The extracellular matrix proteoglycan lumican improves survival and counteracts cardiac dilatation and failure in mice subjected to pressure overload

Left ventricular (LV) dilatation is a key step in transition to heart failure (HF) in response to pressure overload. Cardiac extracellular matrix (ECM) contains fibrillar collagens and proteoglycans, important for maintaining tissue integrity. Alterations in collagen production and cross-linking are associated with cardiac LV dilatation and HF. Lumican (LUM) is a collagen binding proteoglycan with increased expression in hearts of patients and mice with HF, however, its role in cardiac function remains poorly understood. To examine the role of LUM in pressure overload induced cardiac remodeling, we subjected LUM knock-out (LUMKO) mice to aortic banding (AB) and treated cultured cardiac fibroblasts (CFB) with LUM. LUMKO mice exhibited increased mortality 1–14 days post-AB. Echocardiography revealed increased LV dilatation, altered hypertrophic remodeling and exacerbated contractile dysfunction in surviving LUMKO 1–10w post-AB. LUMKO hearts showed reduced collagen expression and cross-linking post-AB. Transcriptional profiling of LUMKO hearts by RNA sequencing revealed 714 differentially expressed transcripts, with enrichment of cardiotoxicity, ECM and inflammatory pathways. CFB treated with LUM showed increased mRNAs for markers of myofibroblast differentiation, proliferation and expression of ECM molecules important for fibrosis, including collagens and collagen cross-linking enzyme lysyl oxidase. In conclusion, we report the novel finding that lack of LUM attenuates collagen cross-linking in the pressure-overloaded heart, leading to increased mortality, dilatation and contractile dysfunction in mice.

overexpressing LUM or vehicle, before harvest of RNA. Non-treated cells served as negative controls, and cells treated with transforming growth factor (TGF)β1 (10ng/ml, GF111, Merck Millipore) served as positive controls. Experiments were performed in three separate cell culture isolations. CFB were passaged only once before stimulation with conditioned medium.Fibroblasts were transferred to fibroblast plating medium (Dulbecco's modified Eagle's medium supplemented with penicillin/streptomyocin and FBS) and maintained in culture for up to 1 week, before being passaged and seeded onto six-well culture plates at a density of 1.89 × 10 5 /ml.

Proliferation assay
Cell proliferation was assessed using the CyQUANT Proliferation Assay Kit (C7026, Thermo Scientific) according to the manufacturer's instructions. Briefly, neonatal CFBs were seeded in 12-well plates at a density of 50000 cells/well, serumdeprived and treated for 24h with conditioned medium containing LUM or vehicle. Cells were detached with trypsin-EDTA (T3925, Sigma), the cell suspension centrifuged for 5 min at 1500 rpm, the supernatant removed and the cell pellet frozen at -80°C for 96h. Subsequently, the cell pellet was suspended in 500 μl CyQuant solution to obtain a cell density within the linear range of the assay (50-50000 cells per 200 µl sample). The fluorescence intensity was measured with excitation at 485 nm and emission at 535 nm.

