Cathepsin A contributes to left ventricular remodeling by degrading extracellular superoxide dismutase in mice

In the heart, the serine carboxypeptidase cathepsin A (CatA) is distributed between lysosomes and the extracellular matrix (ECM). CatA-mediated degradation of extracellular peptides may contribute to ECM remodeling and left ventricular (LV) dysfunction. Here, we aimed to evaluate the effects of CatA overexpression on LV remodeling. A proteomic analysis of the secretome of adult mouse cardiac fibroblasts upon digestion by CatA identified the extracellular antioxidant enzyme superoxide dismutase (EC-SOD) as a novel substrate of CatA, which decreased EC-SOD abundance 5-fold. In vitro , both cardiomyocytes and cardiac fibroblasts expressed and secreted CatA protein, and only cardiac fibroblasts expressed and secreted EC-SOD protein. Cardiomyocyte-specific CatA overexpression and increased CatA activity in the LV of transgenic mice (CatA-TG) reduced EC-SOD protein levels by 43%. Loss of EC-SOD–mediated antioxidative activity resulted in significant accumulation of superoxide radicals (WT 4.54 vs. CatA-TG 8.62 μmol/mg tissue/min), increased inflammation, myocyte hypertrophy (WT 19.8 vs. CatA-TG 21.9 μm), cellular apoptosis, and elevated mRNA expression of hypertrophy-related and profibrotic marker genes, without affecting intracellular detoxifying proteins. In CatA-TG mice, LV interstitial fibrosis formation was enhanced by 19%, and the type I:type III collagen ratio was shifted toward higher abundance of collagen I fibers. Cardiac remodeling in CatA-TG was accompanied by an increased LV weight:body weight ratio and LV enddiastolic volume (WT 50.8 vs. CatA-TG 61.9 μl).


INTRODUCTION
Cardiac function depends on structural and functional integrity of the extracellular matrix (ECM). ECM components are synthesized and secreted by cardiac fibroblasts (CFs) (1) and their proper composition and turnover is controlled by proteolysis (2)(3)(4). Degradation of ECM proteins occur either within the cell after fusion of ECM-containing phagosomes with lysosomes or in the extracellular space by secreted proteolytic enzymes (4). Cathepsins are lysosomal proteases which target a broad range of intraand extracellular proteins like laminin, fibronectin, elastin and fibrillar collagens (2,3). Increased activation of cathepsins results in remodeling of subcellular organelles and the ECM, and is associated with cardiac complications including hypertrophic cardiomyopathy, diabetic cardiomyopathy, dilated cardiomyopathy, and myocardial infarction (2,3). Cysteine protease cathepsins like cathepsin B, K, L and S play pathophysiological roles in cardiac structural changes and progression of heart failure (2,3). However, the role of the serine protease cathepsin A (CatA) during cardiac disease is unclear. CatA is widely distributed in mammalian tissues, with highest expression found in kidney, lung, endothelium, liver, placenta, and heart (5,6). Besides its catalytic function as a protease, lysosomal CatA forms a protein complex with neuraminidase 1 and βgalactosidase, which prevents proteolysis of its binding partners thereby regulating and stabilizing lysosomal activity and function (7). In humans, disruption of this protein complex by CatA deficiency or mutations in the gene coding for CatA results in the lysosomal storage disease galactosialidosis (8). CatA is also localized on the cell surface and in the extracellular space, where it has been suggested to be involved in ECM formation, possibly by degradation of extracellular peptides (5,6,9). In animal models of myocardial infarction, type 2 diabetes, and angiotensin II-stimulated hypertrophy, cardiac expression of CatA is upregulated, and pharmacological inhibition of CatA-activity exhibited cardio-protective, antihypertrophic, and anti-fibrotic effects in these conditions (10-13) but its mechanistic role in cardiovascular disease is unknown. The present study was designed to identify potential mechanisms of CatA-mediated ECM remodeling processes in the heart using stateof-the-art proteomic analysis of the secretome of adult mouse CFs upon digestion by CatA, and supportive in vivo investigations in a transgenic mouse model with cardiomyocytespecific overexpression of CatA (CatA-TG).

Cathepsin A (CatA) processes extracellular matrix (ECM) proteins.
