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

The extracellular matrix proteoglycan fibromodulin is upregulated in clinical and experimental heart failure and affects cardiac remodeling

<div><p>Pressure overload of the heart leads to cardiac remodeling that may progress into heart failure, a common, morbid and mortal condition. Increased mechanistic insight into remodeling is instrumental for development of novel heart failure treatment. Cardiac remodeling comprises cardiomyocyte hypertrophic growth, extracellular matrix alterations including fibrosis, and inflammation. Fibromodulin is a small leucine-rich proteoglycan that regulates collagen fibrillogenesis. Fibromodulin is expressed in the cardiac extracellular matrix, however its role in the heart remains largely unknown. We investigated fibromodulin levels in myocardial biopsies from heart failure patients and mice, subjected fibromodulin knock-out (FMOD-KO) mice to pressure overload by aortic banding, and overexpressed fibromodulin in cultured cardiomyocytes and cardiac fibroblasts using adenovirus. Fibromodulin was 3-10-fold upregulated in hearts of heart failure patients and mice. Both cardiomyocytes and cardiac fibroblasts expressed fibromodulin, and its expression was increased by pro-inflammatory stimuli. Without stress, FMOD-KO mice showed no cardiac phenotype. Upon aortic banding, left ventricles of FMOD-KO mice developed mildly exacerbated hypertrophic remodeling compared to wild-type mice, with increased cardiomyocyte size and altered infiltration of leukocytes. There were no differences in mortality, left ventricle dilatation, dysfunction or expression of heart failure markers. Although collagen amount and cross-linking were comparable in FMOD-KO and wild-type, overexpression of fibromodulin in cardiac fibroblasts <i>in vitro</i> decreased their migratory capacity and expression of fibrosis-associated molecules, i.e. the collagen-cross linking enzyme lysyl oxidase, transglutaminase 2 and periostin. In conclusion, despite a robust fibromodulin upregulation in clinical and experimental heart failure, FMOD-KO mice showed a relatively mild hypertrophic phenotype. In cultured cardiac fibroblasts, fibromodulin has anti-fibrotic effects.</p></div

Fibromodulin is upregulated in hearts of mice in response to pressure overload.

<p>(A) FMOD mRNA in the left ventricle (LV) of wild-type (WT) mice subjected to aortic banding (AB) for 24 hours (h) -18 weeks (w), compared to sham-operated controls, n sham = 3–10, n AB = 7–10. Ribosomal protein L32 (RPL32) was used as reference gene. (B) Representative immunoblots and (C) quantitative levels of extracellular FMOD (FMODext, ~60kDa) protein in mouse LVs 3w and 16w post-AB or sham operation, n = 5–7. Culture medium of FMOD-expressing HEK293 cells was used as positive control (Pos cntr), see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201422#pone.0201422.s001" target="_blank">S1 Fig</a> for details. Coomassie staining was used for loading control. (D and E) Pearson regression analysis of FMOD mRNA and (D) left ventricular weight (LVW)/tibia length, or (E) lung weight (LW)/tibia length in sham- and AB-operated mice, n = 87. (F) FMOD, lumican (LUM), biglycan (BGN) and decorin (DCN) mRNA normalized to RPL32 in LVs of mice 24h-18w post-AB, relative to the average of sham-operated controls, n sham = 3–10, n AB = 7–10. Mouse characteristics are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201422#pone.0201422.s012" target="_blank">S4 Table</a>. Data are shown as mean±SEM. Statistical differences were tested using an unpaired t-test vs. respective sham-operated controls (A and C), Pearson regression analysis (D and E) and one-way ANOVA with Bonferroni post-hoc test vs. FMOD mRNA (F). *p≤0.05; ** p≤0.01; ***p≤0.005.</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

Fibromodulin affects the cardiac immune response after pressure overload.

<p>mRNA expression of immune cell adhesion molecules (ICAM1 and VCAM1) and signature molecules of macrophages (ADGRE1/F4.80), T-lymphocytes (CD3), or leukocytes (CD11a and CD45) in the left ventricle of FMOD knock-out (FMOD-KO) and wild-type (WT) mice 12 weeks (w) post-aortic banding (AB), relative to respective sham-operated controls, n sham = 10, n AB = 4–7. Ribosomal protein L32 (RPL32) was used as reference gene. Data are shown as mean±SEM. Statistical differences were tested using one-way ANOVA with Dunn's post-hoc test vs. WT sham, **p≤0.01, or vs. WT AB, #p≤0.05.</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

Schematic illustration of the proposed role of fibromodulin in the failing heart.

<p>The schematic illustrates the main findings of our study. (A) Fibromodulin (FMOD) expression in the heart is increased during cardiac remodeling and heart failure progression in patients and mice. (B) FMOD is an extracellular matrix (ECM) proteoglycan produced by both cardiomyocytes and cardiac fibroblasts, the two major cell types in the heart. (C) FMOD knock-out (FMOD-KO) mice show mildly exacerbated hypertrophic remodeling of the heart with attenuated infiltration of leukocytes in response to experimental pressure overload. (D) FMOD overexpression (FMOD-OE) in cultured cardiac fibroblasts decreases their migratory capacity and reduces the expression of fibrosis-related ECM molecules such as periostin (POSTN), transglutaminase 2 (TGM2), and the collagen-cross linking enzyme lysyl oxidase (LOX). Thus, we propose that FMOD has anti-hypertrophic, anti-fibrotic and pro-inflammatory effects in the pressure-overloaded heart.</p

FMOD-KO mice show mildly exacerbated hypertrophic remodeling compared to wild-type, associated with increased ERK signaling upon aortic banding.

