Disparate Remodeling of the Extracellular Matrix and Proteoglycans in Failing Pediatric Versus Adult Hearts

Figures

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  Figure 1. High‐confidence interaction among extracellular matrix (ECM)–related proteins and greater fibrosis in adult dilated cardiomyopathy (DCM) hearts compared with pediatric DCM hearts. A, STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis shows high‐confidence interaction clusters (k‐means=5 clusters indicated by node color) formed by ECM‐related proteins. B through D, Representative images of Picrosirius Red (PSR) (B) and Masson trichrome–stained (C) left ventricular free wall from pediatric and adult control and DCM myocardium. Scale bar: 100 μm. D, Averaged quantification of collagen content (from PSR‐stained sections) in indicated groups (mean±SEM). n=6 hearts per group. *P<0.05 compared with the corresponding control, † P<0.05 compared with the corresponding pediatric group. ADAM indicates a disintegrin and metalloprotease; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; AFU, arbitrary fluorescence units; BGN, biglycan; COL, collagen; DCN, decorin; LUM, lumican; MMP, matrix metalloprotease; NFC, nonfailing control; OGN, osteoglycan; SGCG, sarcoglycan gamma; SPARC, secreted protein acidic and cysteine‐rich; THBS1, thrombosondin‐1;…

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  Figure 2. Remodeling of glycosaminoglycans in pediatric versus adult dilated cardiomyopathy (DCM) hearts. Quantification of total sulfated glycosaminoglycan (A), and its constituents, chondroitin sulfate (B) and heparan sulfate (C) in indicated groups. D, Representative images of Alcian blue–stained myocardium showing total proteoglycans/glycosaminoglycans in pediatric and adult nonfailing control (NFC) and DCM myocardium. Scale bar: 100 μm. n=6 per group. E, (i) Relative affinity of glycosaminoglycans extracted from pediatric and adult NFC and DCM hearts, for transforming growth factor (TGF)‐β1. (ii) Percent decrease in glycosaminoglycans–TGF‐β affinity in pediatric and adult DCM hearts compared with the corresponding NFC hearts. Data represent mean±SEM. F, Representative Western blots (i), and averaged quantification for phosphorylated Smad2/3 (p‐Smad2/3) (ii), and phospho‐to‐total ratio for Smad2/3 (iii). GAPDH served as the loading control. n=12 to 16 per group. Data represent median±interquartile range. *P<0.05 compared with the corresponding control, † P<0.05 compared with the corresponding pediatric group. AU indicates arbitrary units.

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  Figure 3. Proteoglycans are differentially regulated in pediatric and adult hearts. A, Representative Western blots for versican, biglycan, decorin, osteoglycin, and lumican. Black arrowheads indicate the core protein, while arrows indicate the glycosylated forms. B, Quantification of the protein levels for (i) versican core protein (black border box and whisker) and glycosylated form (gVersican, grey border box and whisker), (ii) biglycan (black border box and whisker) and glycosylated form (gBiglycan, grey border box and whisker), (iii) decorin, (iv) osteoglycin, and (v) lumican. GAPDH served as the loading control. n=12 to 16 per group. Data represent median±interquartile range. *P<0.05 compared with the corresponding control, † P<0.05 compared with the corresponding pediatric group. AU indicates arbitrary units; DCM, dilated cardiomyopathy; NFC, nonfailing control.

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  Figure 4. Protein levels of matricellular/glycoproteins in pediatric and adult nonfailing control (NFC) and dilated cardiomyopathy (DCM) hearts. A, Representative Western blots for thrombosondin‐1 (THBS1), tenascin‐x, secreted protein acidic and cysteine‐rich (SPARC), osteopontin, and γ‐sarcoglycan. B, Quantification of protein levels for indicated proteins (i through v). GAPDH serves as loading control. n=12 to 16 per group. Data represent median±interquartile range. *P<0.05 compared with the corresponding control, † P<0.05 compared with the corresponding pediatric group.

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  Figure 5. A disintegrin and metalloproteinases (ADAMs) in pediatric and adult control and failing hearts. A, Representative Western blots for myocardial ADAMs 12, 15, 17, and 19. B, Averaged quantification of protein levels for each ADAM (i through iv). GAPDH represents the loading control. n=12 to 16 per group. Data represent median±interquartile range. *P<0.05 compared with corresponding control, † P<0.05 compared with the corresponding pediatric group. AU indicates arbitrary units; DCM, dilated cardiomyopathy; NFC, nonfailing control.

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  Figure 6. Alterations in a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) protein expression in pediatric and adult control and failing hearts. Representative Western blots (A) and averaged protein quantification (B) for ADAMTSs 1, 2, 4, 5, 7, and 9. GAPDH served as the loading control. n=12 to 16 per group. Data represent median±interquartile range. *P<0.05 compared with corresponding control, † P<0.05 compared with corresponding pediatric group. AU indicates arbitrary units; DCM, dilated cardiomyopathy; NFC, nonfailing control.

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  Figure 7. Proteolytic activities of matrix metalloproteases (MMPs) in control and failing hearts of pediatric and adult populations. A, Total MMP activity, measured using a fluorescent‐based in vitro activity assay, in the indicated groups. B, A Representative gelatin zymogram showing MMP‐9 and MMP‐2 levels in nonfailing control (NFC) and dilated cardiomyopathy (DCM) hearts from pediatric and adult groups. Coomassie blue (CBB)–stained SDS‐PAGE was used as the loading control. C, Quantification of gelatinolytic activities for (i) proMMP9, (ii) proMMP2, and (iii) MMP2. n=12 to 16 per group. Data represent mean±SEM. *P<0.05 compared with the corresponding control, † P<0.05 compared with the corresponding pediatric group. AU indicates arbitrary units.

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  Figure 8. Altered tissue inhibitors of metalloprotease (TIMP) levels in nonfailing control (NFC) and dilated cardiomyopathy (DCM) hearts from pediatric and adult populations. Representative Western blots (A) and averaged protein quantification (B) for TIMP1, TIMP2, TIMP3, and TIMP4. n=12 to 16 per group. Data represent median±interquartile range. (C) A collective array showing a summary of protein expression levels for the molecules analyzed in this study in pediatric and adult NFC and DCM hearts. The color scale indicates the relative expression level. *P<0.05 compared with the corresponding control, † P<0.05 compared with the corresponding pediatric group. ADAMS indicates A disintegrin and metalloproteinases; ADAMTSs, a disintegrin and metalloproteinase with thrombospondin motifs; AU, arbitrary units; MMPs, matrix metalloproteases; sGAGs, sulfated glycosaminoglycans; SPARC, secreted protein acidic and cysteine‐rich; THBS1, thrombosondin‐1.