To identify novel ECM-related candidate substrates of CatA, we performed a proteomic analysis of the secretome of adult mouse CFs, which produce and secrete ECM-proteins. Proteins in the conditioned medium of CFs treated with and without human recombinant CatA (n=4 each) were analyzed by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) after filtering using 3kDa columns to recover only small cleavage products rather than intact proteins (14). Using Progenesis ® LC-MS software (Non-linear Dynamics), the ion intensities of all detected peptides in the <3kDa fraction were compared between control and CatA-treated CFs (Fig. 1A). CatA digestion significantly affected abundance of protein degradation products of collagens (CO5A1 (p=0.001), CO5A2 (p=0.0001), CO3A1 (p=0.002), CO1A1 (p=0.003)) and other ECM proteins (i.e. PGS2 (Decorin; p=0.005), LAMA4 (Laminin Subunit Alpha 4, p=0.028), PGBM (Basement membrane-specific heparan sulfate proteoglycan core protein, also known as perlecan; p=0.025), and FINC (Fibronectin; p=0.002) compared to control. The antioxidant enzyme extracellular superoxide dismutase (SODE or EC-SOD) was one of the most significantly affected extracellular proteins after incubation with CatA (p=0.0001) (Fig.  1B, Table S1 Supporting information. The proteomics data are also available via ProteomeXchange with identifier PXD019895). EC-SOD is an essential antioxidant enzyme, which is exclusively located in the ECM, catalyzing the dismutation of superoxide to hydrogen peroxide and oxygen (15). Three fragment peptides and possible cleavage sites were detected for EC-SOD in control conditions but were undetectable upon treatment with CatA (Fig. 1C). This finding added a novel and highly relevant aspect on the role of CatA in cardiac disease, because EC-SOD provides the only direct defensemechanism against superoxide radicals within the ECM (15) and CatA-mediated loss of antioxidant protection may facilitate ECM remodeling.

Differential expression pattern of cathepsin A (CatA)
and extracellular superoxide dismutase (EC-SOD) in cardiomyocytes (CMs) and cardiac fibroblasts (CFs). An initial gene expression analysis comparing isolated primary adult mouse CFs with CMs demonstrated mRNA transcription of CatA in both cell lines, whereas EC-SOD mRNA was only detectable in CFs (Fig. S1 Supporting information). These mRNA data were confirmed at the protein level by analyzing cultured neonatal rat CMs and rat CFs. While CatA protein was expressed and secreted by both cell types ( Fig. 2A and 2B), the presence and secretion of EC-SOD protein was only evident in CFs ( Fig. 2C and 2D).

Cardiomyocyte-specific cathepsin A (CatA) overexpression in mice results in posttranslational downregulation of extracellular superoxide dismutase (EC-SOD), increased oxidative stress and enhanced inflammation in the left ventricle (LV).
To further understand the function of CatA in ECM remodeling in vivo, transgenic mice with a cardiomyocyte-specific postnatal overexpression of active human CatA were generated (CatA-TG) using the alpha myosin heavy chain promoter ( Fig. S2 Supporting  information). CatA-TG mice developed normally and showed no apparent abnormal phenotype. Quantitative Real-Time PCR, Western blot analysis and immuno-histological staining confirmed overexpression of human CatA in the myocardium of CatA-TG mice ( Fig. 3A-C). Detoxification of reactive oxygen species (ROS) are catalyzed by anti-oxidative enzymes, including catalase and superoxide dismutases (SODs). Beside the extracellular isoform EC-SOD, two intracellular isozymes of SODs exist, comprising cytosolic Cu/Zn-SOD (SOD1) and mitochondrial Mn-SOD (SOD2) (15). Western blot analysis demonstrated that overexpression of CatA did not affect protein expression of catalase, SOD1 and SOD2 which are located within the cell. (Fig. 3D and 3E). Using an EC-SOD antibody that specifically targets the Nterminal region demonstrated a significant reduction of EC-SOD protein in the LV of CatA-TG compared to their wild type littermate controls (WT) (Fig. 3D, 3E). Interestingly, EC-SOD mRNA levels were unaltered (Fig. 3F), strongly suggesting a posttranslational regulation, i.e. consistent with proteolysis of EC-SOD. To characterize downstream consequences of reduced EC-SOD and its loss of antioxidant protection (15), we determined the levels of superoxide radicals in LV tissue of CatA-TG and in WT mice. Using electron spin resonance spectroscopy measurements, CatA-TG mice demonstrated increased LV oxidative stress, as indicated by accumulation of superoxide radicals (Fig. 4A). Oxidative stress was associated with elevated gene expression of connective tissue growth factor (CTGF), an important redox-sensitive inducer of fibrosis (16) (Fig. 4B). Increased gene expression of tumor necrosis factor alpha (TNF), interleukin 6 (IL6), and interleukin 2 (IL2) as well as repressed transcription of interleukin 10 (IL10) demonstrated an enhanced inflammatory response in CatA-TG mice (Fig. 4C). Interleukin 1 beta (IL1b) was not regulated. Of note, mRNA levels of IL2 and IL10 were considerably lower compared to gene expression of TNF or IL6, and immunohistological stainings for infiltration of macrophages or neutrophils in the heart of CatA-TG and WT mice showed no significant inflammatory infiltration (Fig. S3. Supporting information). CatA-TG demonstrated a higher proportion of apoptotic cells, as detected by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining (Fig. 4D), independent of a differential expression of proenzyme caspase 1 and caspase 3 protein