Gene expression analysis
Gene expression analyses were performed by qRT-PCR, dd-PCR and RNA sequencing (RNAseq) on RNA extracted from cells and LV tissues. For qRT-PCR and dd-PCR, total RNA including microRNA was extracted using the miRNeasy Mini Kit (#217004, Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNA concentration was measured using the Nanodrop ND-1000 Spectrophotometer (Thermo Scientific, Waltham, MA). cDNA was made using iScript cDNA Synthesis Kit (BioRad Laboratories, Inc., Hercules, CA) according to the manufacturer's protocol. Gene expression levels were measured using pre-designed TaqMan assays (Table S7) by 7900HT Fast Real Time PCR System (Applied Biosystems, Foster city, CA) or dd-PCR (Q x200 TM Droplet digital PCR system). Results were analyzed using the Sequence Detection System (SDS) 2.3 software (Applied Biosystems). Housekeeping genes RPL32, RPL4 or U6 were used for normalization of gene expression (Fig. S4). RNAseq was performed as described 7 8 on total RNA extracted from LV tissue using Trizol (Thermo Scientific). RNA concentration and quality was measured on Tapestation 2200 (Agilent Technologies, Santa Clara, CA) with RIN >7 accepted. LV libraries were constructed from 3 μg of RNA pooled from the LV of three LUMKO and three WT mice 2w post-AB, for cDNA synthesis using random hexamer priming, without fragmentation or duplex-specific nuclease digestion 9 , followed by Nextera XT DNA sample preparation (Illumina, San Diego, CA) according to protocol, including tagmentation and PCR amplification. Libraries were quantified and diluted to 20 nM cDNA and run on the Illumina HighSeq 2500. Data were processed as previously described 9 , normalized to total number of reads per sample and expressed as number of fragments per kilobase of exon per million fragments mapped (FPKM). FPKM <2 was considered not expressed. Reads were aligned to the mouse genome (mm10) with a total of 31920 transcripts using Tophat (version 2.0). Cut-off filters used for differentially expressed (DE) transcripts were fold change >1.33 and <0.75 with p<0.001. Ingenuity pathway analysis (IPA, Qiagen), KEGG Pathways and AmiGO gene ontology (GO) analyses were performed on DE transcripts. For IPA predicted upstream regulators (USR), Z-score cut-offs used were ±2 (>2 predicted activation, <-2 predicted inhibition).
Wheat Germ Agglutinin (WGA) staining and quantification 4 µm mid ventricular sections obtained from all 4 groups (WT SHAM, WT AB, LUMKO SHAM and LUMKO AB) were stained with Wheat Germ Agglutinin (WGA) to examine the average size of cardiomyocyte cross-sectional area (CSA). The histological sections were deparaffinized, hydrated to dH 2 O (5min in each: Xylene, Xylene, 100% alcohol, 96% alcohol, 80% alcohol, 70% alcohol and dH 2 O). The sections then were rinsed in PBS and boiled for 20min in citrate buffer PH6 (98⁰C) (2.1 g citrate acid monohydrate to 1 L dH2O. Adjust pH to 6.0 by NaOH). Then, sections were cooled down at RT for 20min, rinsed with PBS and incubated with WGA 1:200 in PBS over night at 4⁰C. After 24h incubation, sections were rinsed in PBS 1 minx3 and mounted under a coverslip with ProLong™ Gold Antifade Mountant with DAPI (#P36931, Thermo Scientific, Waltham, MA). ImageJ was used to preprocess and threshold the WGA staining to best visualize clear compartments corresponding to cardiomyocytes. One image (i.e. one mid-ventricular section) was analyzed per mouse. The "analyze particles" plugin was used to generate regions of interests, ROIs, of putative cardiomyocytes. Inclusion criteria were defined with ROI size and circularity (a measure of how close to a perfect circle a ROI is). ROI size was set between 90-5000 µm 2 , meaning that almost all the ROIs were selected except nuclei which were less than 90µm 2 . To exclude longitudinal and strange connected ROIs, we defined a circularity range. With circularity criteria, you only select transverse ROIs (i.e. transverse cross-sectional areas). We have used circularity between 0.6-1.
Picrosirius Red (PSR) staining and quantification 4 µm mid ventricular sections obtained from all 4 groups (WT SHAM, WT AB, LUMKO HAM and LUMKO AB) were stained with Picrosirius Red for the quantification of collagen fibrils. The histological sections were deparaffinized and hydrated to dH 2 O (5min in each: Xylene, Xylene, 100% alcohol, 96% alcohol, 80% alcohol, 70% alcohol and dH 2 O). The sections then were incubated in solution A (Phosphomolybdic acid hydrate & water) for 2min, and rinsed in dH 2 O. Following, the sections incubated in F3BA solution B (2, 4, 6-Trinitrophenol, water & direct Red 80) for 60min, and in hydrochloride solution C (Hydrogen chloride & water) for 2min and in 70% ethanol (45sec). The sections were then dehydrated through alcohol, cleared in xylene and mounted under a cover slip. Fibrosis and cross-linking quantification of PSR stained heart tissue sections was done in ImageJ, using the color threshold plugin to determine the red area corresponding to fibrillar collagens and orange/green area corresponding to cross-linked collagens.