<p>Cardiac phenotype assessed by serial echocardiography at 2–12 weeks (w) (A-B and D-E, n sham = 5–10, n AB = 5–24) and organ weights at 2w and 12w (C and F, n sham = 5–14, n AB = 9–14) post aortic banding (AB). (A) Interventricular septum thickness in diastole (IVSd), (B) left ventricular posterior wall thickness in diastole (LVPWd), (C) heart weight-to-body weight (HW/BW) ratio, (D) LV interior diameter in diastole (LVIDd), (E) fractional shortening (FS) and (F) lung weight-to-body weight (LW/BW) ratio in fibromodulin knock-out (FMOD-KO) and wild-type (WT) control mice subjected to AB. (G) Representative histology image of a whole heart section at low magnification(scale bar 1000μm) and insets showing representative histology images at high magnification (scale bar 50μm) of the different groups at 12w post operation, green (WGA staining) shows outline of cardiomyocyte cross sectional area (CSA) and blue (DAPI staining) shows nucleus. (H) Quantitative changes in cardiomyocyte (CM) cross-sectional area (CSA) respective to average sham controls, measured on midventricular sections of FMOD-KO and WT mice 2, 4, and 12w post-AB, n = 2191–14485 CMs from n sham = 1–4 mice, and n AB = 2–4 mice. (I) Representative immunoblots and (J) quantitative phosphorylated (p) and total levels of the extracellular signal–regulated kinase 1 and 2 (ERK1 and ERK2, respectively) in LVs of FMOD-KO and WT mice 4w post-AB (n sham = 2, n AB = 3–5). Coomassie staining was used as loading control. Data are shown as mean±SEM. Statistical differences were tested using an unpaired t-test vs. WT AB, #p≤0.05; ###p≤0.005 (A, B, D and E 4-10w, H and J), one-way ANOVA with Dunn's post-hoc test vs. WT sham, *p≤0.05; **p≤0.01; ***p≤0.005, or vs. WT AB (A-F 2w and 12w).</p

FMOD-KO and wild-type mice have comparable levels of fibrillar collagens and collagen cross-linking in vivo, but fibromodulin decreases the migration and alters the expression of fibrosis-associated genes in cultured cardiac fibroblasts in vitro.

<p>(A) COL1A2 and COL3A1 mRNA in the left ventricle (LV) of fibromodulin knock-out (FMOD-KO) and wild-type (WT) mice at baseline, 2, 4, or 12 weeks (w) post-aortic banding (AB), relative to average of sham-operated controls (n sham = 2–10, n AB = 3–8). Ribosomal protein L32 (RPL32) was used as reference gene. (B) Collagen protein levels assessed by HPLC from whole LV, in mice 2w and 12w post-AB or sham-operation, n sham = 5, n AB = 5–6. (C) Amount of collagen cross-linking quantified from short axis midventricular histological sections of hearts of FMOD-KO and WT mice 2, 4 and 12w after sham or AB operation, n sham = 5–7, n AB = 2–4. (D) mRNA expression of collagens (COL1A2, COL3A1), cell signature proliferation marker PCNA, myofibroblast differentiation signature marker ACTA2, and fibrosis-associated transcripts in cardiac fibroblast (CFB) cultures from neonatal rats transduced with adenovirus encoding FMOD (AdFMOD), or adenovirus-vehicle (AdVeh) as control, n = 6–7. Ribosomal protein L4 (RPL4) was used as reference gene. (E) Representative images of CFB with AdVeh or AdFMOD 24h post-scratching, and (F) quantification of CFB migration 6-24h post-scratching, n = 8–10. Scale bar 250μm. Data are shown as mean±SEM. Statistical differences were tested using one-way ANOVA with Dunn's post-hoc test vs. WT sham, *p≤0.05; ***p≤0.005, or vs. WTAB (A, B and C), unpaired t-test vs. AdVeh, *p≤0.05; **p≤0.01 (D), or two-way ANOVA (F), #p≤0.05; ##p≤0.01; ###p≤0.005.</p

Fibromodulin is expressed in cardiomyocytes and cardiac fibroblasts and regulated by inflammatory pathways.

<p>(A) FMOD mRNA in cultured cardiomyocytes (CM) and cardiac fibroblasts (CFB) from neonatal rats, n = 10–11. Ribosomal protein L4 (RPL4) was used as reference gene. (B) Quantitative levels of extracellular FMOD (FMODext) protein in culture medium from CM and CFB, n = 6–10, with (C) representative immunoblots. (D) CM and (E) CFB cultures were treated with the innate immunity mediator lipopolysaccharide (LPS) or the nuclear factor (NF)κB-inhibitor SM7368, alone or in combination. CM, n = 9–18, and CFB, n = 10–19. Data are shown as mean±SEM. Statistical differences were tested using an unpaired t-test, CM vs. CFB (A-B), *p≤0.05; ***p≤0.005, and (D-E) one-way ANOVA with Holm-Sidak post-hoc test vs. Cntr or co-treatment with SM7368 vs. LPS alone, *** p≤0.005.</p