CatA-TG mice also demonstrated increased LV interstitial fibrosis formation ( Fig. 6A and 6C) and a shift in the ratio of collagen type I (redyellow fibers) to collagen type III (green fibers) as assessed by polarized light microscopy ( Fig.  6B and 6D). In the LV of CatA-TG mice mRNA-expression of pro-fibrotic transforming growth factor beta 1 (TGF-1) and of the ECM components collagen 1a2 (Col1a2), Col3a, Col5a1, fibronectin (FN) and ECM stabilizing protein lysyl oxidase (Lox), which catalyzes cross-linking of collagen fibrils and elastin (18), were elevated (Fig. 6E). The development of perivascular fibrosis in the heart was unaffected by cardiomyocyte-specific cathepsin A overexpression (WT: 31.3±5.9 vs. CatA-TG: 34.3±5.4 % p=0.398; Fig. S6. Supporting information). To asses LV function in this unchallenged phenotype of CatA-TG, we used an isolated working heart preparation to measure functional parameters under standardized hemodynamic conditions. At 6 months of age isolated CatA-TG hearts demonstrated significant increased LV enddiastolic volume (LVEDV) (WT: 50.8±5.8 vs. CatA-TG: 61.9±6.2 µl; p=0.018), while other functional parameters where unchanged ( Table 1). Noteworthy, CatA-TG mice developed a significantly increased left ventricular endsystolic pressure at an age of 18 months (Fig. S7. Supporting information).

DISCUSSION
The composition and turnover of the ECM is tightly controlled by proteolysis (2)(3)(4). Pathophysiological remodeling of the ECM contributes critically to LV dysfunction and progression of heart failure (19,20). Here, we provide new evidence for the involvement of the serine carboxypeptidase CatA in the degradation of the extracellular antioxidant enzyme EC-SOD and regulation of subsequent ECM remodeling processes. These findings highlight proteolysis as a potential target in cardiovascular diseases and heart failure. Proteomic analysis of the secretome of adult mouse CFs identified EC-SOD as a novel target and substrate of CatA at slightly acidic pH5.5. As a multifunctional enzyme, CatA exhibits a strong carboxypeptidase activity in an acidic milieu and a de-amidase as well as a weakened enzymatic carboxypeptidase activity at neutral pH (5,6,21). At neutral pH, the ECM is protected against extracellular degradation by secreted or membrane-bound cathepsins. However, in the LV myocardium of CatA-TG mice the imbalanced expression pattern of pro-and antiinflammatory marker genes suggests an inflammatory state. Under inflammatory conditions acidification of the peri-and extracellular space increases the proteolytic activity of cathepsins and further promotes the secretion of lysosomal proteases. Moreover, within the ECM the acidic pH facilitates the processing of secreted inactive pro-cathepsins into catalytically active mature proteases (22). Additionally, binding of cathepsins to heparin or heparan sulfate has been shown to stabilize its enzyme structure and potentiate peptidase activity even at alkaline pH (23). Taken together, these factors may regulate CatA activity to hydrolyze its target proteins after secretion into the interstitial space (6,7,21). EC-SOD represents the main defensemechanism in the ECM against oxidative stress and is the only extracellular enzyme detoxifying superoxide radicals (15). EC-SOD is anchored to the cell surface and the ECM via its C-terminal region, known as the heparinbinding domain. The heparin-binding domain determines the binding affinity for its ligands including cell surface heparan sulphate proteoglycans, heparin, and type I collagen (15,24,25), thus tightly regulating distribution of EC-SOD in the ECM. Interaction of EC-SOD with ECM-components like collagen and syndecan-1 shield its bindings-partners from oxidative damage, preventing ECM remodeling and attenuate inflammatory responses (15,25). Subsequently, EC-SOD depletion reduces antioxidant protection leading to fragmentation of ECM-components, oxidant-induced fibrosis formation, and myocyte hypertrophy (15,25,26). Here, we demonstrated that both CFs and CMs express and secrete CatA, while EC-SOD is exclusively expressed and secreted by CFs, but not by CMs. In our transgenic mouse model, cardiomyocyte-specific overexpression of CatA induced a significant decrease of LV EC-SOD protein levels. As CMs do not express EC-SOD, our findings suggest a CatA-mediated degradation of EC-SOD protein within the ECM and exclude intracellular digestion of EC-SOD in CMs. Additionally, LV EC-SOD mRNA expression was not affected in CatA-TG mice which indicates a posttranslational depletion of EC-SOD protein in the ECM.
