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

Abstract

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 in vitro. 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.

The Non-Fibrillar Side of Fibrosis: Contribution of the Basement Membrane, Proteoglycans, and Glycoproteins to Myocardial Fibrosis

“…Very little is known about the effects of glypicans on myocardial fibrosis [129,130]. Glypican-6 (GPC6) is produced by both cardiac fibroblasts and cardiomyocytes, and was found to be upregulated in the heart following cardiac pressure overload mice [26]. Overexpression of GPC6 in cardiomyocytes increased hypertrophic growth and ERK1/2 signaling [26].…”

“…Glypican-6 (GPC6) is produced by both cardiac fibroblasts and cardiomyocytes, and was found to be upregulated in the heart following cardiac pressure overload mice [26]. Overexpression of GPC6 in cardiomyocytes increased hypertrophic growth and ERK1/2 signaling [26]. GPC6 expression in cardiac fibroblasts is increased in response to Ang II and BMP4.…”

A novel method for high precision aortic constriction that allows for generation of specific cardiac phenotypes in mice

“…wall thicknesses and ID) and left atrium diameter (LAD) were performed by an experienced researcher blinded to group in lightly anaesthetized mice breathing 1.75% isoflurane through a mask, using the VEVO 2100 (VisualSonics, Toronto, Canada). 24 Doppler-imaging of preand post-stenotic maximal flow velocities were captured 24 h postsurgery and trans-stenotic pressure gradients were calculated using the Simplified Bernoulli equation (DP = 4(v 2 post -v 2 pre )). v 2 pre was not included when lower than 1.5 m/s in accordance with previous recommendations.…”

“…Snap-frozen LV tissue was homogenized using the TissueLyser II (Qiagen), in 1Â PBS-based lysis buffer essentially as described. 24 SDS-PAGE separation was performed before transfer onto PVDF membranes. After incubation with primary and HRP-conjugated secondary antibodies (listed in Supplementary material online), membranes were developed using ECL Prime (Amersham/GE HealthCare, Buckinghamshire, UK) on the Las-4000 (Fujifilm, Tokyo, Japan).…”

“…All presented data are mean ± SEM. Statistical tests were performed in GraphPad Prism 7.02 (GraphPad Software, La Jolla, CA, USA) or IBM SPSS Statistics for Windows, version 24. Statistical tests used were repeated measures two-way analysis of variance (ANOVA) with Tukey's (>2 groups compared) or Sidak's (2 groups compared) post hoc test, one-way ANOVA with Bonferroni or Dunnet's C post hoc test (dependent on Brown-Forsythe test for equal variance), or Student's t-test.…”