Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling

Abstract

Background: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown.

Methods: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5^ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF.

Results: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5^ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin β1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5^ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of β-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that β1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with β-blockers had a distinct cardiac ECM profile.

Conclusions: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that β-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

Keywords: adrenergic beta-agonists; extracellular matrix; heart failure; proteoglycans.

Figure 1.

Extracellular matrix remodeling in human heart failure. A, Histologic characterization demonstrates diffuse fibrosis in patients with nonischemic heart failure (HF) and a pattern of focal replacement fibrosis in patients with ischemic HF. B, Immunoblotting for extracellular and intracellular proteins in 3 different extracts. Extracellular matrix proteins in the 4 M GuHCl extracts: PGS2 (decorin), CLU (clusterin), and DPT (dermatopontin); cellular proteins in the SDS extracts: the cardiac proteins MYL3 (myosin light chain 3) and TNNI3 (cardiac muscle troponin I) as well as the fibroblast marker VIM (vimentin) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as loading control; soluble extracellular proteins in the 0.5 M NaCl extracts: LGALS1 (galectin 1) and APCS (serum amyloid P-component). C, Volcano plot comparing extracellular matrix proteomics profiles from patients with ischemic HF (n=5) with controls (n=6). Extracellular matrix accumulation in patients with ischemic HF includes the proteoglycans ASPN (asporin), BGN (biglycan), DCN (decorin), LUM (lumican), OGN (mimecan, osteoglycin), PRELP (prolargin), and VCAN (versican). These proteoglycans are highlighted in blue.

Figure 2.

Regulation of versican accumulation in the heart. A, Versican can be expressed as 4 isoforms (V0–V3). Versican V1 is the predominant cardiac isoform. GAGα and β are 2 glycosaminoglycan modules. B, Our previous proteomics characterization of the secretome of murine cardiac fibroblasts (CFs) returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases in the secretome of CFs. C, Versikine is generated on cleavage of versican by members of the ADAMTS family and can be detected by an antibody against the versican neoepitope DPEAAE. Versican is expressed by CFs, and versikine is detected in areas surrounding cardiomyocytes (CMs) in mouse hearts. D, Immunoblotting for versikine in left ventricular tissue from patients with heart failure (HF) with ischemic and nonischemic origin, in a porcine model of cardiac ischemia/reperfusion injury (I/R), and in a murine model of angiotensin II–induced cardiac hypertrophy. CS indicates chondroitin sulfate; EGF, epidermal growth factor; and HA, hyaluronic acid.

Figure 3.

ADAMTS5 is responsible for versican cleavage in vivo. A, In mice lacking the catalytic activity of ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5; Adamts5^ΔCat mice), accumulation of intact versican is observed in cardiac tissue after angiotensin II infusion for 2 or 4 weeks. Transforming growth factor β1 (TGFβ1) levels were similar to Adamts5^+/+ controls. B, Gene expression level of Vcan V1 and Adamts5 in the left ventricle of Adamts5^+/+ and Adamts5^Δcat mice at baseline (n=5 and n=9, respectively) and after angiotensin II treatment for 2 weeks (n=6 in both cases) or 4 weeks (n=7 and n=9, respectively). P values were calculated using a 2-way ANOVA with Bonferroni corrections. C, Versikine, the main fragment produced on ADAMTS-mediated versican cleavage, was undetectable in hearts of Adamts5^ΔCat mice at baseline and after angiotensin II infusion for 2 or 4 weeks. D, Picrosirius Red staining for collagen. A similar increase in collagen content was observed in Adamts5^+/+ and Adamts5^ΔCat mice after angiotensin II for 2 weeks.

Figure 4.

Effect of versican accumulation on cardiac function. A, Angiotensin II infusion induced a similar increase of left ventricular (LV) mass in both Adamts5^+/+ and Adamts5^ΔCat mice (n=12 and n=8, respectively). B, No differences in LV ejection fraction % (LVEF%) were observed at baseline. After 2 weeks of angiotensin II treatment, however, a reduced LVEF% was observed in Adamts5^ΔCat compared with Adamts5^+/+ mice. C, Speckle tracking echocardiography revealed impaired global longitudinal strain (GLS) in Adamts5^ΔCat mice in response to angiotensin II infusion. D, Versican binds and coats hyaluronic acid (HA). Biotinylated HABP (HA-binding protein) can be used to assess HA abundance in tissues. E, HA accumulation in Adamts5^ΔCat mice. HA was stained in cardiac tissue sections using HABP. F, Immunoblotting for ITB1 (integrin β1), FLNA (filamin A), and CNX43 (connexin 43). Levels increased after angiotensin II infusion in Adamts5^+/+ mice but not in Adamts5^ΔCat mice. G, Pericellular accumulation of versican and HA was associated with impaired LV function and a decrease in proteins implicated in cell–cell communication. P values were calculated using paired 2-way ANOVAs with Bonferroni corrections.