Oxidative stress highly contributes to the development of cardiac hypertrophy and fibrosis (17). In CatA-TG mice, reduction of EC-SOD was associated with increased oxidative stress, elevated expression of the redox-sensitive pro-fibrotic CTGF (16) and development of a hypertrophic and pro-fibrotic phenotype with increased LV weight and elevated LVEDV, known hallmarks for heart failure (summarized in Figure 7). Comparable phenotypes have been described previously in EC-SOD knockout mice (25, 26, 27, 28). Lack of EC-SOD was associated with exacerbated oxidative stress-induced myocardial apoptosis, LV fibrosis formation and inflammatory cell infiltration, demonstrating an important protective role of EC-SOD against extracellular oxidative stress (25, 26, 27, 28). Vice versa, overexpression of EC-SOD reduced interstitial fibrosis and ventricular dysfunction in a murine model of ischemic cardiomyopathy (29).
In the failing heart, major sources of cellular ROS comprise the xanthine oxidase and NAD(P)H oxidases (like Nox2 and Nox4). Dysregulation of these enzymes is involved in cardiovascular disease, hypertension and heart failure (17). However, in CatA-TG mice, increased superoxide radicals could not be linked to a differential expression of cellular ROS-producing oxidases. Therefore, ECM remodeling in CatA-TG is likely a consequence of CatA-mediated loss of antioxidant protection and subsequent accumulation of extracellularly derived ROS in the ECM.
A recent proteomic profiling analyzed the impact of a pharmacological inhibition of CatA in a mouse model of myocardial-infarction (MI), demonstrating a partial rescue of left ventricular proteome-alterations associated with MI and attenuated elevated levels of cardiac stress response proteins (13). Furthermore, a quantitative proteome comparison of whole heart lysates from CatA transgenic mice and their littermates linked CatA to cardiac oxidative stress response, and CatA-overexpression in cultured rat cardiomyoblasts resulted in higher sensitivity to oxidative stress (30).
Potential limitations of this study are as follows: Secretome analysis and transgenic overexpression in mice might not recapitulate physiological levels or cell-type specific expression patterns. While certain proteins may be inaccessible to the protease in a tissue environment, proteomics returned a plethora of other potential substrates of CatA, including decorin. Processed forms of decorin protein core have been shown to regulate the local bioavailability of pro-fibrotic growth factors, like CTGF (31). Analysis of our proteomics data also indicated a possible participation of CatA in the processing of other structural ECM proteins, which may have contributed to the cardiac phenotype observed in CatA-TG mice. A comparable ECM-proteolytic activity involving the degradation of laminin, fibronectin, elastin and fibrillar collagens has also been described for the larger family of cysteine-cathepsins (2, 3). Although our findings in CatA-TG mice are consistent with digestion of EC-SOD by CatA, future biochemical studies are needed to investigate whether CatA degrades ECM proteins directly or via other intermediate molecules being activated or inhibited by CatA.

CONCLUSION
The present study sheds first light on a previously unrecognized role of CatA outside the lysosome in the proteolysis of the extracellular antioxidant enzyme EC-SOD. In the heart, CatA-mediated reduction of EC-SOD levels resulted in oxidative stress due to insufficient removal of reactive oxygen species. Thus, EC-SOD degradation represents a plausible link between CatA activation and LV ECM remodeling and implicates CatA as a potential therapeutic target to prevent cardiac ECM remodeling.

