls (A), n = 3–26. Immunoblot and quantification of the N-terminal GPC6 (GPC6N; Mw ≈35kDa) analyzed under reducing conditions (+dithiothreitol) in NIH 3T3 cells stimulated with BMP4 for 24 h compared to non-stimulated controls (B and C), n = 3. Recombinant human GPC6 (Rec.hGPC6) and protein lysate from NFB overexpressing GPC6 (AdhGPC6) were used as positive controls and β-2-microglobulin was used for loading control. Relative left ventricular BMP4 mRNA after 24h, 1, 3, 16 and 18 weeks of aorta banding (AB) or sham-operation in male mice (D; n = 3–10, see Table 1 for animal characteristics). BMP4 mRNA in left ventricular biopsies from end-stage heart failure patients (n = 17), compared to controls (n = 5) (E). BMP4 mRNA was normalized to ribosomal protein L32 (RPL32) in D and E. Data are presented as mean ± S.E.M. Unpaired Student’s t-test were used to test for statistical significance. *P<0.05; **P<0.01; ***P<0.001; experimental/disease group different from control.
Fig 5.
Glypican-6 enhances BMP4-dependent ERK1/2 signaling in cultured cardiac fibroblasts.
Representative immunoblots and quantification of phospho-extracellular signal-regulated kinase (ERK)1 (pERK 44) relative to total ERK1 (totERK 44; Mw ≈44 kDa) and phospho-ERK2 (pERK 42) relative to total ERK2 (totERK 42; Mw ≈42 kDa) in bone morphogenetic factor (BMP)4-treated human endothelial kidney (HEK)293 cells transfected with a plasmid encoding human GPC6 (pcGPC6) or vehicle (A-C), n = 3. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Representative immunoblots and quantification of pERK/totERK1 and 2 in BMP4- and non-treated rat cardiac neonatal fibroblasts (NFB), transduced with an adenovirus encoding human GPC6 (AdhGPC6) or empty vector (AdNull; D-F), n = 4. Vinculin was used as loading control. Immunoblots in A and D were run under reducing conditions (+dithiothreitol) revealing the unbound N-terminal domain of GPC6 (GPC6N; Mw ≈35kDa). The full length GPC6 (GPC6FL; Mw ≈62kDa) band represents non-glycanated GPC6 where N- and C-terminal domains are held together by disulfide bonds. Relative mRNA levels of α-smooth muscle actin (ACTA2; G), collagen I (COL1A2; H), collagen III (COL3A1; I), lysyl oxidase (LOX; J) and proliferating cell nuclear antigen (PCNA; K) normalized ribosomal protein L4 (RPL4) in BMP4- and non-treated NFB, transduced with AdhGPC6 or empty vector, n = 6–9 from three separate cell cultures. Migration (%) of BMP4- and non-treated NFB transduced with AdhGPC6 or empty vector, 12–96 h after scratch (L), n = 1–3 images of n = 3–6 wells per condition. Data are presented as mean ± S.E.M. Unpaired Student’s t-test (B, C) and one-way ANOVA with Bonferroni post-hoc test (E-L) were used to test for statistical significance. *P<0.05; **P<0.01; group significantly different from vehicle-transfected control, empty vector control or non-stimulated control.
Fig 6.
Glypican-6 enhances ERK1/2 signaling and hypertrophic responses in cultured cardiomyocytes.
Representative immunoblots and quantification of phospho-extracellular signal-regulated kinase (pERK)1 (pERK 44) relative to total ERK1 (totERK 44; Mw ≈44 kDa) and phospho-ERK2 (pERK 42) relative to total ERK2 (totERK 42; Mw ≈42 kDa) in neonatal rat cardiomyocytes (NCM) transduced with an adenovirus encoding human GPC6 (AdhGPC6) or empty vector (AdNull; A-C), n = 5. Vinculin was used as loading control. Immunoblots in A were run under reducing conditions (+dithiothreitol) revealing the N-terminal domain of GPC6 (GPC6N; ≈35kDa). The full length GPC6 (GPC6FL; Mw ≈62kDa) band represents non-glycanated GPC6 where N- and C-terminal domains are held together by disulfide bonds. Relative mRNA levels of atrial and brain natriuretic peptides (NPPA and NPPB, respectively, D and E) normalized to ribosomal protein L4 (RPL4) in NCM transduced with AdhGPC6 or empty vector, and treated with the dual specificity kinase (MEK1/2) inhibitor U0126 or vehicle control, n = 9–18 from three separate cell cultures. [3H] leucine incorporation in NCM transduced with AdhGPC6 or empty vector and treated with U0126 or vehicle control (F), relative to AdNull, non-treated control, n = 6–12. Serum was used as a positive control. Relative mRNA levels of α-skeletal actin (ACTA1, G) normalized to ribosomal protein L4 (RPL4) in NCM transduced with AdhGPC6 or empty vector, and treated with U0126 or vehicle control, n = 8–18 from three separate cell cultures (t-test AdNull vs. AdhGPC6, p = 0.0063). Data are presented as mean ± S.E.M. Unpaired Student’s t-test (B and C) and one-way ANOVA with Bonferroni post-hoc test (D-G) were used to test for statistical significance. *P<0.05; **P<0.01; ***P<0.001; AdhGPC6-transduced NCM significantly different from empty vector control or U0126-treated groups.