Figure 5.

Expression of ADAMTS5 and versican in human hearts. A, Single-cell RNA sequencing data in human cardiac tissue. Versican expression was restricted to cardiac fibroblasts (CFs) and a subset of myeloid cells. ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) was expressed by CFs (FB). B, FB1 showed high ADAMTS5 but low versican expression. The opposite was the case for FB2. Thus, there was an inverse association between the expression of ADAMTS5 and of its main substrate versican in CFs of human hearts. C, Aggregated expression for ADAMTS5 and VCAN in the cardiac fibroblast populations FB1 and FB2, displaying both levels of expression and the percentage of cells in which each transcript expression was detected. CM indicates cardiomyocyte.

Figure 6.

Extracellular matrix remodeling in ischemic heart failure. A, Compared with other medications, the use of β-blockers (44 of 65 patients) was associated with the highest number of significant changes in SDS and the extracellular matrix (ECM)–rich GuHCl extracts. B, Term enrichment analyses on proteins changing significantly in patients with ischemic heart failure with the use of different medications. DAVID functional annotation tool was used for the analysis. The horizontal axis represents the number of proteins quantifiable proteins in our proteomics analysis for a specific Gene Ontology/Reactome/Kyoto Encyclopedia of Genes and Genomes term. The vertical axis represents the number of proteins significantly altered in that term. Only terms accounting for <20% of the background list and >10 proteins are displayed. A list of all proteins per term is provided in Table S8. EASE score threshold was set to 0.1, minimum number of proteins for each corresponding term was set to 2, and P values were corrected for multiple testing using the Benjamini-Hochberg method. P<0.05 was considered significant. C, Volcano plot showing a reduction of ECM and ECM-associated proteins in the proteomics analysis of GuHCl extracts in patients with ischemic HF with β-blockers. Proteoglycans (labeled) account for the majority of changes. D, CFs isolated from wild-type mice (n=8 male and n=12 female) were stimulated with noradrenaline (NA). Compared with untreated controls, Versican V1 (Vcan V1) expression was increased after incubation with NA. P values were derived from 2-tailed paired t tests with Bonferroni corrections for multiple comparisons. *P<0.05; **P<0.01; ***P<0.001. The effect of β-blockers on CFs stimulated with NA was assessed using bisoprolol (biso). Bisoprolol prevented the NA-induced increase in Vcan V1 expression in CFs. CFs were obtained from male and female mice (n=8 each, black and white dots, respectively). P values were derived from a 2-tailed ANOVA with Bonferroni correction for multiple comparisons. ***P<0.001. E, Compared with controls (n=6), administration of bisoprolol in vivo for 2 weeks (n=5) reduced cardiac expression of Vcan V1. P value was derived from a 2-tailed paired t test. *P<0.05. AC indicates anticoagulant; ACEI, angiotensin-converting enzyme inhibitor; APT, antiplatelet therapy; ASPN/Aspn, asporin; BB, β-blocker; BGN/Bgn, biglycan; Ddr2, discoidin domain receptor 2; LUM/Lum, lumican; OGN/Ogn, mimecan; Postn, periostin; and PRELP/Prelp, prolargin.

Figure 7.

Effect of β-blockers on cardiac extracellular matrix remodeling. A, Abundance pattern-based clustering of extracellular matrix (ECM) proteins. A dense cluster of interstitial, fibroblast-secreted ECM proteins comprised the majority of those altered with β-blocker usage (circled in black). Serum-derived proteins were excluded from this analysis. B, Principal component analyses (PCAs) based on clinical variables or ECM proteomics. Patients with or without β-blocker administration shared similar clinical characteristics. Their ECM proteomic profiles, however, were distinct. C, A comparison of PCA values for ECM proteomics profiles between patients with or without β-blocker administration detected significant differences according to PCA1. The limma package was used for P value calculation using the Ebayes algorithm and correcting for age and sex.