EXPERIMENTAL PROCEDURES
Study approval -All animal studies were performed in accordance to the German law for the protection of animals. The investigation conforms with "The guide for the care and use of laboratory animals" published by the United States National Institutes of Health (Eighth edition; revised 2011), the Declaration of Helsinki and was approved by the local animal ethics committee of the University of the Saarland (#21/2014).

Identification of candidate substrates of cathepsin A (CatA) by a proteomics approach -
Five-week old male C57BL/6N mice (n=4) were anesthetized with 5% isoflurane in 95% O2 and sacrificed by intraperitoneal (i.p.) injection of ketamine hydrochloride (100mg/kg body weight) and xylazine hydrochloride (10mg/kg body weight). Isolation and culture of primary cardiac fibroblasts (CFs): Primary mouse CFs were isolated from the hearts of 5 weeks old male C57BL/6N mice (n=4). Hearts were extracted, washed and diced into small pieces, carefully washed in ice-cold phosphate buffered saline (PBS, Sigma-Aldrich) to remove plasma contaminants. The pieces were pre-digested in collagenase II solution (5mg/mL) for 10min. The collagenase II solution was replaced and the tissue pieces were incubated for 45min-60min at 37ºC. The digested tissue pieces were washed in complete medium (DMEM supplemented with 10% FBS, 2mM L-glutamine, 100U/mL penicillin, 100ug/mL streptomycin) before plating. CFs were cultured on 0.1% gelatin-coated T25 flasks in complete medium at 37C in a humidified incubator with 5% CO2 until 80% confluence. Cells were washed 3 times with serum-free medium then incubated in serumfree medium for 3 days. The conditioned medium was centrifuged at 4000rpm (3200 x g) for 10min to remove cell debris. The supernatants were transferred into new tubes and stored at -80°C until further analysis. Digestion by CatA: For digestion with CatA, conditioned media were concentrated and the buffer was exchanged to assay buffer (25mM MES, 5mM DTT, pH=5.5) with 3KDa MWCO spin column (Merck Millipore). Control samples (n=4) were incubated in buffer with protease inhibitor only (for inhibition of endogenous proteases other than CatA). Treated samples (n=4) were incubated in buffer with protease inhibitor only (for inhibition of endogenous proteases other than CatA) plus human recombinant CatA (1µg/mL, Sanofi-Aventis, Deutschland) (10). Both groups were incubated at 37°C for 24 hours with agitation (reaction took place inside the spin column). After reaction, the samples were centrifuged. The degradation products in the <3kDa fractions were collected and purified with C18 spin columns (Thermo Fisher Scientific). Analysis by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS): Peptides were analyzed by LC-MS/MS as previously described (32,33). Peptides were separated by reverse phase chromatography (Acclaim PepMap100 C18, 75μm Å x 25cm) on a nanoflow LC system (Ultimate3000 RSLCnano, Thermo Fisher Scientific) equipped with a trap column (Acclaim PepMap100 C18, 300μm Å x 5mm). The chromatographic separation was performed with a mobile phase of HPLC grade water containing 2% acetonitrile and 0.1% formic aid (eluent A) and a mobile phase of 80% acetonitrile, 20% HPLC grade water and 0.1% formic acid (eluent B) with a 70-minute gradient (2% to 10%B in 3 minutes, 10% to 30%B in 34 minutes, 30% to 40%B in 3 minutes, 99%B for 10 minutes, 2%B for 20 minutes) at a flow rate of 350 nL/min. The column was coupled to an LTQ OrbitrapXL mass spectrometer (Thermo Fisher Scientific) with a nanospray source (Picoview, New Objective, Inc.). The mass spectrometry acquisition involved 1 full MS scan over a mass-to-charge range encompassing m/z 400-1600 using the Orbitrap analyzer, followed by data-dependent collision-induced dissociation MS/MS scans of the 6 most intense ions detected in the full scan, with dynamic exclusion enabled and rejection of singly charged ions. The data were analysed using Progenesis LC-MS software (version 4.1, Nonlinear Dynamics). Peaklist of MS2 spectra from features with intensity > 2-fold change, p < 0.05 between the CatA treated group and control group was exported and identified by Mascot (Matrix Science; version 2.3.01) with the following parameters: enzyme: none; database: UniProt/SwissProt mouse database (release 2012_03, 16520 protein entries); precursor mass tolerance: 10ppm; fragment mass tolerance: 0.8Da; fixed modification: none; variable modification: none. Search results were loaded back to Progenesis to match with the significantly changed features. Peptides with Mascot score > 10 were used for quantification and final protein table was exported (Supplemental Table 1).
