The Heparan Sulfate Proteoglycan Glypican-6 Is Upregulated in the Failing Heart, and Regulates Cardiomyocyte Growth through ERK1/2 Signaling

<div><p>Pressure overload is a frequent cause of heart failure. Heart failure affects millions of patients worldwide and is a major cause of morbidity and mortality. Cell surface proteoglycans are emerging as molecular players in cardiac remodeling, and increased knowledge about their regulation and function is needed for improved understanding of cardiac pathogenesis. Here we investigated glypicans (GPC1-6), a family of evolutionary conserved heparan sulfate proteoglycans anchored to the extracellular leaflet of the cell membrane, in experimental and clinical heart failure, and explored the function of glypican-6 in cardiac cells <i>in vitro</i>. In mice subjected to pressure overload by aortic banding (AB), we observed elevated glypican-6 levels during hypertrophic remodeling and dilated, end-stage heart failure. Consistently, glypican-6 mRNA was elevated in left ventricular myocardium from explanted hearts of patients with end-stage, dilated heart failure with reduced ejection fraction. Glypican-6 levels correlated negatively with left ventricular ejection fraction in patients, and positively with lung weight after AB in mice. Glypican-6 mRNA was expressed in both cardiac fibroblasts and cardiomyocytes, and the corresponding protein displayed different sizes in the two cell types due to tissue-specific glycanation. Importantly, adenoviral overexpression of glypican-6 in cultured cardiomyocytes increased protein synthesis and induced mRNA levels of the pro-hypertrophic signature gene ACTA1 and the hypertrophy and heart failure signature genes encoding natriuretic peptides, NPPA and NPPB. Overexpression of GPC6 induced ERK1/2 phosphorylation, and co-treatment with the ERK inhibitor U0126 attenuated the GPC6-induced increase in NPPA, NPPB and protein synthesis. In conclusion, our data suggests that glypican-6 plays a role in clinical and experimental heart failure progression by regulating cardiomyocyte growth through ERK signaling.</p></div

Glypican-6 expression is upregulated in the failing human heart.

<p>Relative GPC6 mRNA levels in left ventricular biopsies from end-stage heart failure patients (n = 18) compared to controls (n = 7) (A). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165079#pone.0165079.t002" target="_blank">Table 2</a> for patient characteristics. GPC6 mRNA levels were normalized to ribosomal protein L32 (RPL32) expression and data presented as individual data points. Unpaired Student’s <i>t-test</i> 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).</p

Glypican-6 is mainly produced by cardiac fibroblasts in the heart.

<p>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 <i>E</i>.<i>coli</i> 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 <i>t-</i>test (A and C) was used to test for statistical significance. **<i>P<</i>0.01; ***<i>P<</i>0.001; significantly different from other cell type.</p

Glypican-6 expression is increased in the failing mouse heart.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165079#pone.0165079.t001" target="_blank">Table 1</a> 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165079#pone.0165079.ref008" target="_blank">8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165079#pone.0165079.ref036" target="_blank">36</a>](G and H; n = 3 at all time-points). Recombinant human GPC6 produced in <i>E</i>.<i>coli</i> was used as positive control (Rec.hGPC6). Data are presented as mean ± S.E.M. Unpaired Student’s <i>t-test</i> was used for statistical testing vs. controls at respective time-points. *<i>P<</i>0.05; **<i>P<</i>0.01; ***<i>P<</i>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).</p

Glypican-6 enhances ERK1/2 signaling and hypertrophic responses in cultured cardiomyocytes.

<p>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 (<i>t-test</i> AdNull vs. AdhGPC6, p = 0.0063). Data are presented as mean ± S.E.M. Unpaired Student’s <i>t-test</i> (B and C) and one-way ANOVA with Bonferroni post-hoc test (D-G) were used to test for statistical significance. *<i>P<</i>0.05; **<i>P<</i>0.01; ***<i>P<</i>0.001; AdhGPC6-transduced NCM significantly different from empty vector control or U0126-treated groups.</p

Characterization of patients with end-stage heart failure.

<p>Characterization of patients with end-stage heart failure.</p

Glypican-6 enhances BMP4-dependent ERK1/2 signaling in cultured cardiac fibroblasts.

<p>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 <i>t-test</i> (B, C) and one-way ANOVA with Bonferroni post-hoc test (E-L) were used to test for statistical significance. *<i>P<</i>0.05; **<i>P<</i>0.01; group significantly different from vehicle-transfected control, empty vector control or non-stimulated control.</p