Lumican Accumulates with Fibrillar Collagen in Fibrosis in Hypertrophic Cardiomyopathy

Figures

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  Figure 1. Lumican is increased in the myocardium of HOCM patients. Left ventricular (LV) myectomy samples were obtained from HOCM patients and compared with LV samples from control hearts (Ctrl). (A) Patient lumican (LUM) mRNA expression relative to control (n = 3 Ctrl, n = 14 HOCM). (B) Cryosections were immunostained for lumican (green) and scanned at 20×, from control (left) and HOCM (right) tissues. Negative antibody control displayed in Figure S1A . Scale bars have a width of 1 mm. (C) Quantification of lumican protein via mean signal intensity in control and HOCM tissue sections (n = 3 Ctrl, n = 3 HOCM). (D) Immunoblotting of total lysates for lumican protein in control and HOCM tissues, displaying lumican in its 50–75 kDa glycosylated state (blot displayed in i and quantified as ‘glycosylated’ in iii). These samples were deglycosylated using PNGaseF to measure total lumican protein via the 38‐kDa core protein (blot displayed in ii and quantified as ‘deglycosylated’ in iv). Representative bands are shown, with complete blots and loading controls displayed in Figure S2 . Schematic of the small leucine‐rich proteoglycan lumican is displayed with its four N‐glycan sites where keratan…

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  Figure 2. Lumican co‐localizes with collagen I in fibrotic regions of the HOCM myocardium. Correlation and co‐localization between lumican and collagen I were examined in left ventricular (LV) myectomy samples from HOCM patients and compared with control hearts (Ctrl). (A) mRNA expression levels of collagen types I and III were analysed via COL1A2 (left) and COL3A1 (right), shown relative to control (n = 5 Ctrl, n = 14 HOCM). (B) Correlations (R2) for collagen I or III mRNA expression with lumican mRNA expression in HOCM samples (n = 14). (C) Levels of total lumican protein via Western blotting correlated against fibrosis (%), quantified via histological examination of HOCM patient myectomy tissues (n = 14) by an experienced pathologist. Total fibrosis percentage in the tissue is displayed (left panel), with interstitial fibrosis (right panel) correlated separately. R2 and P values displayed within the graph. (D) Cryosections were binarized to assess degree of red/green overlap through Fiji/ImageJ (n = 3 Ctrl, n = 3 HOCM, lumican in green, collagen I in red, and overlap in yellow). Scale bars have a width of 1 mm and representative images follow Pearson's R quantification. (E) Pearson's R…

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  Figure 3. Lumican and collagen are upregulated and localized across left ventricles of mice with HCM. Heterozygous Myh6 R403Q and wild‐type (WT) littermate male mice were subjected to 3 weeks of cyclosporine A (CsA) or vehicle (Veh) administration (n = 8–10). Myh6 R403Q mice have an accelerated phenotype upon treatment with CsA. (A) mRNA expression levels of collagens I (Col1a1, left, Col1a2, centre) and III (Col3a1, right) shown relative to WT Veh control. (B) mRNA expression levels of collagen cross‐linking enzyme lysyl oxidase (Lox) shown relative to WT Veh control. (C) mRNA expression levels of lumican (Lum) normalized to Rpl32 and relative to WT Veh control. (D) Lumican protein in its native 50–75 kDa glycosylated form as detected in two protein lysate fractions; left lane = cellular, membrane and soluble/loosely bound ECM fraction (cell + sol. ECM); right lane = ECM‐enriched fraction containing insoluble ECM protein (insol. ECM). Representative bands shown, complete blots and loading controls displayed in Figure S4 . Quantification was performed between 50 and 75 kDa, shown relative to WT Veh control. (E) Lumican protein localization across the LV displayed in transverse,…

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  Figure 4. Lumican and collagen I in areas with and without apparent fibrosis in HCM. Confocal imaging of lumican and collagen I deposition in heterozygous Myh6 R403Q and wild‐type (WT) littermate male mice administered 3 weeks of cyclosporine A (CsA) or vehicle (Veh). Transverse mid‐ventricular sections were used for imaging, where Ctrl = WT Veh (n = 5), HCM = Myh6 R403Q with CsA (n = 5). Negative antibody control displayed in Figure S1B . (A) Representative image displaying typical organization of lumican (green) and collagen I (red) in the ECM in control. Cardiomyocytes are outlined with collagen 1 and WGA. The scale bar indicates a width of 25 μm. (B) Representative image of lumican and collagen I distribution across an HCM mouse LV, following progressive fibrotic remodelling (white dashed arrow indicates increase in severity of fibrosis from left to right). The pink arrow displays an area of HCM tissue that resembles control tissue, which is defined as an area ‘without apparent fibrosis’ for the purposes of this study. The scale bar indicates a width of 100 μm. (C) Representative images from HCM mouse LV displaying lumican deposition between cardiomyocytes in areas with either little…

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  Figure 5. Collagen I deposits reach larger sizes more frequently and co‐localize more with lumican in areas without apparent fibrosis in HCM. Super‐resolution imaging via dSTORM and electron microscopy (EM) of lumican and collagen I in Myh6 R403Q CsA and wild‐type (WT) littermate male mice administered 3 weeks of cyclosporine A (CsA) or vehicle (Veh). Transverse mid‐ventricular sections were used for imaging, where Ctrl = WT Veh (n = 3), HCM = Myh6 R403Q with CsA (n = 3). (A) Lumican (green) and collagen I (red) in confocal images of the LV (box labelled 1), with control (Ctrl) tissue in upper panels and HCM tissue in lower panels. Interstitial areas without apparent fibrosis were selected for dSTORM imaging (dashed box, labelled 2). Cardiomyocyte indicated by CM. Negative antibody control displayed in Figure S1B . The scale bar indicates a width of 10 μm on the left (confocal) and 5 μm on the right (dSTORM). (B) Binarized dSTORM images of lumican and collagen deposits (dotted box, labelled 3) and overlap between them (yellow). The scale bar indicates a width of 0.5 μm. (C) Frequencies of collagen deposits above 1 μm2 are displayed via histogram for HCM (teal) and control (superimposed,…

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  Figure 6. Exogenous lumican increases collagen fibre width in cultured human foetal cardiac fibroblasts. Confocal imaging of lumican (green) and collagen I (red) in human foetal cardiac fibroblasts (hfCFBs) cultured for 5 days with recombinant lumican (Lum) or vehicle (Veh), n = 3 biological replicates. (A) Representative confocal images of collagen I fibres (right panels, red), lumican (middle panels, green) and merged image (left panels). DAPI (light blue) displays cell nuclei. Seven images were chosen at random from each culture for quantification. Negative antibody control displayed in Figure S1C . Scale bar indicates a width of 100 μm. (B) Mean width of all collagen fibres in images. (C) Mean length of all collagen fibres in images. (D) The average number of collagen fibres in images. All data are shown as mean ± SD; statistical comparisons performed by t‐test. * P < 0.05, **P < 0.01, ***P < 0.001.