Isolation of primary neonatal rat cardiac myocytes and cardiac fibroblasts -Neonatal rat CMs and CFs were isolated from 5 days old Sprague-Dawley rat hearts (Charles River, Germany) of mixed sex. Hearts were removed, the ventricles dissected and digested in ADS buffer containing (in mmol/L) NaCl 116, HEPES 20, Na2HPO4 0.8, Glucose 5.6, KCl 5.4 and MgSO4 x 7 H2O 0.8, pH 7.35, 0.6mg/ml Pankreatin (Sigma, P-3292) and 0.5 mg/ml Collagenase Type 2 (Worthington Biochemical; #LS004176) at 37°C in a water bath with constant stirring at 80-100rpm for 5min. Supernatant (not tissue) was transferred in a 50 ml Falcon tube containing 2 ml neonatal calf serum (NCS; Gibco #16010-159) to stop enzymatic reaction. Procedure was repeated 6 times, all supernatants were collected and the cells were pre-plated on 6-Well plates (Falcon #353846, BD, Franklin Lakes, NJ, USA) in F10 medium (Gibco; +10% horse serum, 5% FCS and 1% penicilline/streptomycine) to allow separation of cardiac fibroblasts by adhesion. After 45 min, the still-suspended neonatal cardiomyocytes were removed from the attached cardiac fibroblasts, counted (Neubauer counting chamber) and plated at a density of 1.65 -1.75 x 10 6 cells per 60 mm culture dish (#93060, TPP, Switzerland) in complete F10 medium at 37°C in a humidified incubator with 5% CO2 until 80% confluence. The attached neonatal cardiac fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, gentamicin (0.08mg/ml), and penicillin (100IU/ml) at 37°C in a humidified incubator with 5% CO2 until 80% confluence. Afterwards both cell lines were kept in serum-free medium for 48h. Medium and cells were harvested and stored at -80°C until further processed.
CatA-transgenic mice (CatA-TG)-mouse model: For the generation of transgenic mice that overexpress the human CatA specifically in cardiomyocytes a vector containing the mouse alpha MHC promoter (5.7kb) driving the human CatA minigene (cDNA clone ID: CLN16325899) was constructed and described elsewhere (11). Kozak translation initiation sequence and SV40pA (249bp) was introduced to enhance transgene expression. LoxP-hUBp-em7-neo-loxP cassette (2648 bp) was inserted downstream of stop codon for selection. VelociMouse® technology (Regeneron) was used to target embryonic stem cells and microinject them into mouse embryos (For details refer to reference article (11,36)). In brief, F1H4 (129S6SvEv/C57BL6F1) embryonic stem cells were electroporated with the linearized vector construct and positive clones were microinjected into 8-cell stage mouse C57BL/6N embryos. Pseudopregnant recipient female mice were anesthetize with Avertin (2,2,2-tribromoethanol; Fluka 90710, Aldrich Chemical) 2.5% in tert-amyl alcohol administered i.p. at a dose of 0.1 ml/10 g body weight and embryos were transferred to uteri by microinjection, weaned pups were scored, and high percentage chimera males were selected for mating with flp-positive C57B/L6N females to remove the selection cassette, to prove germline transmission, and to generate F1 animals for further breeding. Hemizygote animals were identified by genomic tail DNA probed with PCR primers 5'-AATCTCTATGCCCCGTGTGC-3' (F) and 5'-GGCAGGCGAGTGAAGATGTT-3'(R). The copy number of the transgene (4 copies) was estimated by a quantitative PCR assay for the inserted transgene that determines the normalized average difference in threshold cycle among the transgenic ES cell clones and the mice derived from them. Mice were kept and bred under specific pathogen-free conditions (SOPF) in the animal care facility.
Working heart -Working heart experiments with isolated hearts of six months old male CatA-TG mice and their male littermates were performed as previously described (37). Mice were anesthetized with 5% isoflurane in 95% O2 and sacrificed by intraperitoneal (i.p.) injection of ketamine hydrochloride (100mg/kg body weight) and xylazine hydrochloride (10mg/kg body weight). The aorta was cannulated with an 18-G metal cannula for a Langendorff retrograde perfusion mode (baseline, 80mmHg perfusion pressure) with Krebs-Henseleit buffer (KHB) in a working heart apparatus (IH-SR Perfusion system type 844/1 Hugo Sachs Elektronik). After establishing coronary perfusion in the Langendorff mode, "working heart" preparation was continued by cannulating the left atrium through the pulmonary vein with a 16-G steel cannula. This cannula was connected to a preload column, which was water-jacketed and heated to 38°C, resulting in a myocardial temperature of 37°C when the heart was operating in the working mode (preload 10mmHg, afterload 60mmHg). Two platinum pacing electrodes embedded in polyester resin were attached to the right atrium to pace the hearts at about 400bpm. LV systolic and diastolic function was recorded via a high fidelity pressure-conductance catheter (Millar 1.4 F SPR-835, Millar) inserted into the LV cavity through a small puncture in the apex made with a 22 1/4 gauge needle. Cardiac inflow and aortic flow were recorded continuously by inline ultrasonic transit time probes and these measurements were used to calibrate volume measurement of the conductance catheter signal gain. Parallel conductance (conductance signal offset) was determined by the saline dilution method by injecting a 5ml bolus of hypertonic (5%) saline into the left atrial cannula, causing a transient change in the conductivity of KHB in LV cavity.
Histology and immunohistochemical staining -Hearts were rapidly removed, trimmed free from non-cardiac tissues and weighed. Thereafter, the heart was fixed in buffered 4% formaldehyde for 24h and imbedded in paraffin for histological evaluation. Tissue sections of 4μm were fixed at 56°C overnight, deparaffinized, rehydrated and stained with hematoxylin and eosin (HE) (Hematoxylin: #10228.01000, Morphisto GmbH, Frankfurt am Main, Germany and Eosin Y; #1.15935.0025, Merck, Darmstadt, Germany) to determine cardiomyocyte diameter. To visualize tissue fibrosis amount, the sections were stained with Picro-Sirius Red (#13422.00500, Morphisto GmbH, Germany). The percentage of the LV consisting of interstitial collagen was calculated as the ratio of Picro-Sirius Red positively stained area over total LV tissue area, excluding blood vessels and the epi-and endocardial plane. For LV stained with Picro-Sirius Red, polarization microscopy was performed to visualize collagen type I (yellow-red fibers) and type III (green fibers) based on the birefringence properties of collagen (39). Perivascular fibrosis was evaluated as the ratio of fibrosis surrounding the vessel wall to total vessel area. For the analysis Nicon Instruments Software (NIS)-Elements (BR 3.2, Nikon instruments) was used.
For immunostaining of CatA, heart tissue was fixed in buffered 4% formaldehyde for 24h and imbedded in paraffin for histological evaluation. Tissue sections of 4µm were mounted on glass slides, deparaffinized with xylene and hydrated in descending concentrations of ethanol. Following hydration, sections were incubated for 1 hour in 0.05% Citraconic anhydrid (Sigma-Aldrich) at 98°C in a water bath and washed afterwards in 1xPBS-Tween (Phosphate-Buffered Saline: 137mmol/L NaCl; 2.7mmol/L KCl; 4.3mmol/L Na2HPO4; 1.47mmol/L KH2PO4, pH7.4 containing 0.1% Tween) for 10 minutes at room temperature. Slides were incubated with anticathepsin A antibody (R&D Systems #AF1049; dilution 1:30) in 1xPBS-Tween overnight in a moisture-chamber at 4°C. Sections were washed 3x5 minutes with 1xPBS-Tween and secondary antibody (biotin-labeled anti-goat-IgG, dilution 1:30) was added for 2hours and incubated in the moisture-chamber at 37°C. Slides were washed 3x5 minutes with 1xPBS-Tween and incubated with a tertiary streptavidin-peroxidase antibody (#SA202; Chemicon/Millipore) in 1xPBS-Tween and incubated for 20min in a moisture-chamber. After washing as before, slides were incubated shortly with AEC-chromogen (DAKO; #K3464) at room temperature and rinsed with Aqua dest. followed by staining with hematoxylin for 5min and blued with tap water for 15min to allow staining. Slides were mounted with Aquatex (Merck) and analyzed.
TUNEL assay -To assess the amount of apoptotic cells we performed TUNEL (terminal deoxyribonucleotidyl transferase (TdT)mediated dUTP nick end labeling; ApopTag®, Chemicon) assay following the manufacturer´s protocol. In short: Tissue sections of 4µm were mounted on glass slides, deparaffinized with xylene and hydrated in descending concentrations of ethanol. Slices were treated with proteinase K solution (20µg/ml) for 30min and washed twice with bi-distillated water (ddH2O). Afterwards, slices were incubated with 3% hydrogen peroxide solution for 5min, washed twice with phosphate buffered saline solution (1xPBS: 137mmol/l NaCl; 2.7mmol/l KCl; 4.3mmol/l Na2HPO4; 1.47mmol/l KH2PO4, pH 7.4) and incubated with "Working Strength TdT Enzyme" solution for 60min at 37°C in a moisture-chamber. Slices were washed for 10min with a "Stop/Wash" buffer and incubated with anti-digoxygenin conjugate peroxidase for 30min. Afterwards slices were stained with AEC-chromogen (3-amino-9ethylcarbazole; #K3464 DAKO), washed with ddH2O, and staining with hematoxylin for 5min and blued with tap water for 15min to allow staining. Slides were afterwards mounted with Aquatex (Merck) and analyzed.
Cathepsin A activity assay -CatA activity was measured using the fluorogenic peptide substrate V (R&D Systems; #ES005) according to the manufacturer´s protocol. Degradation of the fluorogenic peptide substrate Mca-R-P-P-G-F-S-A-F-K(Dnp)-OH by CatA was monitoring in a spectrofluorometer (Tecan®; Germany) at 330nm excitation and 390nm emission.
Statistical analysis -All data are expressed as mean±SD. Statistical analysis was carried out using Prism software (Graph Pad Version 7) or Progenesis® LC-MC software (Non-linear Dynamics) for proteomics. An unpaired Student´s t-test (two-tailed) was used for statistical analyses comparing 2 groups. Pvalues of <0.05 and fold-change >2 (for proteomics) were considered statistically significant.  Figure 1: Proteomics of cathepsin A (CatA) degradation products in conditioned media from cardiac fibroblasts (CFs). (A) Proteomics-workflow of degradation products in conditioned media from CFs after digestion with CatA. Isolation and culture of primary mouse CFs: Primary mouse CFs were isolated from the hearts of male C57BL/6N mice (n=4). For secretome analysis, 80% confluent CFs were cultured in serum-free medium for 3 days. The secreted proteins from the conditioned media were either incubated with human recombinant CatA (1µg/mL) or without (control group) in assay buffer (pH5.5). Degradation products in the <3kDa fractions were analyzed by LC-MS/MS and significantly changed peptides (>2-fold intensity change, p<0.05) were identified by Mascot (Matrix Science; version 2.3.01) in a no-enzyme search against the UniProt/SwissProt mouse database. (B) Proteins with differential abundance in the conditioned medium after filtering using 3kDa columns to recover protein fragments (collagens (CO5A1, CO5A2, CO3A1, CO1A1), Decorin (PGS2), Laminin Subunit Alpha 4 (LAMA4), Basement membrane-specific heparan sulfate proteoglycan core protein (PGBM), Fibronectin (FINC) and extracellular superoxide dismutase (SODE or EC-SOD). (C) Three peptides were detected for EC-SOD. The Figures display the retention time (RT, y-axis) versus the mass-to-charge ratio (m/z, x-axis) for the three EC-SOD peptides. Quantification based on normalized abundance is shown for each of the peptides (n=4 per group). All values are presented as mean±SD.      In the ECM, EC-SOD protects against reactive oxygen species by dismutation of . O2to H2O2 and O2. The carboxypeptidase cathepsin A (CatA) is expressed by both CFs and cardiomyocytes (CMs) and secreted into the extracellular space where it proteolytically degrades EC-SOD, thereby regulating EC-SOD distribution in the ECM. Cardiomyocyte-specific overexpression of CatA leads to enhanced EC-SOD protein degradation and accelerated depletion from the ECM, subsequently increasing vulnerability to reactive oxygen species followed by cardiac fibrosis formation with a higher quantity of collagen type I fibers (red fibers) and myocardial hypertrophy.